CN109097333B - It resists cell ageing and extends the mescenchymal stem cell and its preparation method and application of blood glucose-control effect time-histories - Google Patents
It resists cell ageing and extends the mescenchymal stem cell and its preparation method and application of blood glucose-control effect time-histories Download PDFInfo
- Publication number
- CN109097333B CN109097333B CN201810785793.0A CN201810785793A CN109097333B CN 109097333 B CN109097333 B CN 109097333B CN 201810785793 A CN201810785793 A CN 201810785793A CN 109097333 B CN109097333 B CN 109097333B
- Authority
- CN
- China
- Prior art keywords
- cell
- gene
- stem cell
- aav
- sgrna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 44
- 210000004369 blood Anatomy 0.000 title claims abstract description 17
- 239000008280 blood Substances 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 230000032677 cell aging Effects 0.000 title claims abstract description 11
- 230000000694 effects Effects 0.000 title abstract description 12
- 210000004027 cell Anatomy 0.000 claims abstract description 72
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 50
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 32
- 230000005611 electricity Effects 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000003780 insertion Methods 0.000 claims abstract description 21
- 230000037431 insertion Effects 0.000 claims abstract description 21
- -1 RNP compound Chemical class 0.000 claims abstract description 19
- 239000000725 suspension Substances 0.000 claims abstract description 18
- 108091033409 CRISPR Proteins 0.000 claims abstract description 14
- 210000002845 virion Anatomy 0.000 claims abstract description 13
- 238000001890 transfection Methods 0.000 claims abstract description 12
- 230000006698 induction Effects 0.000 claims abstract description 9
- 230000004069 differentiation Effects 0.000 claims abstract description 8
- 230000003321 amplification Effects 0.000 claims abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 6
- 241000700605 Viruses Species 0.000 claims description 24
- 108020004414 DNA Proteins 0.000 claims description 23
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 claims description 7
- 238000007385 chemical modification Methods 0.000 claims description 7
- 239000006285 cell suspension Substances 0.000 claims description 6
- 210000002242 embryoid body Anatomy 0.000 claims description 6
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 230000006801 homologous recombination Effects 0.000 claims description 5
- 238000002744 homologous recombination Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 206010022489 Insulin Resistance Diseases 0.000 claims description 4
- 230000003915 cell function Effects 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 4
- 238000002054 transplantation Methods 0.000 claims description 4
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims description 4
- 102100022464 5'-nucleotidase Human genes 0.000 claims description 3
- 102100037241 Endoglin Human genes 0.000 claims description 3
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 claims description 3
- 101000881679 Homo sapiens Endoglin Proteins 0.000 claims description 3
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 claims description 3
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 claims description 3
- 210000004204 blood vessel Anatomy 0.000 claims description 3
- 230000001413 cellular effect Effects 0.000 claims description 3
- 208000037976 chronic inflammation Diseases 0.000 claims description 3
- 230000006020 chronic inflammation Effects 0.000 claims description 3
- 230000006378 damage Effects 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 230000005684 electric field Effects 0.000 claims description 3
- 108010082117 matrigel Proteins 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- 230000008929 regeneration Effects 0.000 claims description 3
- 238000011069 regeneration method Methods 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 3
- 238000002560 therapeutic procedure Methods 0.000 claims description 3
- 238000012136 culture method Methods 0.000 claims description 2
- 238000000684 flow cytometry Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 210000003716 mesoderm Anatomy 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims 1
- 101150094574 GPX7 gene Proteins 0.000 abstract description 2
- 208000002267 Anti-neutrophil cytoplasmic antibody-associated vasculitis Diseases 0.000 description 28
- 241000702421 Dependoparvovirus Species 0.000 description 16
- 239000000047 product Substances 0.000 description 15
- 239000001963 growth medium Substances 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 11
- 230000014509 gene expression Effects 0.000 description 9
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 9
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 8
- 210000003954 umbilical cord Anatomy 0.000 description 8
- 230000032683 aging Effects 0.000 description 7
- 101000910035 Streptococcus pyogenes serotype M1 CRISPR-associated endonuclease Cas9/Csn1 Proteins 0.000 description 6
- 108010076089 accutase Proteins 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 230000001079 digestive effect Effects 0.000 description 6
- 238000004043 dyeing Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 238000004806 packaging method and process Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 108010005774 beta-Galactosidase Proteins 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000003198 gene knock in Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 4
- 229960004359 iodixanol Drugs 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 241001164825 Adeno-associated virus - 8 Species 0.000 description 3
- 238000010354 CRISPR gene editing Methods 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 101000819074 Homo sapiens Transcription factor GATA-4 Proteins 0.000 description 3
- 102100021380 Transcription factor GATA-4 Human genes 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 102100034229 Citramalyl-CoA lyase, mitochondrial Human genes 0.000 description 2
- 101000710917 Homo sapiens Citramalyl-CoA lyase, mitochondrial Proteins 0.000 description 2
- 101001000998 Homo sapiens Protein phosphatase 1 regulatory subunit 12C Proteins 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
- 102100035620 Protein phosphatase 1 regulatory subunit 12C Human genes 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 2
- 210000001789 adipocyte Anatomy 0.000 description 2
- 102000005936 beta-Galactosidase Human genes 0.000 description 2
- 239000008004 cell lysis buffer Substances 0.000 description 2
- 238000003889 chemical engineering Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 101150101112 7 gene Proteins 0.000 description 1
- 241000580270 Adeno-associated virus - 4 Species 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000006587 Glutathione peroxidase Human genes 0.000 description 1
- 102100036755 Glutathione peroxidase 7 Human genes 0.000 description 1
- 108700016172 Glutathione peroxidases Proteins 0.000 description 1
- 101001071391 Homo sapiens Glutathione peroxidase 7 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108010034546 Serratia marcescens nuclease Proteins 0.000 description 1
- 108091028113 Trans-activating crRNA Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000013412 genome amplification Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004968 inflammatory condition Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000012666 negative regulation of transcription by glucose Effects 0.000 description 1
- 230000006780 non-homologous end joining Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000008943 replicative senescence Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Gynecology & Obstetrics (AREA)
- Immunology (AREA)
- Diabetes (AREA)
- Rheumatology (AREA)
- Hematology (AREA)
- Reproductive Health (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Transplantation (AREA)
- Physics & Mathematics (AREA)
- Endocrinology (AREA)
Abstract
Present disclose provides a kind of methods for preparing and resisting the mescenchymal stem cell of cell ageing and extension blood glucose-control effect time-histories, and this method comprises the following steps: S1, will be mixed to form RNP compound for sgRNA the and Cas9 albumen for knocking in site of gene insertion safety island；S2, the AAV virion to be transfected for carrying GPX7 gene is formed；S3, the suspension of stem cell is mixed with the suspension of the RNP compound and carries out electricity turn；S4, transfection of the suspension of AAV virion to be transfected to carry out is added；S5, it carries out monoclonal culture and filters out to have carried out the monoclonal cell strain that gene orientation is knocked in；S6, amplification and Induction of committed differentiation become MSC.The disclosure additionally provides the mescenchymal stem cell and application thereof that the above method is prepared.Through the above technical solutions, present invention obtains a kind of resistance cell ageing and the multipotential stem cell preparation methods of extension blood glucose-control effect time-histories.
Description
Technical field
This disclosure relates to which technical field of pharmaceutical biotechnology, more particularly to a kind of resistance cell ageing and there is adjusting blood glucose to make
Mescenchymal stem cell and its preparation method and application.
Background technique
Type-1 diabetes mellitus be by beta Cell of islet autoimmune inflammation caused by β cytoclasis, patient's body is due to pancreas islet
Cell function missing, patient need lifelong progress injection of insulin treatment.Latest result shows, China have every year 13000 it is new
Type-1 diabetes mellitus is sent out, between past 20 years, child morbidity increases nearly 4 times within 15 years old or less.
Research finds that the miRNA in mescenchymal stem cell (MSCs) excretion body can be relieved insulin resistance.In addition, MSCs is also
It can promote β cell autophagy, promote α cell to β cells switch, protect β cell, promote β cytothesis.It is total at above-mentioned two aspect
Under same-action, hypoglycemic effect is generated.The cell origin of Current Domestic mescenchymal stem cell therapy is broadly divided into autologous (bone
Marrow, adipose tissue etc.) and two kinds of xenogenic origin (umbilical cord, placenta etc.).But the mescenchymal stem cell in this two kinds of sources, generally
There is following problems: (1) being difficult to draw materials (especially marrow)；(2) pot-life is shorter；(3) increase mesenchyma with the age to do
Cell quantity and expansion potential significantly reduce, and amplification in vitro but will accelerate mescenchymal stem cell aging；(4) with the age increase between fill
Matter stem cell differentiation potential substantially fails, such as: the mescenchymal stem cell for being derived from young man is easily divided into the cells such as cartilage, and takes
Tend to fat cell from the Derived from Mesenchymal Stem Cells of old man；(5) preparation process is not easy Quality Control, such as: Different Individual and same
The mescenchymal stem cell difference in one individual different tissues organ sites source is very big, can not predict.These disadvantages are largely
On result in the mescenchymal stem cell of adult origin as stem cell drugs, quality control process lacks controllability, and curative effect exists
Unstability, to limit its clinical application as cell transplantation drug.That is, the replicability of mescenchymal stem cell
The defect of aging is the technology barrier for limiting its clinical application.
CRISPR gene editing technology forms DNA double by carrying out accurate targeting shearing in genome specific site
Chain notch (Double-strand breaks, DSBs) links (Non-homologous end by intracellular nonhomologous end
Joining, NHEJ) it realizes to the knockout of gene in situ, pass through homologous recombination (Homology directed repair, HDR)
In the presence of external source gene template, the introducing of point mutation and inserting for reparation and large fragment gene (such as reporter gene) are realized
Enter.As described in patent document CN105985985A, slow virus is constructed using CRISPR/Cas9 system, to umbilical cord source
Immunizing antigen B2M, inflammatory factor TNF-α in MSCs are knocked out, so that realizing reduces allosome mescenchymal stem cell immunogen
The purpose of property.But in mammalian cells, NHEJ revision points account for dominant pattern, realize gene knockout than realizing gene knock-in
It is easy very much.And the segment of gene knock-in is bigger, it is lower to integrate probability, and technology is realized more difficult.It has had not yet to see
The report of large fragment gene knock-in is carried out in MSCs.
Summary of the invention
The purpose of the disclosure is to overcome the defect of easy aging existing for mescenchymal stem cell, and cell can be resisted by obtaining one kind
Aging and the cell therapy product for extending blood glucose-control effect time-histories.
To achieve the goals above, present disclose provides a kind of resistance cell ageings and extension blood glucose-control to act on time-histories
The preparation method of mescenchymal stem cell, this method comprises the following steps: S1, will knock in site for gene insertion safety island
SgRNA and Cas9 albumen is mixed to form RNP compound；S2, AAV disease will be packaged into inserted with template DNA homologous recombination vector
In poison, AAV virion to be transfected is formed；The template DNA includes knocking in the left same of site for gene insertion safety island
Source arm sequence and right homology arm sequence, inserted with GPx7 gene between the left homology arm sequence and the right homology arm sequence；
S3, the suspension of iPSC cell or embryonic stem cell is mixed to the suspension of the RNP compound and is carried out electricity turn, after obtaining electricity turn
Culture；S4, in 1-30 minutes, AAV virus to be transfected is added in the material after turning to the electricity after electricity turns
Culture of the suspension of grain to carry out transfection in 4-30 hours, after being transfected；It is S5, the culture limit after the transfection is dilute
Monoclonal culture is carried out after releasing, and the monoclonal cell strain for having carried out gene orientation and having knocked in is filtered out by PCR and sequencing；
S6, the gene that carried out is oriented into the monoclonal cell strain amplification knocked in and Induction of committed differentiation as mescenchymal stem cell.
On the other hand, the disclosure additionally provides the mescenchymal stem cell that the above method is prepared.
In another aspect, the disclosure additionally provides the purposes of above-mentioned mescenchymal stem cell, the purposes be it is following any one:
Preparation improves the product of insulin resistance and/or chronic inflammation；Prepare the product of cellular transplantation therapy；Preparation improves β cell function
The therapeutic products of energy；Preparation regeneration and/or the product for repairing damage blood vessel.
Through the above technical solutions, the present invention mescenchymal stem cell gene insertion safety island (including rDNA 28s,
RDNA 18S, rDNA45s, CLYBL and AAVS1) position is accurate, is efficiently inserted into GPx7 Expression element, Lai Tisheng GPx7 expression quantity
To delay the replicative senescence of mescenchymal stem cell, to extend the time of the adjusting blood glucose of mescenchymal stem cell.
Other feature and advantage of the disclosure will the following detailed description will be given in the detailed implementation section.
Specific embodiment
The specific embodiment of the disclosure is described in detail below.It should be understood that described herein specific
Embodiment is only used for describing and explaining the disclosure, is not limited to the disclosure.
Present disclose provides a kind of resistance cell ageing and the systems for the mescenchymal stem cell for extending blood glucose-control effect time-histories
Preparation Method, this method comprises the following steps: S1, sgRNA the and Cas9 egg for knocking in site that will will be directed to gene insertion safety island
It is white to be mixed to form RNP compound；S2, the homologous recombination vector inserted with template DNA is packaged into AAV virus in, formed to
Transfect AAV virion；The template DNA includes the left homology arm sequence for knocking in site and the right side for gene insertion safety island
Homology arm sequence, inserted with GPx7 gene between the left homology arm sequence and the right homology arm sequence；S3, by iPSC cell
Or the suspension of embryonic stem cell mixes with the suspension of the RNP compound and carries out electricity and turns, and obtains the culture after electricity turns；S4,
Electricity turn after 1-30 minute in, to it is described electricity turn after material in be added the suspension of AAV virion to be transfected with into
Row transfection in 4-30 hours, the culture after being transfected；S5, Dan Ke will be carried out after the culture Method of Limited Dilution after the transfection
Longhua culture, and the monoclonal cell strain for having carried out gene orientation and having knocked in is filtered out by PCR and sequencing；S6, by the progress
The monoclonal cell strain amplification and Induction of committed differentiation that gene orientation is knocked in become mescenchymal stem cell.
SgRNA and Cas9 albumen is transferred in cell in such a way that electricity turns by the present invention RNP compound, and after electricity turns
The suitable time in selection AAV virus by be inserted into GPx7 gene homologous recombination vector be transfected into cell, finally make
SgRNA, Cas9 albumen and carrier inserted with template DNA carry out the external source base so that large fragment commonly through CRISPR gene editing
Target position is knocked in because being able to orientation.
Wherein, the definition of the gene insertion safety island is the safe insertion point of foreign gene, after foreign gene insertion simultaneously
It will not influence the gene expression of cell itself；The gene insertion safety island can be RNA45SN4, rDNA 28S (NCBI
Gene ID be 106632264), (NCBI Gene ID is by rDNA 18S (NCBI Gene ID is 106631781), rDNA 45S
109864271), CLYBL (NCBI Gene ID is 171425) or AAVS1 (NCBI Gene ID is 17)；The insertion gene
GPx7, NCBI Gene ID are 2882.Preferably, the gene insertion safety island is RNA45SN4, NCBI Gene ID
It is 109864271.
Wherein, optionally, the sgRNA has chemical modification group；The chemical modification group is preferably methyl chemistry
Modification group or phosphorothioate chemical modification group；SgRNA the and Cas9 albumen for knocking in site of safety island is inserted into for gene
Dosage molar ratio is 1:1 to 1:5.Wherein it is possible to using online sgRNA design tool (http://crispr.mit.edu/) according to
The sequence for knocking in site design sgRNA of safety island is inserted into according to gene and is synthesized.Wherein, the 5 ' ends and 3 ' of sgRNA sequence
That holds end can be added with methyl (- O-Me) chemical modification group or phosphorothioate (- phosphorothioate) chemistry respectively
Modification group.
Wherein, optionally, the time that will be mixed for sgRNA the and Cas9 albumen for knocking in site of gene insertion safety island
It is 5-20 minutes, temperature is 10-40 DEG C.
Wherein, optionally, in step S3, the suspension of the iPSC cell or embryonic stem cell and the RNP compound are mixed
When conjunction, relative to every 106A iPSC cell or embryonic stem cell, with the meter of sgRNA, the dosage of the RNP compound
For 1-50 μm of ol；In the suspension of 1016 cell, cell concentration is (1-5) × 107A/mL；It is described with the meter of sgRNA
The final concentration of 0.1-1.5 μm of ol/ μ L of RNP compound.
Wherein, optionally, in step S3, the condition that electricity turns includes: that electric field strength is 50-250V/cm, single pulse time
For 2-15ms, the time interval between adjacent two subpulse is 10-60s, and total pulses are 2-10 times.
Wherein, optionally, it in step S4, after electricity turns in 5-20 minutes, is added in the material after turning to the electricity
Culture of the suspension of AAV virion to be transfected to carry out transfection in 8-24 hours, after being transfected.
Wherein, optionally, in step S4, the additional amount of the suspension of AAV virion to be transfected makes AAV to be transfected
The MOI value of virion is 104-106.The ratio of virus and cell quantity when MOI value is infection.
Wherein it is preferred to.The serotype of the AAV virus can for AAV-1 virus, AAV-2 virus, AAV-3 virus,
AAV-4 virus, AAV-5 virus, AAV-6 virus, AAV-7 virus, AAV-8 virus, AAV-9 virus, AAV-DJ virus and AAV-
DJ/8 virus, preferably AAV-8 virus, AAV-6 virus, AAV-1 virus or AAV9 virus, the blood of the more preferable AAV virus
Clear type is AAV8 virus, under the preferable case, can further increase the efficiency of large fragment gene knock-in stem cell.
Wherein, the GPx7 gene refers to that 7 gene of endoplasmic reticulum glutathione peroxidase, NCBI Gene ID are
2882。
Wherein, the template DNA may include left homology arm sequence, knock in segment and right homology arm sequence；Preferably, institute
It states and is inserted with cmv enhancer, CMV promoter, GPx7 gene between left homology arm sequence and the right homology arm sequence
Expression cassette and bGH poly A；Preferably, the sequence being inserted between the left homology arm sequence and the right homology arm sequence is such as
Shown in SEQ ID NO.4.
Wherein it is preferred to which the sgRNA is as shown in SEQ ID NO.1；Correspondingly, the left homology arm sequence such as SEQ
Shown in ID NO.2；Correspondingly, the right homology arm sequence is as shown in SEQ ID NO.3.
Wherein, optionally, the frame sequence of the carrier is as shown in SEQ ID NO.7.The frame sequence of the carrier is
Refer to the sequence for being not inserted into the carrier of template DNA.
Wherein, optionally, the condition of amplification and Induction of committed differentiation includes: that the gene that carried out is oriented the list knocked in
Clonal cell line forms embryoid body after continuing culture in low adherency culture plate 18-100 hours；Then the embryoid body is inoculated with
With flow cytometry sorting, wherein CD73, CD90 and CD105 are positive cell after continuing culture 10-20 days on matrigel
Group.
On the other hand, the disclosure additionally provides the mescenchymal stem cell that the above method is prepared.
In another aspect, the disclosure additionally provides the purposes of above-mentioned mescenchymal stem cell, the purposes be it is following any one:
Preparation improves the product of insulin resistance and/or chronic inflammation；Prepare the product of cellular transplantation therapy；Preparation improves β cell function
The therapeutic products of energy；With preparation regeneration and/or the product for repairing damage blood vessel.
Present invention be described in more detail by the following examples:
Embodiment 1
Construct the insertion in the safe site (RNA45SN4) of 1016 cell strains (being purchased from Harvard University's stem cell bank)
The cell model of GPX7 gene expression Cassette.
1, sgRNA design and synthesis
(1) safety island is inserted into gene by online sgRNA design tool (http://crispr.mit.edu/)
(RNA45SN4) RNA45SN4sgRNA of targets identification is designed on, preferred result is as follows: 5 '-acgccgcggcggccgucggg
guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc
uuuuuuu-3'(SEQ ID NO.1).Wherein 1-20 sequences are identification motif, remaining sequence is tracrRNA.
(2) sgRNA is synthesized and by Integrated DNA Technologies US company the 5 ' of the sgRNA sequence
The modification of O-Me, phosphorothioate are not added on No. two positions of three bases at end and 3 ' end ends point and third place.
2, the activity of in vitro method detection sgRNA
(1) target sequence segment (2000bp) is identified from genome amplification, primer is had by giving birth to work bioengineering (Shanghai) share
The synthesis of limit company.
GPx7FW:5 '-gaggaattcccagtaagtgc-3 ' (SEQ ID NO.5),
GPx7REV:5 '-gcgctcccccgaccctctct-3 ' (SEQ ID NO.6).
(2) pcr amplification product 200ng, sgRNA 100ng, SpCas9 200ng (are purchased from Sigma-Aldrich, goods
Number: TGEN-CP-500UG), 10 × buffer, 2 μ L is configured to 20 μ L reaction systems.Reaction system keeps the temperature one hour at 37 degree,
70 degree keep the temperature one hour.
(3) using TAE configuration 1% BioWest agarose gel, the electrophoresis 30min under the voltage of 90V, using gel at
As instrument observes result.
3, the selection of AAV packaging system and plasmid construction
(1) cell to be edited is 1016 cell strains, and according to the AAV tissue affinity table of comparisons, preferably serotype is AAV-8's
Packaging system.
(2) the overall package capacity of AAV-8 is 4.7Kb.The segment being inserted into the carrier loaded of AAV packaging system should wrap
Containing left homology arm (500bp or so), Insert Fragment and right homology arm (500bp or so).
Left homology arm sequence is SEQ ID NO.2.
Right homology arm sequence is SEQ ID NO.3, wherein 4-7 bit base is the site PAM by mutation.
Insert Fragment is that a complete Cassette includes that CMV increases son, CMV promoter, GPX7 CDS and bGH
Poly A, total 1296bp, sequence is as shown in SEQ ID NO.4, wherein 1-507 are promoter, 508-1071 are
EGFP, 1072-1296 are bGH pol yA.(it is limited to be purchased from NEB (Beijing) using NheI and BsmI restriction enzyme
Company) Cassette is inserted on pAAV carrier, carrier sequence is SEQ ID NO.7.In order to avoid SpCas9/sgRNA
RNP compound influences the efficiency of gene editing on the secondary cut of recombination site, poly- by PrimerStar high-fidelity DNA
Synthase (being purchased from precious bioengineering (Dalian) Co., Ltd, article No.: R044A) is introduced using the method that PCR carves ring in the site PAM
Point mutation (TGG sports TAG), the cutting so as to avoid SpCas9/sgRNA RNP compound to recognition site.Point is prominent
Become primer to be synthesized by Sangon Biotech (Shanghai) Co., Ltd..
GPx7-PAM-mut-FW:5'-gcggcggccgtcgggtaggggctttacccggcg-3'(SEQ ID NO.8)；
GPx7-PAM-mut-REV:5’-cgccgggtaaagcccctacccgacggccgccgc-3’(SEQ ID
NO.9)。
4, AAV virus packaging and purifying
(1) virus packaging on the day before by HEK293T cell according to every ware 5 × 106Quantity plant to diameter 10cm's
Containing 10mL complete medium (DMEM+10%FBS+1%P/S is dual anti-), (DMEM culture medium, is purchased from ThermoFisher
Scientific, Inc., article No.: C11995500BT；FBS, is purchased from ThermoFisher Scientific, Inc., article No.:
sv30087.02；P/S is dual anti-, is purchased from ThermoFisher Scientific, Inc., article No.: SV30010) CORNING training
It supports in ware, plants 30 wares altogether.In 37 degree, 5%CO2Cell incubator in cultivate 24 hours.
(2) the virus packaging same day checks whether HEK293T cell confluency degree reaches 80%.Every ware is independent according to the following steps
Configure rotaring redyeing system: by the pAAV-Cassette of 10 μ g, use is without blood after the pAAV9-RC mixing of the pHelper of 10 μ g, 10 μ g
It is mixed after clear DMEM adjustment volume to 910 μ L through vortex oscillator, the PEI that 90 μ L are added (is purchased from Polysciences Asia
Pacific, Inc., article No.: 23966-2) after reuse vortex oscillator mixing, stand 15 minutes.By CORNING culture dish
In complete medium be substituted for 9mL serum free medium, then will be dropped evenly before by the DNA-PEI compound stood
In culture dish, softly shakes and even be placed on 37 degree, 5%CO2Cell incubator in cultivate 6 hours.CORNING is trained after transfection 6h
The serum free medium supported in ware is substituted for complete medium, in 37 degree, 5%CO2Cell incubator in continue to cultivate.
(3) after transfection 60 hours, supernatant cell is harvested respectively.
Obtained supernatant will be collected and discard impurity after ten minutes through 4 degree of centrifugations of 4000rpm.It will go on deimpurity to reset and add
Enter Amicon Ultra-15 and surpass and (be purchased from Merck Chemical Engineering Technology (Shanghai) Co., Ltd., article No.: UFC 905096) from column, passes through
Volume concentration to 10 was arrived into 15mL in centrifugation 30 minutes after 4000rpm several times, 4 degree.The HEK293T scraped with cell scraper is thin
Born of the same parents blow even and are transferred in 50mL centrifuge tube with appropriate culture medium, abandon supernatant, Suo Youchen after 1500rpm, 4 degree of centrifugation 10min
It forms sediment in total plus 3mL cell lysis buffer solution (150mM NaCl, 20mM tris pH8.0) makes its resuspension.Cell will be resuspended -80
Multigelation is three times in DEG C alcohol bath and 37 DEG C of water-baths.The cell suspension of the supernatant freeze thawing of concentration is mixed, 1M is added
MgCl2To final concentration of 1mM.Addition Benzonase (is purchased from Merck Chemical Engineering Technology (Shanghai) Co., Ltd., article No.: 70746-
1kU) to final concentration of 25U/mL, 37 DEG C of reaction 40min after mixing.50mL centrifuge tube is taken out, 4 DEG C, 4000rpm is centrifuged 20min,
Take supernatant.
(4) Sigma-Aldrich (is purchased from, article No.: D1556- using Iodixanol density-gradient centrifugation method purified virus
250mL).Configure Iodixanol gradient 17%:5mL 10 × PBS, 0.05mL 1M MgCl2,0.125mL 1M KCl,10mL
5M NaCl, 12.5mL Optiprep, adds water to supply 50mL.25%:5mL 10 × PBS, 0.05mL 1M MgCl2,
0.125mL 1M KCl, 20mL Optiprep, 0.1mL 0.5%phenol red, adds water to supply 50mL.40%:5mL 10
×PBS,0.05mL 1M MgCl2, 0.125mL 1M KCl, 33.3mL Optiprep adds water to supply 50mL.60%:
0.05mL 1M MgCl2, 0.125mL 1M KCl, 50mL Optiprep, 0.025mL 0.5%phenol red.To hypervelocity from
The Iodixanol of 3.5mL 60%, 3.5mL 40%, 4mL 25%, 4mL 17% are successively slowly added in heart pipe from the bottom to top.
The supernatant cell pyrolysis liquid of concentration is slowly added in centrifuge tube top layer, fills centrifuge tube with cell lysis buffer solution.It uses
Beckman L-80XP landing ultracentrifuge, 70Ti determine angle rotor, accelerate 6, slow down 4 degree of 9,60000rpm and are centrifuged 2 hours.With
Tack syringe draws 40% concentration layer Iodixanol, is transferred to Amicon Ultra-15 and surpasses from column, 10mL PBS is added
4 DEG C of 4000rpm are centrifuged 20 minutes, are repeated 3 times.By viral centrifugal concentrating to 1mL.
(5) titre detection is carried out to AAV8 using qPCR.According to EGFP primers, make the length of qPCR product about
200bp.QPCR primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
AAV-GPx7-F:5’-actggtgtcgctggagaagt-3’(SEQ ID NO.10)。
AAV-GPx7-R:5’-caatctccttgttgctgtcag-3’(SEQ ID NO.11)。
7 AAV8 recombinant plasmid standard items are prepared, gradient dilution is carried out with the ratio of 1:10.It is below dense from 10ng/ μ L
Degree start to dilute, respectively 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, 0.0001ng/ μ L and
0.00001ng/μL。
Diluted DNA copy number is calculated according to the following formula:
Virus Sample is pre-processed using DNaseI (it is purchased from precious bioengineering (Dalian) Co., Ltd, article No.:
2270A).(Japan is purchased from and spins (Shanghai) Biotechnology Co., Ltd, article No.: QPS-201) configuration using 2 × SYBR PCR mix
QPCR reaction system.Use Roche480II real-time fluorescence quantitative PCR system carries out quantitative PCR.According to
Ct value draws standard curve, and calculates the titre of AAV9.
5, the assembling of RNP compound and the pretreatment of cell to be edited
(1) by SpCas9 (1 μm of ol/ μ L of final concentration) and RNA45SN4sgRNA (1 μm of ol/ μ L of final concentration) rubbing according to 1:3
You are than carrying out being blended in incubation at room temperature 10min, to form SpCas9/sgRNA RNP compound.
(2) 1016 cell strains of observation, which converge rate and reach 80%, moves back except mTeSR culture medium (is purchased from StemCell
Article No.: 85850) Technologies, Inc., are rinsed primary with PBS.Accutase digestive ferment is added (to be purchased from
ThermoFisher Scientific, Inc., article No.: A1110501) it is allowed to that ware bottom is completely covered.37 DEG C after incubation 3-5 minutes
Stop digestion, sucks Accutase digestive ferment.At once fresh mTeSR culture medium is added, blows and beats culture dish with 1mL rifle sector
Bottom, the stem cell colonies for attaching ware/bottom of bottle fall off, and soft slowly pressure-vaccum mixes, and stem cell suspension is made.Use blood count
Plate counts cell density.It (is purchased from using Opti-MEM (magnesium chloride of ATP, 23.6mM of 14.5mM)
Article No.: 11058021) ThermoFisher Scientific, Inc. adjust cell density to 5 × 107/mL。
(3) (with the meter of sgRNA, the RNP is multiple for the SpCas9/sgRNA SNP compound by 10 μ L by incubation at room temperature
It closes the content of object and passes through 1016 cell suspension (the wherein cell numbers for counting and being resuspended using Opti-MEM for 7.5 μm of ol) and 10 μ L
Amount is 5 × 105It is a) it mixes, 20 μ L mixed liquors are transferred in 16 hole electricity rotating plate items.It is selected using Lonza 4D nuclear transfection system
Selecting CB150 mode, (electric field strength 150V/cm, single pulse time are 10ms, and the time interval between adjacent two subpulse is
20s, total pulses are 5 times) carry out electricity turn.Cell is transferred at once after the completion of turning and (is purchased from by Geltrex coating by electricity
ThermoFisher Scientific, Inc., article No.: A1413202) and it is added to 500 μ L mTeSR and 10 μM of Y-
27632 (are purchased from Stem Cell Technologies, Inc., article No.: 72304) in 37 degree, 5%CO in 24 orifice plates2It is thin
Continue to cultivate in born of the same parents' incubator.
(4) electricity has turned to start in the 5 to 20th minute according to 1 × 105MOI value be gently added dropwise AAV-DJ, and turn in electricity
It is added dropwise in complete 20 minutes.
(5) electricity removes old culture medium after having turned 24 hours, is changed to new mTeSR culture medium (containing 10 μM of Y-
27632)
6, the culture of monoclonal cell strain:
(1) electricity has turned to rinse a culture dish using PBS on the 48th hour.Accutase digestive ferment is added to be allowed to be completely covered
Ware bottom.37 DEG C stop digestion after incubation 3-5 minutes, suck Accutase digestive ferment.At once fresh mTeSR culture medium is added,
Culture dish/bottom of bottle is blown and beaten with 1mL rifle sector, the stem cell colonies for attaching ware bottom fall off, and soft slowly pressure-vaccum mixes, and guarantee thin
Intercellular does not have adhesion, and stem cell suspension is made.Cell density is counted using blood counting chamber.By each 10cm culture dish
The ratio of 15000 cells cell seeding to by Geltrex be coated with and be added to 10mL mTeSR and 10 μM of Y-
In 37 degree, 5%CO in 27632 10cm CORNING culture dish2Cell incubator in continue to cultivate.
(2) it is observed and is removed old culture medium within every 24 hours, be changed to new mTeSR culture medium (containing 10 μM
Y-27632).It can be in microscopic observation to the formation of clone after 72 hours.
(3) 12 to 14 days sizes that can see clone under ten times of object lens have been equivalent to a coin after electricity turns.
Clone cannot be allowed to continue to become larger or intersect.120 μ L are added in by each hole on coated 96 orifice plate of Geltrex
MTeSR (contains 10 μM of Y-27632), marking plate O.It is observed in super-clean bench by microscope, P200 pipettor is adjusted
It uses the pipette tips with filter core to scrape broken clone to 45 μ L, collect cell with pipettor and is transferred in the aperture of 96 orifice plates.
(4) old culture medium is removed after every 24 hours after clone's picking, is changed to new mTeSR culture medium and (contains
10 μM of Y-27632) cell confluency rate reaches 80% after four days.
(5) (contain 10 μM by 133 μ LmTeSR are added in each hole on coated 96 orifice plate of Geltrex at two pieces
Y-27632), it is respectively labeled as plate A, plate B.Every hole of plate O is rinsed using the PBS of 150 μ L.It is added 35 μ L's in every hole
The mTeSR of 165 μ L is added in Accutase digestive ferment, digestion 5 into every hole after ten minutes (containing 10 μM of Y-27632).From hole
The cell suspension for pipetting 66 μ L respectively is transferred in the respective apertures of plate A and plate B.Plate A is extracted for genome, and plate B is spare.?
It is directly added in each hole of plate O after the mFreSR (being purchased from Stem Cell Technologies, Inc.) of 165 μ L as deep low
Temperature refrigerator storage.
7, gene editing Efficiency testing (allele insertion detection)
(1) design primer (nonconformity site PCR product about 2000bp or so, the integration site PCR product outside homology arm
3400bp).Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
GPx7FW:5 '-gaggaattcccagtaagtgc-3 ' (SEQ ID NO.5),
GPx7REV:5 '-gcgctcccccgaccctctct-3 ' (SEQ ID NO.6).
(2) picked clones from 96 orifice plates, extracting postgenome carry out PCR amplification.
(3) PCR product is subjected to electrophoresis and analyzed.Only 2000bp or so band is non-editor's cell, existing
It is editor's cell that 2000bp or so band, which also has 3400bp,.It marks respectively and counts the ratiometric result of these two types of editing types such as
Shown in table 1.
Table 1
| Non- editor's cell proportion | Gene editing cell | |
| Embodiment 1 | 25% | 75% |
8, multipotential stem cell Induction of committed differentiation becomes MSC
1016 cell strains of GPx7 insertion are subjected to embryoid tire (EB) differentiation
Prepare 300-500 cell, uniform 1016 clone of GPx7 insertion is cleaned once with room temperature PBS, used
Digestion 3-5 minutes of 37 degree of Accutase digestive ferment.After 1016 Clone formation spheres, after being resuspended with CDF12 culture medium, it is added to low
It adheres in culture plate (article No. 3471 is purchased from CORNING company).37 degree, 5%CO2Cell incubator in continue culture 1 to 3
Embryoid body is formed after it.
The embryoid body of acquisition is inoculated in matrigel (geltrex is purchased from ThermoFisher Scientific, Inc.)
It is cultivated in coated six orifice plate, continues to cultivate 2 weeks to fibrous cell's appearance.After primary passage, streaming is utilized
Wherein CD73, CD90, CD105 are positive cell colony for cell art sorting, this is the enhanced MSC of GPx7.
Comparative example 1: umbilical cord mesenchymal stem cells
1 cultural method of comparative example method referring to described in " the human umbilical cord mesenchymal stem cells cultural method of improvement ".
Testing example:
Aging state after the enhanced MSC subculture in vitro separately of the GPx7 that embodiment 1 obtains detects (relative to comparative example 1)
The dyeing of cell ageing beta galactosidase be it is a kind of based on aging when SA- β-Gal activity level up-regulation and to declining
The method that old cell or tissue carry out dyeing detection.In the case where passing on for 10,15 generations in vitro, obtained respectively to from embodiment 1
The umbilical cord mesenchymal stem cells of the enhanced MSC of GPx7 and comparative example 1 that obtain carry out SA- β-Gal dyeing, and aobvious in common optics
The aging situation of cell or tissue is observed under micro mirror, and the SA- β-Gal positive cell dyeing ratio in two groups of cells is carried out
Quantitative statistical analysis.
Staining procedure: washed once donorcells with PBS, add dyeing fixer (+0.2% glutaraldehyde of 2% formaldehyde),
Room temperature fixes 5 minutes.Fixer is discarded, is washed 1 time with PBS, 1ml dyeing working fluid is added in every hole.Using X-Gal as substrate,
Navy blue product can be generated in the beta galactosidase of senescence-specific.Experimental result: senile cell positive rate: comparative example 1 >
Embodiment 1, the results are shown in Table 2.
Table 2
The umbilical cord mesenchymal stem cells of the enhanced MSC of the GPx7 obtained in embodiment 1 and comparative example 1 are passed on 10 in vitro
Protein immunoblotting inspection is carried out to the protein expression situation of aging cance high-expression gene p16, p21, GATA4 respectively after to 15 generations
It surveys.Primary antibody be respectively p16 (be purchased from BD, article No.: 550834), p21 (be purchased from Cell Signaling, article No.: 2947), GATA4
(being purchased from Santa Cruz, article No.: SC-1237), Actin (being purchased from Sigma-Aldrich, article No.: A1978), the HRP mark used
It is respectively sheep anti-Mouse, article No.: ZDR5307, goat-anti rabbit, article No.: ZDR5306, rabbit-anti sheep, goods that the secondary antibody of note, which is purchased from Zhong Shan Golden Bridge,
Number: ZDR5308.
Experimental result: the gene expression abundance of p16, p21, GATA4 in the enhanced MSC of GPx7 is lower than it in the navel of comparative example 1
With the gene expression abundance in mescenchymal stem cell.Corresponding protein expression abundance=corresponding albumen gray value/actin band gray value,
The results are shown in Table 3.
Table 3
3, the enhanced MSC of GPx7 that embodiment 1 obtains and the umbilical cord mesenchymal stem cells of comparative example 1 persistently adjust blood glucose
The detection of effect
It is injected into STZ modeling rat body, the modeling of STZ rat is referring to " according to fat mesenchymal stem cell and its excretion body pair
The effect and mechanism of inflammatory conditions fat cell and diabetes B rat " described in method.The GPx7 that embodiment 1 is obtained increases
The umbilical cord mesenchymal stem cells of strong type MSC cell and comparative example 1, according to 1ml concentration dose 2x106The cell concentration of cell/ml is logical
It crosses in tail vein injection to STZ rat body, respectively in injection cell 5 days, 10, day, tail vein takes blood at 15 days, 20 days, detects
Fasting plasma glucose concentration.As a result such as table 4
Table 4
| Fasting blood-glucose (mM) | d5 | d10 | d15 | d20 |
| Embodiment 1 | 4.6 | 6.1 | 7.2 | 10.1 |
| Comparative example 1 | 5.1 | 11.1 | 15.6 | 20.8 |
As shown in table 4, embodiment 1 has the ability for maintaining to adjust blood glucose effect for a long time compared to comparative example 1.
The preferred embodiment of the disclosure is described in detail above, still, during the disclosure is not limited to the above embodiment
Detail a variety of simple variants can be carried out to the technical solution of the disclosure in the range of the technology design of the disclosure, this
A little simple variants belong to the protection scope of the disclosure.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the disclosure to it is various can
No further explanation will be given for the combination of energy.
In addition, any combination can also be carried out between a variety of different embodiments of the disclosure, as long as it is without prejudice to originally
Disclosed thought equally should be considered as disclosure disclosure of that.
Sequence table
<110>Hangzhou Guan Zi health Science and Technology Ltd.
<120>it resists cell ageing and extends the mescenchymal stem cell and its preparation method and application of blood glucose-control effect time-histories
<130> 10996-K-HZGZ
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 103
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 1
acgccgcggc ggccgucggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103
<210> 2
<211> 500
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gggagcggtc cccggccgcg gccccgacga cgtgggtgtc ggcgggcgcg ggggcggttc 60
tcggcggcgt cgcggcgggt ctgggggggt ctcggtgccc tcctccccgc cggggcccgt 120
cgtccggccc cgccgcgccg gctccccgtc ttcggggccg gccggattcc cgtcgcctcc 180
gccgcgccgc tccgcgccgc cgggcacggc cccgctcgct ctccccggcc ttcccgctag 240
ggcgtctcga gggtcggggg ccggacgccg gtcccctccc ccgcctcctc gtccgccccc 300
ccgccgtcca ggtacctagc gcgttccggc gcggaggttt aaagacccct tggggggatc 360
gcccgtccgc ccgtgggtcg ggggcggtgg tgggcccgcg ggggagtccc gtcgggaggg 420
gcccggcccc tcccgcgcct ccaccgcgga ctccgctccc cggccggggc cgcgccgccg 480
ccgacgccgc ggcggccgtc 500
<210> 3
<211> 500
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gggtaggggc tttacccggc ggccgtcgcg cgcctgccgc gcgtgtggcg tgcgccccgc 60
gccgtggggg cgggaacccc cgggcgcctg tggggtggtg tccgcgctcg cccccgcgtg 120
ggcggcgcgc gcctccccgt ggtgtgaaac cttccgaccc ctctccggag tccggtcccg 180
ttcgctgtct cgtctggccg gcctgaggca accccctctc ctcttgggcg gggggggggg 240
gacgtgccgc gccaggaagg gcctcctccc ggtgcgtcgt cgggagcgcc ctcgccaaat 300
cgacctcgta cgactcttag cggtggatca ctcggctcgt gcgtcgatga agaacgcagc 360
tagctgcgag aattaatgtg aattgcagga cacattgatc atcgacactt cgaacgcact 420
tgcggccccg ggttcctccc ggggctacgc ctgtctgagc gtcgcttgcc gatcaatcgc 480
ccccgggggt gcctccgggc 500
<210> 4
<211> 1296
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60
gacgtcaata atgacgtatg ttcccatagt aacgtcaata gggactttcc attgacgtca 120
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 360
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttgcacca aaatcaacgg 420
gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta 480
cggtgggagg tctatataag cagagctatg gtggcggcga cggtggcagc ggcgtggctg 540
ctcctgtggg ctgcggcctg cgcgcagcag gagcaggact tctacgactt caaggcggtc 600
aacatccggg gcaaactggt gtcgctggag aagtaccgcg gatcggtgtc cctggtggtg 660
aatgtggcca gcgagtgcgg cttcacagac cagcactacc gagccctgca gcagctgcag 720
cgagacctgg gcccccacca ctttaacgtg ctcgccttcc cctgcaacca gtttggccaa 780
caggagcctg acagcaacaa ggagattgag agctttgccc gccgcaccta cagtgtctca 840
ttccccatgt ttagcaagat tgcagtcacc ggtactggtg cccatcctgc cttcaagtac 900
ctggcccaga cttctgggaa ggagcccacc tggaacttct ggaagtacct agtagcccca 960
gatggaaagg tggtaggggc ttgggaccca actgtgtcag tggaggaggt cagaccccag 1020
atcacagcgc tcgtgaggaa gctcatccta ctgaagcgag aagacttata accatagagc 1080
ccaccgcatc cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc 1140
ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat 1200
tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg 1260
ggcaaacaac agatggctgg caactagaag gcacag 1296
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gaggaattcc cagtaagtgc 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gcgctccccc gaccctctct 20
<210> 7
<211> 3024
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgcg aatgctgcgg cgcgcggcta gcttaacaac 180
aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaact 240
agacccagct ttcttgtaca aagttggcat tagagctcgc ggccgcagga acccctagtg 300
atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 360
gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 420
ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 480
atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 540
tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 600
tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 660
tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg 720
gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 780
agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 840
cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 900
agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat 960
ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 1020
caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 1080
ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 1140
cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 1200
tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 1260
ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 1320
aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 1380
tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 1440
atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 1500
agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 1560
tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 1620
tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 1680
atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 1740
ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 1800
tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 1860
acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 1920
ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 1980
aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 2040
ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 2100
cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 2160
gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 2220
actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 2280
agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 2340
cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 2400
tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 2460
agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 2520
tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 2580
acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 2640
ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 2700
gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 2760
gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 2820
gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 2880
tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 2940
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 3000
tttgctggcc ttttgctcac atgt 3024
<210> 8
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gcggcggccg tcgggtaggg gctttacccg gcg 33
<210> 9
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cgccgggtaa agcccctacc cgacggccgc cgc 33
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
actggtgtcg ctggagaagt 20
<210> 11
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
caatctcctt gttgctgtca g 21
Claims (10)
1. a kind of prepare resists cell ageing and extend the method that blood glucose-control acts on the mescenchymal stem cell of time-histories, feature exists
In this method comprises the following steps:
S1, RNP compound will be mixed to form for sgRNA the and Cas9 albumen for knocking in site of gene insertion safety island；
S2, the homologous recombination vector inserted with template DNA is packaged into AAV virus, forms AAV virion to be transfected；Institute
Stating template DNA includes the left homology arm sequence and right homology arm sequence for knocking in site for gene insertion safety island, the left side
Inserted with GPx7 gene between homology arm sequence and the right homology arm sequence；
The sequence being inserted between the left homology arm sequence and the right homology arm sequence is as shown in SEQ ID NO. 4；
The sgRNA is as shown in SEQ ID NO. 1；Correspondingly, the left homology arm sequence is as shown in SEQ ID NO. 2；Phase
Ying Di, the right homology arm sequence is as shown in SEQ ID NO. 3；
S3, the suspension of iPSC cell or embryonic stem cell is mixed to the suspension of the RNP compound and is carried out electricity turn, obtain electricity
Culture after turning；
S4, in 1-30 minutes, the outstanding of AAV virion to be transfected is added in the material after turning to the electricity after electricity turns
Culture of the liquid to carry out transfection in 4-30 hours, after being transfected；
S5, will after the culture Method of Limited Dilution after the transfection carry out monoclonal culture, and by PCR and sequencing filter out into
Gene of having gone orients the monoclonal cell strain knocked in；
S6, by it is described carried out monoclonal cell strain amplification that gene orientation is knocked in and Induction of committed differentiation to become mesenchyma dry thin
Born of the same parents.
2. according to the method described in claim 1, wherein, the sgRNA has chemical modification group；The chemical modification group
Preferably methyl chemistry modification group or phosphorothioate chemical modification group；Site is knocked in for gene insertion safety island
The dosage molar ratio of sgRNA and Cas9 albumen is 1:1 to 1:5.
3. method according to claim 1 or 2, wherein by for gene insertion safety island the sgRNA for knocking in site and
The time of Cas9 albumen mixing is 5-20 minutes, and temperature is 10-40 DEG C.
4. according to the method described in claim 1, wherein, in step S3, the iPSC cell or embryonic stem cell suspension and institute
When stating the mixing of RNP compound, relative to every 106A iPSC cell, with the meter of sgRNA, the dosage of the RNP compound
For 10-50 μm of ol, in the suspension of the iPSC cell, cell concentration is (1-5) × 107A/mL；With the meter of sgRNA, institute
State the final concentration of 0.1-1.5 μm of ol/ μ L of RNP compound.
5. method according to claim 1 or 4, wherein in step S3, the condition that electricity turns includes: that electric field strength is 50-
250V/cm, single pulse time are 2-15ms, and the time interval between adjacent two subpulse is 10-60s, and total pulses are
2-10 times.
6. according to the method described in claim 1, wherein, in step S4, after electricity turns in 5-20 minutes, turning to the electricity
Culture of the suspension of AAV virion to be transfected to carry out transfection in 8-24 hours, after being transfected is added in material afterwards
Object.
7. method according to claim 1 or 6, wherein in step S4, the suspension of AAV virion to be transfected adds
Entering amount makes the MOI value of AAV virion to be transfected be 104-106。
8. according to the method described in claim 1, wherein, the iPSC cell is 1016 cell strains, the serum of the AAV virus
Type is AAV-8 virus, AAV-6 virus, AAV-1 virus or AAV9 virus；
It expands and the condition of Induction of committed differentiation includes: that the gene that carried out is oriented the monoclonal cell strain knocked in low viscous
Embryoid body is formed after continuing culture in attached culture plate 18-100 hours；Then the embryoid body is inoculated in and continues to train on matrigel
With flow cytometry sorting, wherein CD73, CD90 and CD105 are positive cell colony after supporting 10-20 days.
9. the mescenchymal stem cell that method described in any one of claim 1-8 is prepared.
10. the purposes of mescenchymal stem cell as claimed in claim 9, which is characterized in that the purposes be it is following any one:
Preparation improves the product of insulin resistance and/or chronic inflammation；
Prepare the product of cellular transplantation therapy；
Preparation improves the therapeutic products of β cell function；
Preparation regeneration and/or the product for repairing damage blood vessel.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810785793.0A CN109097333B (en) | 2018-07-17 | 2018-07-17 | It resists cell ageing and extends the mescenchymal stem cell and its preparation method and application of blood glucose-control effect time-histories |
| PCT/CN2018/119852 WO2020015279A1 (en) | 2018-07-17 | 2018-12-07 | Method for gene-directed-knock-in in stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810785793.0A CN109097333B (en) | 2018-07-17 | 2018-07-17 | It resists cell ageing and extends the mescenchymal stem cell and its preparation method and application of blood glucose-control effect time-histories |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109097333A CN109097333A (en) | 2018-12-28 |
| CN109097333B true CN109097333B (en) | 2019-07-23 |
Family
ID=64846629
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810785793.0A Expired - Fee Related CN109097333B (en) | 2018-07-17 | 2018-07-17 | It resists cell ageing and extends the mescenchymal stem cell and its preparation method and application of blood glucose-control effect time-histories |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109097333B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117448379A (en) * | 2023-12-22 | 2024-01-26 | 上海元戊医学技术有限公司 | Construction method and application of iPSC-derived IL-10 protein over-expression MSC cell strain |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636305A (en) * | 2016-11-16 | 2017-05-10 | 宁夏医科大学总医院 | Method for detecting antioxidant capacity of human placenta fetal side mesenchymal stem cells |
| CN107988256A (en) * | 2017-12-01 | 2018-05-04 | 暨南大学 | Human Huntington gene knock-in recombinant vector and its construction method and the application in swine model structure |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101135636B1 (en) * | 2009-10-27 | 2012-04-17 | 서울대학교산학협력단 | Method for producing mesenchymal stem cells from human pluripotent stem cells and mesenchymal stem cells produced by thereof |
| CN106591228B (en) * | 2016-12-21 | 2019-06-18 | 中国科学院生物物理研究所 | A kind of preparation method of human pluripotent stem cells that are while resisting cell ageing and vicious transformation |
-
2018
- 2018-07-17 CN CN201810785793.0A patent/CN109097333B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636305A (en) * | 2016-11-16 | 2017-05-10 | 宁夏医科大学总医院 | Method for detecting antioxidant capacity of human placenta fetal side mesenchymal stem cells |
| CN107988256A (en) * | 2017-12-01 | 2018-05-04 | 暨南大学 | Human Huntington gene knock-in recombinant vector and its construction method and the application in swine model structure |
Non-Patent Citations (3)
| Title |
|---|
| Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA;Xiquan liang等;《Journal of Biotechnology》;20161111(第241期);第136-146页 |
| Loss of the oxidative stress sensor NPGPx compromises GRP78 chaperone activity and induces systemic disease;Pei-Chi Wei等;《Mol Cell》;20121101;第48卷(第5期);第747-759页 |
| Metformin alleviates human cellular aging by upregulating the endoplasmic reticulum glutathione peroxidase 7;Jingqi Fang等;《Aging Cell》;20180416;第17卷(第4期);第e12765页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109097333A (en) | 2018-12-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111269888B (en) | Adipostem cell modified by adipsin gene, preparation method and application thereof | |
| CN112480264B (en) | Chimeric antigen receptor taking TIGIT and PD-1 as targets, CAR-T cell and preparation method thereof | |
| CN112680466B (en) | Animal model for expressing humanized ACE2 and application thereof | |
| CN113453702A (en) | Cell reprogramming to reverse senescence and promote tissue and tissue regeneration | |
| CN112522271B (en) | sgRNA and application thereof | |
| CN109055373B (en) | A method of it carrying out gene orientation in stem cell and knocks in | |
| AU2024216450A1 (en) | Methods and compositions for non-myeloablative bone marrow reconstitution | |
| CN109055428A (en) | A kind of recombined glandulae correlation viral vectors and the preparation method and application thereof | |
| CN117715660A (en) | Gene therapy compositions and treatments for right ventricular arrhythmogenic cardiomyopathy | |
| CN112210543B (en) | CAR-T cell for overcoming TGF-beta immunosuppression aiming at solid tumor | |
| CN108379597B (en) | A kind of genophore and its gene therapy medicament for treating retinal ganglion cells degeneration | |
| CN114107390B (en) | rAAV vector for expressing antibody IgG1 and application thereof | |
| CN109097333B (en) | It resists cell ageing and extends the mescenchymal stem cell and its preparation method and application of blood glucose-control effect time-histories | |
| CN117136232A (en) | Chimeric Antigen Receptor (CAR) NK cells and uses thereof | |
| CN112301058B (en) | Recombinant adeno-associated virus vector and preparation method and application thereof | |
| CN104403998A (en) | Improved fat stem cell for epidermis damage repair | |
| CN112266935A (en) | Human iPS cell gene editing and screening method | |
| CN106755095A (en) | Drug screening cell model and its structure and application that targeting HBC dimers are formed | |
| CN108159434B (en) | Gene vector and gene therapy medicine for treating glaucoma | |
| CN114277190B (en) | Specific DNA fragment, primer, kit and detection method for detecting exogenous gene residues in hiPSC | |
| CN113025655B (en) | Gene vector | |
| CN108277208A (en) | The vesicular stomatitis virus infection clones and preparation method and application of Carrying Green Fluorescent Protein and transferrins | |
| CN115335523A (en) | Molecular system and therapy using the same | |
| CN112301057B (en) | Recombinant adeno-associated virus vector and preparation method and application thereof | |
| CN112342228B (en) | AAV viral vector for expressing VEGF fusion protein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190723 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |