CN108486060A - It is a kind of to be used to treat excretion body of tumour and its preparation method and application - Google Patents
It is a kind of to be used to treat excretion body of tumour and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of excretion bodies and its preparation method and application for treating tumour.Specifically, the present invention provides the excretion bodies and preparation method thereof of a kind of growth that can significantly inhibit brain glioblastoma cell strain and one-tenth knurl ability.The excretion body of the present invention can significantly inhibit growth and the one-tenth knurl ability of brain glioblastoma cell strain.Therefore, it can be used for the treatment of glioma.
Description
Technical field
The invention belongs to molecular biology and biomedicine field, in particular it relates to a kind of for treating tumour
Excretion body and its preparation method and application.
Background technology
Tumor tissues are made of tumour cell and interstitial, tumour cell be by normal cell turnover Lai paraplasm it is thin
Born of the same parents.Tumour can be happened at many organs and tissue of human body.Divided according to the size of tumour harm to the human body and its growth characteristics
For two class of benign tumour and malignant tumour.
Benign tumour is slow-growing, is in expansive growth, and often there is complete coating on surface, the less whole body disease in addition to local symptom
Shape, to surrounding tissue infiltration also not to whole body shift, operation excision after it is uneasy to recur, to body endanger it is smaller, as lipoma,
Hemangioma, adenoma, tumour etc..
Malignant growth is rapid, and growth is infiltrated to surrounding tissue often, and there are few coatings on surface, often there is whole body transfer, disease
Reason checks visible atypical karyokinesis, and in addition to local symptom, constitutional symptom is apparent, and end-stage patients have more existing cachexia, and operation is cut
Except rear high recurrence rate, body is endangered greatly, such as osteocarcinoma, the cancer of the esophagus, liver cancer, lung cancer, leukaemia, osteosarcoma, glioma.
Malignant tumour is a kind of disease that current serious threatens human health and life, has division under the influence of carcinogenic factor
The cell of potential, occurs vicious transformation and clonal proliferation is formed by neoformation.Tumour is often body heredity and ring
Border carcinogenic factor is accompanied by multiple oncogenes successively or in a manner of collaboration, to cause damage, the mutation of hereditary material DNA
The inactivation of activation and tumor suppressor gene, makes the continuous hyperplasia of normal cell, converts final canceration.
Glioma is most common neural tumor in neurosurgery, since the malignant proliferation ability and invasion of tumour are strong
And cause current all treatment means (such as aggregate measures of operation, chemicotherapy, gene therapy, immunization therapy or combinations thereof) equal
Glioma cannot be effected a radical cure and reduce the high relapse rate of the disease, therefore ground as the problem and hot spot of neurosurgery clinical treatment
Study carefully project.The biological nature of glioma depends on inherent gene alteration, finds reliable tumor marker gene and to brain
The characteristics such as glioma proliferative, invasion, differentiation judge, and are Diagnosing Gliomas, prognosis evaluation and determining effectively treatment
The important prerequisite of measure.
There is serious influence on people's health in view of tumour, it is new more that those skilled in the art are dedicated to exploitation
Effective oncotherapy technology.
Invention content
The purpose of the present invention is to provide a kind of excretion bodies and its preparation method and application for treating tumour.
The first aspect of the present invention provides a kind of excretion body for treating tumour, wherein the excretion of the excretion body
Contain CD47 molecules on body film.
In another preferred example, the excretion body is enclosed with siRNA molecule or its precursor.
In another preferred example, the siRNA molecule is the siRNA molecule or its precursor of tumour-specific.
In another preferred example, the siRNA molecule targets BPTF genes.
In another preferred example, such as SEQ ID NO. in the siRNA molecule targeting BPTF genes:Sequence shown in 3.
In another preferred example, the tumour is glioma.
The second aspect of the present invention provides a kind of method preparing excretion body, the method includes the steps：
(1) Lentiviral is built
Wherein, the Lentiviral includes the polynucleotide sequence of coding CD47；
(2) slow virus is packed
The Lentiviral that step (1) is built is packaged as virion；
(3) cell strain is built
Infestation with virus particles host cell is obtained in practical step (2), to build the cell strain for stablizing expression CD47；
(4) excretion body is prepared
The cell strain built in incubation step (3) collects cell supernatant extraction excretion body, to obtain the excretion
Body.
In another preferred example, the method further includes step：
(5) excretion body-siRNA compounds are prepared.
In another preferred example, the siRNA molecule is the siRNA molecule of tumour-specific.
In another preferred example, the siRNA molecule targets BPTF genes.
In another preferred example, such as SEQ ID NO. in the siRNA molecule targeting BPTF genes:Sequence shown in 3.
In another preferred example, the tumour is glioma.
In another preferred example, in the step (5), in such a way that electricity is transduceed, the siRNA molecule is wrapped up into institute
Excretion body is stated, to which the excretion body-siRNA compounds be made.
In another preferred example, in the step (5), excretion body particle and siRNA amount ratios are 10 in electric transductive process8
~1010A excretion body particle：0.1~10ug siRNA；Preferably 108~1010A excretion body particle：1ug siRNA；It is optimal
Selection of land is 109A excretion body particle：1ug siRNA.
In another preferred example, in the step (1), the expression vector is using FUGW viral vectors as skeleton.
In another preferred example, in the step (2), the helper plasmid used in slow virus packaging process is pCMV-
DR8.2dvpr carriers and pCMV-VSV-G carriers.
In another preferred example, in the step (2), incasing cells is 293T cells.
In another preferred example, in the step (3), host cell is HEK293 cells.
In another preferred example, described in first aspect present invention for treating the excretion body of tumour through the invention second
Method described in aspect is prepared.
In the third aspect of the present invention, a kind of pharmaceutical composition is provided, including pharmaceutically acceptable carrier and effectively
The active constituent of amount, wherein the active constituent is the excretion body described in first aspect present invention.
In another preferred example, the pharmaceutical composition is for preventing or treating tumour.
In another preferred example, the tumour is glioma.
The fourth aspect of the present invention provides the purposes of the excretion body described in first aspect present invention, for preventing or controlling
Treat tumour.
In another preferred example, the tumour is glioma.
In the fifth aspect of the present invention, a kind of method of external non-therapeutic inhibition tumour cell, including step are provided：
Described in first aspect present invention for treat existing for the excretion body of tumour under the conditions of, cultivate tumour cell, to inhibit
Inhibit tumour cell.
In another preferred example, the tumour is glioma；Preferably, the tumour cell is brain glioblastoma cell
It is (such as U-87 cell lines and U-251 cell lines).
In another preferred example, the inhibition tumour cell be inhibit tumour cell growth or inhibit tumour cell at
Tumor.
In another preferred example, compared with control tumor cell, the work of BPTF gene coded proteins in the tumour cell
Property reduce by 10% or more, preferably reduce by 20% or more, more preferably reduce by 30% or more, more preferably reduce by 40% or more, more preferably
Ground reduces by 50% or more, more preferably reduces by 60% or more, more preferably reduces by 70% or more, more preferably reduces by 80% or more, more preferably
Ground reduces by 90% or more, the most preferably activity completely without BPTF gene coded proteins.
In another preferred example, compared with control tumor cell, the expression reduction of BPTF genes in the tumour cell
10% or more, 20% or more is preferably reduced, more preferably reduces by 30% or more, 40% or more is more preferably reduced, more preferably reduces
50% or more, 60% or more is more preferably reduced, more preferably reduces by 70% or more, 80% or more is more preferably reduced, more preferably reduces
90% or more, the most preferably expression completely without BPTF genes.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Description of the drawings
Following drawings is for illustrating specific embodiments of the present invention, rather than limits and be defined by the claims
The scope of the invention.
Figure 1A shows that the siRNA for ATM gene difference target position strikes decreasing effect rate.
Figure 1B shows that the siRNA for BPTF gene difference target position strikes decreasing effect rate.
Fig. 1 C show that the proliferation for the cell for knocking out BTPF and ATM genes respectively is horizontal.
Fig. 2A shows identified by immunofluorescence CD47-HEK293 cells.
Fig. 2 B show the CD47-siRNA-Exosome of Electronic Speculum detection.
Fig. 3 shows the droplet measurement result of CD47-siRNA-Exosome.
Fig. 4 is that Western blot detect the significant albumen of CD47-siRNA-Exosome.
Fig. 5 is that excretion body is incubated fluorescence microscope testing result altogether with glioma cell.
Fig. 6 is that excretion body is incubated MTT testing results altogether with glioma cell.
Fig. 7 shows that excretion body is incubated the result of Clone formation detection with glioma cell altogether.
Fig. 8 shows that excretion body is incubated invasive ability testing result altogether with glioma cell.
Fig. 9 shows that excretion body injects tumor formation experiment of nude mouse result.
Specific implementation mode
The present inventor after extensive and in-depth study, has found that BPTF gene inhibitors can significantly inhibit brain colloid for the first time
The growth of tumor cell strain and one-tenth knurl ability.Therefore BPTF genes play the role of very important in glioma, are one new
The glioma treatment target spot of type.The present invention is completed on this basis.
Term
Excretion body
Excretion body (exosome) is a kind of to be widely present and be distributed in the film property that can be secreted by various kinds of cell in various body fluid
Vesicles have double-layer of lipoid membrane structure, and diameter is typically in the range of between 30~120nm, includes albumen, the lipid of cell-specific
And nucleic acid, important signaling molecule can be carried and transmit, a kind of completely new cell-tocell transmission system of formation is to change
Become outside the function of other cells, and plays an important role on many physiological and pathologicals.
BPTF genes and its coding albumen
BPTF genes (NCBI sequence number Gene ID:2186) it is located on 17q24 chromosomes, is chromosome remodeling complex
The maximum subunit of NURF middle-molecular-weihydroxyethyls can recruit the other subunits of NURF complexs to the promoter or Enhancer district of gene, lead to
The sliding of regulation and control nucleosome is crossed, genetic transcription is promoted.It is existing the study found that BPTF is played during the zebra fish nerve rear portionization
Very important effect.BPTF functionally interacts in structure with Smad2.BPTF and Smad2 synergic adjustments are tied
The nucleosome of the Wnt8a promoter regions of conjunction slides to regulate and control expression of target gene, to play a role in nervous system development.
The present invention has carried out genechip detection to the relevant non-small cell lung cancer sample of metastatic encephaloma, by sieving on a large scale
Choosing, has been surprisingly found that BPTF unconventionality expressions in metastatic encephaloma, illustrates that the gene is related with the incidence of metastatic encephaloma；Pass through RNAi
After BPTF genes in technology silence lung embryonic fiber cell, the clonality of cell is suppressed significantly；In liver cancer sample
There is mutation in the middle gene for finding BPTF；In carcinoma of urinary bladder it has also been found that BPTF there are saltant types, struck for 3 plants of bladder cancer cell lines
It can be with the clonality of the reduction cell of conspicuousness except after BPTF.
It is preferably carried out in mode in the present invention, the sequence such as SEQ ID NO. of the BPTF genes:Shown in 1.
It is preferably carried out in mode in the present invention, the sequence such as SEQ ID NO. of the BPTF gene coded proteins:2 institutes
Show.
The BPTF gene coded proteins of the present invention can be recombinant polypeptide, natural polypeptides or synthesis polypeptide.
As used herein, term " RNAi " (RNA interference, RNA interference) refers to that height is protected during evolution
Selective degradation keep, being induced by double-stranded RNA (dsRNA), efficient has the phenomenon that RNA of complementary pairing sequence.Due to making
With RNAi technology can specificity close the expression of specific gene, so the technology has been widely used for exploring gene function and biography
The fields such as the gene therapy of infectious diseases and tumour.The RNAi phenomenons that dsRNA is mediated are in fungi, drosophila, arabidopsis, trypanosome, water
It is found in a variety of eucaryotes such as Xi, turbellarian worm, zebra fish, and the posttranscriptional gene silencing in plant
The disease that (posttranscriptional gene silencing, PTGS), co-suppression (cosuppression) and RNA are mediated
Malicious resistance, inhibition (quelling) phenomenon of fungi also belong to RNAi different plant species the form of expression.
As used herein, term " siRNA " (Small interfering RNA, siRNA) refers to a kind of small RNA molecular
(about 21-25 nucleotide) (can be compared by Dicer (enzyme to double-stranded RNA in III family of RNA enzyme with specificity) from its precursor
Such as dsRNA, shRNA) it is process, it can also be generated by being chemically synthesized or by other Protein processings.SiRNA is siRISC
Major Members, the target RNA of excitation sequence complementation therewith leads to the silence of target gene by rapid cutting degradation, thus at
For the key function molecule in RNAi.In the case where providing specific gene target position sequence, those skilled in the art can lead to
Conventional method is crossed to design and obtain the siRNA for the target position sequence.
As used herein, term " siRNA precursors " refers to that can be processed to generate siRNA in mammalian cell
RNA molecule, specifically, being selectively to be processed to generate ripe siRNA, Jin Ershi by Dicer or other albuminoids
Apply RNAi.
As used herein, term " construction " is the construction for including shRNA of the present invention.
As used herein, term " expression cassette " refer to comprising shRNA of the present invention coded sequence and with the code sequence
The expression cassette of the promoter and termination signal that are operatively connected is arranged, the expression cassette generates the shRNA of the present invention after transcription.
As used herein, term " miRNA " (microRNA) is a kind of length about 20-24 by endogenous gene
The non-coding single strand RNA molecule of nucleotide participates in the expression regulation to lots of genes in animals and plants.Up to the present, dynamic
More than 4,000 kinds of miRNA molecule is had been found that in plant and virus.Most of miR-96 genes are with single copy, multicopy or gene
The form of cluster (cluster) is present in genome.Each miRNA can regulate and control multiple target genes, and several miRNA can also
It is common to participate in adjusting same gene, form complicated regulating networks.It is assumed that miRNA adjusts the mankind more than half gene
Expression.There are diversified forms by miRNA, and most original is pri-miRNA；Pri-miRNA becomes pre- after Drosha is processed
MiRNA, i.e. miRNA precursors, length are about 50-90 nucleotide；After pre-miRNA is using Dicer enzyme digestions, become
It is about the ripe miRNA of 20-24 nucleotide.MiRNA is mainly by inhibiting to translate and the de- polyadenylation of mRNA being accelerated to inhibit
Expression of target gene, mechanism are different from the mRNA degradations of siRNA mediations.
Generating a kind of method of " siRNA " (siRNA) in the living body is, using siRNA sequence as " bob folder "
A part is cloned into plasmid vector.When being sent into animal body, which is expressed, and forms one and carries top
" double-stranded RNA " (shRNA) of ring structure, is identified and is processed by intracellular Dicer albumen, generate functional siRNA.
As used herein, term " shRNA ", " shRNA " are used interchangeably, and are using the precursor of people miR-26b as skeleton
A kind of special shRNA of structure.The shRNA is followed successively by from 5 ' ends to 3 ' ends：(a) 5 ' end flanking sequence area；(b) 5 ' end
Match the regions siRNA；(c) top ring region；(d) 3 ' end pairing siRNA regions, and the 5 ' end pairing regions siRNA with
3 ' ends the regions pairing siRNA form double-stranded region；(e) 3 ' end flanking sequence area；The shRNA generates siRNA, and described
The nucleotide sequence of siRNA corresponds to the 3 ' end pairing regions siRNA or the 5 ' end regions pairing siRNA.
The shRNA of broad sense is the abbreviation of short hairpin RNA, that is, " short hairpin RNA ".ShRNA includes two short
Reverse complementary sequence, it is intermediate by top ring (loop) sequence separates, hairpin structure is formed, usually by the RNA of cellular endogenous
The control transcription of polymerase III (RNApolymeraseIII) promoter, the end of shRNA sequences connect 5-6 T and gather as RNA
The transcription terminator of synthase III.ShRNA can also be generated by the promoter transcription of other RNA polymerases.
Pharmaceutical composition
The present invention provides a kind of pharmaceutical composition, including pharmaceutically acceptable carrier and a effective amount of following activity at
Point：Excretion body described in first aspect present invention.
As used herein, term " effective quantity " or " effective dose " refer to that can generate function or activity to people and/or animal
And the amount that can be received by people and/or animal.
As used herein, the ingredient of " pharmaceutically acceptable " is suitable for people and/or mammal and without excessively bad
Side reaction (such as toxicity, stimulation and allergy), i.e., with rational benefit/risk than substance.Term " can pharmaceutically connect
The carrier received " refers to the carrier for Therapeutic Administration, including various excipient and diluent.
The pharmaceutical composition of the present invention contains the active constituent and pharmaceutically acceptable of the present invention of safe and effective amount
Carrier.This kind of carrier includes (but being not limited to)：Brine, buffer solution, glucose, water, glycerine, ethyl alcohol, and combinations thereof.Usual medicine
Object preparation should match with administering mode, the dosage form of pharmaceutical composition of the invention be injection, oral preparation (tablet, capsule,
Oral solution), transdermal agent, sustained release agent.Such as the aqueous solution with physiological saline or containing glucose and other adjuvants passes through conventional side
It is prepared by method.The pharmaceutical composition preferably aseptically manufactures.
The effective quantity of active constituent of the present invention can be with the pattern of administration and the severity of disease to be treated etc.
And change.Preferred a effective amount of selection can depending on various factors be determined by those of ordinary skill in the art (such as to be passed through
Clinical test).The factor includes but not limited to：The pharmacokinetic parameter of the active constituent such as biological utilisation
Rate, metabolism, half-life period etc.；Patient the severity of disease to be treated, the weight of patient, the immune state of patient, administration
Approach etc..In general, the active constituent as the present invention is (preferable with about 0.00001mg-50mg/kg the weight of animals daily
0.0001mg-10mg/kg the weight of animals) dosage give, satisfactory effect can be obtained.For example, being compeled by treatment situation
It highly necessary asks, dosage separated several times can be given once daily, or dosage is reduced pari passu.
Pharmaceutically acceptable carrier of the present invention includes but is not limited to：Water, brine, liposome, lipid, egg
In vain, Protein-antibody conjugate, peptide matters, cellulose, nanogel, or combinations thereof.The selection of carrier should be with administering mode phase
Matching, these are all known to those skilled in the art.
Using
The present invention provides a kind of methods of the inhibition tumour cell of external non-therapeutic, including step：In the present invention the
Under the conditions of existing for excretion body described in one side, tumour cell is cultivated, to inhibit tumour cell, the tumour cell to be
Brain glioblastoma cell.
In the preference of the present invention, the inhibition tumour cell is to inhibit the growth of tumour cell or inhibit swollen
Tumor formation.
Preferably, compared with control tumor cell, the activity of BPTF gene coded proteins reduces in the tumour cell
10% or more, 20% or more is preferably reduced, more preferably reduces by 30% or more, 40% or more is more preferably reduced, more preferably reduces
50% or more, 60% or more is more preferably reduced, more preferably reduces by 70% or more, 80% or more is more preferably reduced, more preferably reduces
90% or more, the most preferably activity completely without BPTF gene coded proteins.
Or preferably, compared with control tumor cell, the expression of BPTF genes reduces by 10% in the tumour cell
More than, 20% or more is preferably reduced, more preferably reduces by 30% or more, 40% or more is more preferably reduced, more preferably reduces by 50%
More than, 60% or more is more preferably reduced, more preferably reduces by 70% or more, 80% or more is more preferably reduced, more preferably reduces by 90%
More than, the expression most preferably completely without BPTF genes.
Main advantages of the present invention are：
(1) a kind of excretion body of the growth that can significantly inhibit brain glioblastoma cell strain and one-tenth knurl ability is provided for the first time
And preparation method thereof.
(2) a kind of efficient method for preparing excretion body is provided.
(3) excretion body of the invention can significantly inhibit growth and the one-tenth knurl ability of brain glioblastoma cell strain therefore can
For the treatment of glioma.
With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise number and hundred
Divide ratio by weight.
1 genetic chip selective mechanisms of embodiment
After glioma and control tissue total serum IgE sample are analyzed by agilent 2100, using 3 ' IVT of GeneChip
Express Kit prepare aRNA (amplified RNA).It synthesizes to obtain cDNA by a chain, be synthesized further across two chains
Double-stranded DNA template is obtained, then obtains the aRNA (amplified RNA) with biotin labeling by inverting in vitro.By aRNA
It is purified, then will be hybridized with chip probe after its fragmentation.After the completion of hybridization, chip is carried out to wash dye, is finally scanned
To picture and initial data, initial data is carrying out depth analysis, is obtaining Final Report, and concrete outcome is shown in Table 1.
Table 1
The gene expression difference in pathologic group sample and normal group sample is detected by chip, it is aobvious to select processing differential expression
The gene information of work, such as the closely related ATM of tumour are similarly high expression in this detection, in addition, we also send out
Expression of the existing BPTF in glioma samples is similar to the expression of ATM, and abnormal high expression, is a potential target
Mark gene.
For ATM genes, (gene is numbered：NM_000051) and BPTF genes, the present inventor have separately designed multiple siRNA
It is verified.
The targeting information of the typical siRNA for ATM genes is as follows：
ATM siRNA 1#：GCGATTGGCTTATACGCGC(SEQ ID NO.:5)
ATM siRNA 2#：GCGCCTGATTCGAGATCCT(SEQ ID NO.:6)
ATM siRNA 3#：TTACAGTAATTGGAGCATT(SEQ ID NO.:7)
It is directed to the siRNA of different target position by chemical synthesis, then transfects into U-87 glioma cell lines, passes through qPCR
The gene expression abundance of testing goal Gene A TM, to filter out most effective target position.
The selection result is as shown in Figure 1A, the results showed that, target position 2 in selectively targeted ATM genes (ATM siRNA 2#,
KD2) siRNA of sequence is most effective, and 60% or so can be reached by striking decreasing effect rate.
The targeting information of the typical siRNA for BPTF genes is as follows：
BPTF siRNA 1#：CAGGAGAGTTCTCAAGTAGAT(SEQ ID NO.:3)
BPTF siRNA 2#：CAGCACAGAGAAGACCATGAT(SEQ ID NO.:8)
BPTF siRNA 3#：GAGACTGAGAATGACTCTAAA(SEQ ID NO.:9)
It is directed to the different siRNA of target gene by chemical synthesis, then transfects into U-87 glioma cell lines, passes through
The gene expression abundance of qPCR testing goal genes BTPF, to filter out most effective target spot.
The selection result is as shown in Figure 1B, the results showed that, target position 1 in selectively targeted BTPF genes (BPTF siRNA 1#,
KD1) siRNA of sequence is most effective, and 80% or so can be reached by striking decreasing effect rate.
Further, the applicant knocks out BTPF and ATM genes respectively using preferably siRNA, compares two genes
To the proliferation of U87 glioma cells level after knockout.Specifically, to be transferred to glioma thin by the siRNA for first passing through for different genes
Born of the same parents are, then detect influence of the different siRNA target spots to the proliferation level of cell by MTT experiment.
Experimental result is as shown in Figure 1 C, by the comparative analysis of MTT experiment, as can be seen that being directed to from experimental result
After BPTF is knocked out, the proliferation water of cell is flat to be decreased obviously, while being also significantly better than the effect that ATM clpp genes subtract.
The structure of 2 CD47 slow virus carriers of embodiment：
(1) to be cloned as template by CD47cDNA, PCR reactions are carried out by amplimer, amplification obtains size~1Kb
The PCR product of left and right.
(2) double digestion of BamHI and AgeI is carried out to PCR product and FUGW viral vectors (being purchased from Addgene companies).
(3) conversion reaction is carried out after crossing T4DNA ligases (being purchased from Takara companies) connection.
(4) by the identification to transformant, select positive colony and send survey, using sequencing sequence it is consistent with expected sequence as
Correct clone.
CD47cDNA gene orders such as SEQ ID NO.:Shown in 4.
The packaging of 3 CD47 slow virus carriers of embodiment：
(1) DNA solution (CD47 slow virus carriers 20 the μ g, pCMV- of 3 kinds of plasmids in slow virus packaging system are prepared
DR8.2dvpr carriers (be purchased from Addgene companies) 15 μ g, pCMV-VSV-G carriers (being purchased from Addgene companies) 10 μ g, and it is corresponding
The Opti-MEM of volume is uniformly mixed dilution, and adjustment total volume is 2.5ml, is incubated 5 minutes at room temperature.
(2) take 100 μ l Lipofectamine2000 (being purchased from invitrogen) reagent in another Guan Zhongyu 2.4ml
Opti-MEM (being purchased from invitrogen) mixed diluting, incubates 5 minutes at room temperature.
(3) the DNA and Lipofectamine2000 after being diluted described in (2) after being diluted described in (1) is mixed
It closes, mixing is lightly overturned in 5 minutes.20min is incubated at room temperature.
(4) DNA and 2000 mixed liquors of Lipofectamine are transferred in the culture solution of 293T cells (being purchased from ATCC),
Mixing, in 37 DEG C, 5%CO2It is cultivated in cell incubator.The culture medium containing transfection mixture is removed after culture 8h, it is every bottle thin
The PBS liquid of 20ml is added in born of the same parents, and gently once then culture bottle is gone double swerve with washing remaining transfection mixture.
(5) the cell culture medium 25ml containing 10% serum is added in every bottle of cell, in 37 DEG C, 5%CO2Continue in incubator
Culture 48 hours.
(6) the 293T cell supernatants after transfecting 48 hours are collected.In 4 DEG C, 4000g centrifuges 10min, and it is broken to remove cell
Piece.With 0.45 μm of filter filtering supernatant in 40ml ultracentrifugation pipes.Virus crude extract sample is added in filter cup simultaneously
It closes the lid, filter cup is inserted into filtered solution collecting pipe.After combining, balance is carried out, is placed in rotary head.It is centrifuged in 4000g
About 10-15 minutes.After centrifugation, centrifugal device is taken out, filter cup and following filtered solution collection cups are separated.Sample collection
In cup is viral concentration liquid.
(7) viral concentration liquid is removed, is stored in after packing in viral pipe, -80 DEG C of long-term preservations.It is named as LV-
CD47。
Embodiment 4 stablizes strain structure
Stable cell line is built, and experimental procedure is as follows：
(1) before infection 12-18 hours, HEK293 cells (being purchased from ATCC) are inoculated in 6 orifice plates, when cell infection melts
Conjunction rate is about 30%-50%.
(2) virus is taken out from -80 DEG C of refrigerators, is put in and melts on ice.
(3) according to MOI values 100, CD47-GFP-LV slow virus amounts are added.
(4) 37 DEG C, 5%CO2 incubator cultures 72h are put back to after mixing.
(5) monoclonal cell strain is screened after infecting 72 hours.
(6) freeze-stored cell strain after the completion of being screened by GFP is named as CD47-HEK293 stable cell lines.
(7) pass through the expression of immuno-fluorescence assay CD47.
Fig. 2A shows the qualification result of identified by immunofluorescence CD47-HEK293 cells, the results showed that the present embodiment is built
Cell strain can stablize express express target protein.
5 excretion body of embodiment prepares and extraction
Serum free medium is replaced, CD47-HEK293 stable cell lines 2 days or more is persistently cultivated, collects cell supernatant
Excretion body extraction is carried out, experimental procedure is as follows：(refer to the Shanghai bio tech ltd Yu Meibo, excretion body extracts kit
Specification method operates)
(1) cell is removed：4 DEG C/300g of stable cell line culture supernatant is centrifuged 10 minutes, takes supernatant；
(2) dead cell is removed：4 DEG C/the 2000g of supernatant for removing cell is centrifuged 10 minutes, takes supernatant；
(3) cell fragment is removed：4 DEG C/the 10000g of supernatant for removing dead cell is centrifuged 30 minutes, takes supernatant；
(4) supernatant pre-processes：Exosomoe Concentration are added in going deimpurity centrifuged supernatant
The ECS reagents of 5mL are often added in Solution (ECS reagents) in 20mL supernatants；
(5) excretion body is precipitated：4 DEG C/100000g of re-suspension liquid is centrifuged 70 minutes, and precipitation is excretion body, with appropriate PBS weights
Outstanding, packing is stored in -80 DEG C of refrigerators.
(6) solution mixes：Centrifuge tube is covered tightly after ECS reagents are added, by turbula shaker mixing 1min, then is positioned over
2 DEG C to 8 DEG C standing 2h；
(7) excretion body is precipitated：It takes out the centrifuge tube equipped with mixed liquor and 60min is centrifuged with 10000g in 4 DEG C, abandon supernatant, sink
Excretion body particle is rich in shallow lake；(note：Exhaust supernatant as far as possible)
(8) excretion weight is outstanding：It takes 100 1 × PBS of μ L uniformly to blow and beat centrifugal sediment, waits for that it is uniformly suspended in PBS
Afterwards, re-suspension liquid is transferred in new 1.5mL centrifuge tubes；
(9) excretion body particle is harvested：1.5mL centrifuge tubes containing re-suspension liquid are centrifuged into 2min with 12000g in 4 DEG C, are retained
Supernatant is rich in excretion body particle in the supernatant.
(10) excretion body is purified：The excretion body particle crude product of harvest is transferred to Exosomoe Purafication Filter
In (EPF columns) upper chamber, 10min is centrifuged with 3000g in 4 DEG C, the liquid at EPF column tubes bottom is collected after centrifugation, this liquid is to purify
Excretion body particle afterwards；
(11) preservation of excretion body：Excretion body after purification is to be stored in -80 DEG C of low temperature refrigerators, in case subsequent experiment makes
With.
6 excretion body of embodiment is identified
(1) transmission electron microscope detects
By garbled excretion body sample by following processing：
It is fixed：2.5% glutaraldehyde is fixed, 2 hours；It is rinsed with 0.1M phosphoric acid rinsing liquids, three times；
1% osmic acid fixer is fixed, 2 hours；It is rinsed with 0.1M phosphoric acid rinsing liquids, three times；
Dehydration：50% ethyl alcohol, 20 minutes；70% ethyl alcohol, 20 minutes；90% ethyl alcohol, 20 minutes；90% ethyl alcohol：90% the third
Ketone (1：1), 20 minutes；90% acetone, 20 minutes；It is carried out in 4 degree of refrigerators above, 100% acetone room temperature, 20 minutes；
Embedding：Pure acetone+embedding liquid (2：1), room temperature 3 hours；Pure acetone+embedding liquid (1：2), ambient temperature overnight；Pure embedding
Liquid, 37 DEG C 2 hours；
Solidification：In 37 degree of baking ovens, overnight；In 45 degree of baking ovens, 12 hours；In 60 degree of baking ovens, 24 hours；
Slice (ultramicrotome is sliced 50-60nm)：Sample is loaded onto, the edge of a knife is selected, the edge of a knife is made to want parallel with sample；It checks
Each fixture locking device opens locking switch, the adjusting finely tuned by thickness, with making sample be contacted with knife face manually；Add distillation
Water grasps the water surface and wants parallel with knife face by the refractive power of water；Automatic sectioning is converted, dials the slice on the water surface, uses chloroform
It is smoked；Then slice is picked up with copper mesh；
Dyeing：The double dyeing of 3% acetic acid uranium-lead citrate.
Detection：Transmission electron microscope observing is taken a picture, film making.
(2) nanometer laser particle size analyzer detects
By garbled excretion body sample by following processing：(by taking U.S.'s Zetaview particle instruments as an example)
Computer and instrument power source are opened, preheater apparatus 10 minutes, then start-up operation software, sets grain size pattern and ginseng
Number, is put into sample cell by garbled excretion body sample, is positioned over the embedded sample detection device of instrument, closes slip lid, if
Detection time and number are set, then starts to detect sample, observes numerical value before panel, numerical value is preferred in 300HZ or so；Too low or mistake
Height need to stop detecting, and be carried out after suitably diluting to sample, then be measured.After detection, data is preserved.
(3) Western Blot are detected
By garbled excretion body sample by following processing：
Extract proteins：Be added lysate into garbled excretion body, 4 DEG C crack 15 minutes, then 4 DEG C/12000g from
The heart 5 minutes is put into 100 DEG C of water-baths 20 minutes, then proceedes to 12000g at 4 DEG C and centrifuges 2 minutes, -20 DEG C save backup.
Configure SDS-PAGE：Glass plate is rinsed well, is dried.Glass plate after drying is put into fixture, root as required
SDS-PAGE glue is prepared according to the size of albumen, first matches separation gel, 7ml separation gels is added in glass plate, then add 2ml absolute ethyl alcohols,
Match concentration glue after equal separation gels fully solidify after 30min again, the absolute ethyl alcohol in glass plate is discarded, with filter paper remaining anhydrous
Ethyl alcohol blots, and 2ml is added and concentrates glue, is inserted into broach.
Loading electrophoresis：After equal gellings are solid, put it into electrophoresis tank, loading is begun preparing for after filling up enough electrophoresis liquids.With
Both hands catch the both ends of comb gently firmly to take out comb upwards, are gently purged with the pipettor sucking electrophoretic buffer of 1ml
Sample hole, by ready sample loading, each sample takes identical total protein concentration, with the pipettor of 20ul perpendicular to loading hole sample
Product are slowly added in loading hole.Suitable electrophoresis liquid is added in electrophoresis tank lower part
Electrophoresis：Constant current 30mA 2 hours
Immunoblotting：After electrophoresis, using electrophoretic blotting device, electricity turns 120 points under 4 DEG C, 400mA constant current conditions
Clock, will be on protein delivery to pvdf membrane：The electrotransfer buffer solution that 500-800ml is poured into medical tray, glass plate from electricity
Swimsuit takes out in setting, and is shoveled with glue and lightly two pieces of glass plates are pried open in the upper end of glass plate, the lowermost end of glue is shoveled with glue
It gently cuts off, is then gently held up with glue shovel glue to be placed on filter paper, placement order is sequentially placed from cathode to anode：Filter
Paper-glue-pvdf membrane-filter paper, jig is put into electrophoretic blotting device, and the electrotransfer buffer solution that 1L is added carries out transferring film.
Immune colour developing：
Closing：The confining liquid that 40-50ml is poured into culture dish faces up the pvdf membrane to have taken a turn for the better and is put into culture
In ware, to prevent albumen from falling off, pvdf membrane will be completely submerged in confining liquid, and room temperature is closed 1 hour.
Primary antibody is incubated：The pvdf membrane closed is wrapped with PE gloves, the diluted antibody of confining liquid is added, is placed on mixed
4 degree of incubation 12h in clutch.
Wash film：Pvdf membrane is taken out from PE gloves, is put into culture dish, 40-50mlTBST solution is added, is placed on de-
Jog on color shaking table, washes film 3 times, 10 minutes every time.
Secondary antibody is incubated：Corresponding secondary antibody is diluted with confining liquid, is incubated pvdf membrane at room temperature 2 hours.
Colour developing：It is developed the color using Amersham companies ECL+plusTM Western blotting system kits
X-ray develops：It carries out obtaining the film for showing band in dark place.
ECL, exposure, development, fixing is added to be as follows：
Pvdf membrane is placed on tiled preservative film, with 1：40 ratio mixing A liquid and B liquid, being made into total volume is
1ml drops evenly mixed liquor on pvdf membrane, is protected from light 5 minutes.
It takes the film out, extra ECL substrate reactions liquids are fallen in drip, not allow PVDF to kill, and keep PVDF without apparent anti-
Answer drop, so that it may to be put into magazine, spread preservative film, shut magazine, according to the luminous degree of ECL, select the length of time for exposure
It is short that corresponding label is made on preservative film.X-ray is taken out, is put into developer solution, is taken out after about 1min, rinsed in clear water several
Second, after be put into fixing solution 2 minutes.
It wears gloves tweezers X-ray is taken out from fixing solution and be put into 65 degree of baking ovens drying, analysis.The X-ray after drying
Piece is put into magazine, and the albumen positions pre-dyed marker and sample ID are labeled in X-ray with marking pen according to mark before
Then on piece is analyzed result.
Fig. 2 B show the CD47-siRNA-Exosome (excretion body) of Electronic Speculum detection.
Fig. 3 shows the droplet measurement result of CD47-siRNA-Exosome.
Fig. 4 shows the testing result of the Western blot detection significant albumen of CD47-siRNA-Exosome.
It is prepared by 7 excretion body-siRNA compounds of embodiment
1. appropriate siRNA is added in 1.5ml centrifuge tubes, excretion body suspension is added, gently mixing.Ratio used is such as
Under, 109Excretion body particle：1ug siRNA：400uL electricity turns buffer solution).
2. by the Neon equipped with 3ml electrolytic buffers ETMPipe is inserted into pipettor rack.
3. pulse voltage, pulse width, umber of pulse are arranged on instrument.
4. pipette tips are inserted into NeonTM pipettors, excretion body mixed liquor is inhaled with the pipette tips of 10 μ l, it must be without gas in pipette tips
Bubble.NeonTM pipettors with sample are inserted perpendicularly into NeonTM pipes.
5. selecting electroporation, and press the Start on touch screen (beginning) key.
6. after electric pulse release, completion can be shown on touch screen, and electroporation is prompted to complete.
7. the good sample of pulse is immediately transferred into the ready culture plate equipped with preheating culture medium.Culture plate is put
To incubator culture.
8. after experiment, outwelling E liquid in pipettor, electroporation is closed.
By experimental verification, preferably BTFP siRNA sequences are as follows:
CAGGAGAGTTCTCAAGTAGAT(SEQ ID NO.:3)。
8 excretion body of embodiment-cell is incubated experiment altogether
Cultivating the good aim cell of growth conditions, (U-87-GFP stablizes strain and U-251-GFP stablizes strain, is purchased from the Chinese Academy of Sciences
Cell bank), aim cell is divided into 6-well culture plate cultures by the previous day, and it is outer to be incubated the group addition that the same day is empirically designed altogether
Secrete the total incubation experiment that body particle carries out aim cell.Fluorescence microscopy microscopic observation GFP expressions after incubation.
(1) cell Proliferation detects
It is to carry green fluorescence that U-87-GFP, which stablizes strain and U-251-GFP stabilization strains, can be read by Cellomics instruments
It takes the cell with fluorescence and takes pictures, the cell number that different groups contain in orifice plate is then calculated by software analyzing processing.
After continuous detection 3-5 days, cell growth curve figure is drawn out, to show cell growth condition.
A) by after each experimental group cell tryptase enzymic digestion in exponential phase, complete medium is resuspended into cell suspension；
B) with blood counting chamber to cell count；
C) bed board cell density (majority is 2000cell/well) is determined according to cell growth speed.Every group of 3-5 multiple holes,
Per 100 μ l of hole, to ensure that the consistent of cell number is added in every hole during bed board；
D) after completing plate, 37 DEG C of 5%CO2 incubator cultures are set；
E) since after bed board second day, daily CELLOMICS detections read plate is primary, continuous to detect read plate 3-5 days；
F) it by adjusting the input parameter of cellomics arrayscan, is accurately calculated in scanning orifice plate every time
The quantity of cell with green fluorescence；
G) statistics drawing is carried out to data, draws 5 days cell Proliferation curves.
Fig. 5 shows that excretion body is incubated fluorescence microscope testing result altogether with glioma cell.
Fig. 6 shows that excretion body is incubated MTT testing results altogether with glioma cell.Testing result shows using specificity
The RNAi for targeting BPTF genes, can significantly inhibit the proliferation of tumour cell.
(2) cell clonal formation detects
By clonality of the infected cell on tissue culture plate come prompt cell after slow-virus infection at
Tumor ability.
A) by each experimental group cell tryptase enzymic digestion in exponential phase, complete medium is resuspended, and cell suspension is made；
B) blood counting chamber carries out cell count to cell suspension；
C) cell inoculation：Each experimental group is inoculated with 800 cells/wells in 6 orifice plates culture plate, and each experimental group sets 3 again
Hole；
D) cell being inoculated with is continued into culture in incubator cell number is big by 14 days or in most single clone
Until 50, carries out changing liquid every 3day halfway and observe cell state；
E) it takes pictures to cell clone under fluorescence microscope before experiment terminates；
F) PBS washings cell 1 time when experiment terminates；
G) 1ml paraformaldehydes, 30~60min of fixed cell are added per hole；
H) PBS washs cell 1 time；
I) clean, 500 μ L of free from admixture GIEMSA dye liquors, dye cell 20min are added per hole；
j)ddH2O washes cell for several times, until cleaning background on plate, dries；
K) it takes pictures under microscope monoclonal；
L) digital camera is taken pictures whole plate；
M) colony count.
Fig. 7 shows that excretion body is incubated the result of Clone formation detection with glioma cell altogether.Testing result shows to carry
After thering is the excretion body of siRNA and cell to be incubated altogether, after carrying out endocytosis by cell, it can effectively inhibit the proliferation of cell.
(3) cell invasion ability detects
It is an important step of metastases from extracellular matrix invasion, tumour cell is by being attached to vascular wall and edge
Vascular wall stretching, extension and start to invade, proteolytic enzyme such as MMP collagenase digestions basement membrane of blood vessel and allow cancer cell to invade.BD
BiocoatTM MatrigelTMInvasion Chambe provide one effectively for detection tumour cell across substrate membrane modle
System.
A) take out kit from -20 DEG C of refrigerator, with 70% ethanol disinfection tweezers take out needed for number cell to newly
In 24 orifice plates, place room temperature makes it be restored to room temperature for a period of time；
B) respectively add 500 μ l to incubate (37 DEG C of incubations) serum free mediums in cell and lower room, placed in 37 DEG C of incubators
2h makes Matrigel hypothallus rehydration；
C) prepare cell suspension：Each group cell of the pancreatin digestion in exponential phase, is resuspended with serum free medium, is made
At cell suspension；
D) blood counting chamber carries out cell count to cell suspension；
E) after step 3 rehydration, cell is transferred to another aperture, and carefully remove culture medium in cell；
F) plus in 750 μ l 30%FBS culture mediums to lower room；
G) adding the 500 ready cell suspensions of μ l steps 4, (cell density is adjusted according to cell category difference, generally
To contain 5-10*104cell) in each cell；
H) it (is adjusted according to cell category difference) in 37 DEG C of incubator culture 24-48h；
I) back-off cell on blotting paper in, to remove culture medium, non-invasion cell gently being removed with cotton swab
J) plus in 500 μ l dyeing liquors to the emptying aperture of plate；
K) cell is immersed in 30min in Gimesa dyeing liquors, in the lower surface of film dyeing intrusion cell；
L) prepare in a large beaker for filling 3/4ths volume of water, rinsed back and forth with pincet gripping cell, in air
Dry cell；
M) it is integrally taken pictures, is focused when taking pictures critically important to cell with camera；
N) microscope is taken pictures film, each umber of beats of 100X, 400X (>=5)；
O) 10% acetic acid of 200uL is added in the air in 96 orifice plates, counterdie is taken off with scissors and tweezers, in 10% vinegar of 180uL
Acid dissolving (is blown with the suction of 200uL pipette tips and is stirred evenly) after being completely dissolved, draws 100ul in another hole, OD570 detections.
Fig. 8 shows that excretion body is incubated invasive ability testing result altogether with glioma cell.Testing result shows to carry
After the excretion body of siRNA is incubated altogether with cell, after carrying out endocytosis by cell, it can effectively inhibit the invasive ability of cell.
9 excretion body of embodiment injects tumor formation experiment of nude mouse
In tumor research, most conventional zoopery is exactly tumor formation in nude mice, is used due to most of tumor research
It is human cell, so there are the presence that xenogenesis excludes, needs the carrier for using immune-deficient mice as transplantation model, leads to
The injection tumour cell to the mouse is crossed, its tumor formation is made, then injects excretion body particle again, observes the growth of knurl to judge it
Biology changes.
A) by after each experimental group tumor cells pancreatin digestion in exponential phase, complete medium is resuspended outstanding at cell
Liquid；
B) cell is counted with blood counting chamber, and the PBS of certain volume is finally used to be resuspended, make the dense of cell suspension
Degree is 1~2 × 107A cell/ml；
C) with disposable syringe by armpit on the right side of a certain amount of pallium cell injection to nude mice, the cell concentration of injection is general
It is 2 × 106A cell；
D) nude mice is raised after injecting to the visible knurl of naked eyes；(time is 2 weeks or so)
E) after injection excretion body PBS solution (100ug/ml, 100ul/ are only) continues raising 4 weeks daily, experiment terminates, and collects
Data；
F) it takes pictures (including the photo of nude mice lotus knurl and knurl photo after execution)；
G) statistics drawing is carried out to data.
Fig. 9 shows that excretion body injects tumor formation experiment of nude mouse result.Testing result shows to carry the siRNA of the present invention
Excretion body injection nude mice model after, by blood circulation, can effectively inhibit the proliferation of tumour.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of excretion body for treating tumour, which is characterized in that contain CD47 molecules on the excretion body film of the excretion body.
2. excretion body as described in claim 1, which is characterized in that the excretion body is enclosed with siRNA molecule or its precursor.
3. excretion body as described in claim 1, which is characterized in that the siRNA molecule is the siRNA molecule of tumour-specific
Or its precursor.
4. excretion body as described in claim 1, which is characterized in that the siRNA molecule targets BPTF genes.
5. a kind of method preparing excretion body, which is characterized in that the method includes the steps：
(1) Lentiviral is built
Wherein, the Lentiviral includes the polynucleotide sequence of coding CD47；
(2) slow virus is packed
The Lentiviral that step (1) is built is packaged as virion；
(3) cell strain is built
Infestation with virus particles host cell is obtained in practical step (2), to build the cell strain for stablizing expression CD47；
(4) excretion body is prepared
The cell strain built in incubation step (3) collects cell supernatant extraction excretion body, to obtain the excretion body.
6. method as claimed in claim 5, which is characterized in that the method further includes step：
(5) excretion body-siRNA compounds are prepared.
7. method as claimed in claim 5, which is characterized in that, will be described in such a way that electricity is transduceed in the step (5)
SiRNA molecule is wrapped up into the excretion body, to which the excretion body-siRNA compounds be made.
8. a kind of pharmaceutical composition, which is characterized in that including pharmaceutically acceptable carrier and a effective amount of active constituent, wherein
The active constituent is excretion body described in claim 1.
9. the purposes of excretion body described in claim 1, which is characterized in that for preventing or treating tumour.
10. a kind of method that external non-therapeutic inhibits tumour cell, which is characterized in that including step：Described in claim 1
The excretion body for treating tumour it is existing under the conditions of, tumour cell is cultivated, to inhibit tumour cell.
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| CN114369575B (en) * | 2021-12-30 | 2023-08-29 | 上海艾棵颂生物科技有限公司 | Brain glioma cell-derived exosome without tumor promotion function, and preparation method and application thereof |
| CN114292848A (en) * | 2022-01-05 | 2022-04-08 | 多莱泌生物科技(武汉)有限公司 | Engineered exosome with high expression of cancer-suppressing miRNA and targeting lung cancer |
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