CN104232568A - Osteogenic induction method of periodontal ligament stem cells (PDLSCs) - Google Patents
Osteogenic induction method of periodontal ligament stem cells (PDLSCs) Download PDFInfo
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Abstract
The invention provides an osteogenic induction method of periodontal ligament stem cells (PDLSCs), which comprises the following steps: by using a third molar or premolar of a healthy patient as a raw material, separating and culturing the PDLSCs; and inoculating the PDLSCs in an osteogenic induction differentiation culture medium with the inoculum size of 2.0*10<4>/cm<2>, putting in a 37-DEG C cell culture box, and carrying out induction differentiation for 2 weeks, wherein the osteogenic induction differentiation culture medium is prepared by adding 10<-3> mol/L icariin, 10<-6> mol/L emodin, 10<-5> mol/L puerarin, 10<-6> mol/L berberine, 10<-5> mol/L encommiol extract, 2 mg/L drynaria total flavone, 4 mg/ml psoralen, 4 mg/ml oleanolic acid and 10<-6> mol/L Chinese teasel root saponin VI into a basal culture medium. The method can successfully implement osteogenic induction of the PDLSCs without influencing the stem cell characteristics and immunologic characteristics of the PDLSCs.
Description
Technical field
The present invention relates to medical science and technical field of cell biology, be specifically related to a kind of bone of periodontal ligament stem cell to induction method.
Background technology
Periodontitis is the clinical common disease of Stomatological Department, is take periodontal tissue destruction as the infectious diseases of principal character, and clinical main manifestations is that the formation of the inflammation of gum and hemorrhage, periodontal pocket, frontal resorption and odontoseisis, displacement are until lose.Periodontitis is ubiquity in crowd, and wherein chronic periodontitis reaches more than 60%, and aggressive periodontitis is 5%-15%, and periodontitis accounts for the 30%-44% of exodontia reason.Periodontitis is not only the major cause of loss of tooth, and develops relevant, as diabetes, cardiovascular disorder etc. with the generation of some whole body system disease.Once the destruction of periodontal attachment and alveolar bone occurs, optimal mode intactly rebuilds healthy periodontal tissue, formation periodontal new attachment and new bone, but because of the periodontal tissue particularly osseous tissue that periodontitis is lost, the difficulty of its Reparation and Reconstruction is very large, therefore the treatment of periodontitis is one of Focal point and difficult point of clinical oral research always.
At present, the periodontitis treatment method of clinical normal employing comprises: periodental non-surgical treatment (scaling, scrape control, root planing), periodontal flap surgery and paradenlal tissue regeneration art etc., but these methods only can patients in remission, stop the further developing of disease, still can completely does not rebuild healthy periodontal tissue, namely can not cure periodontitis.
American scientist research shows to comprise more mono-clonal stem cell in people's periodontium tissue, be referred to as periodontal ligament stem cell (periodontal ligament stem cells, PDLSCs), PDLSCs has higher clonality and cell proliferation capacity, have and be divided into adipocyte and fibroblastic ability, express mark STRO-1 and CD146 of mescenchymal stem cell.When being migrated to the mouse of immune deficiency, PDLSCs can produce the dental cement spline structure along the direction traveling of periodontium reticular tissue, and prompting PDLSCs is the potential cell derived of the periodontal disease therapeutic of stem cell mediation.
And the allosome application of bone to differentiation PDLSCs will be completed, bone to the differentiation stem cell properties of PDLSCs and immunological characteristic be another must in the face of and the problem of solution of having to.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of bone of periodontal ligament stem cell to induction method, and the method can successfully realize to the bone of PDLSCs to induction, and does not affect stem cell properties and the immunological characteristic of PDLSCs.
The technical solution adopted in the present invention is:
The bone of periodontal ligament stem cell is to an induction method, and it comprises the following steps:
S1: get the third molar pulled out from healthy patients or premolar teeth is raw material, is separated and turns out PDLSCs;
S2: the PDLSCs obtained is inoculated in bone in inductive differentiation medium, inoculum size is 2.0 × 10
4/ cm
2, be then placed in 37 DEG C of cell culture incubators and carry out differentiation-inducing 2 weeks, wherein bone adds 10 to substratum based on the formula of inductive differentiation medium
– 3mol/L icarin, 10
-6mol/L Schuttgelb, 10
-5mol/L puerarin, 10
-6mol/L Berberine, 10
-5mol/L ulmoprenol extract, 2mg/L Process of Total Flavonoids in Drynaria Fortunei, 4mg/mL psoralene, 4mg/mL Oleanolic Acid and 10
-6mol/L asperosaponin VI.
In described S2 step, the formula of basic medium is α-MEM liquid nutrient medium.
Compared with prior art, the present invention has following remarkable advantage and beneficial effect:
(1) the present invention adopts the substratum of special formulation, special Chinese medicinal materials composition is added in basic medium, PDLSCs is realized bone to differentiation-inducing by success, the bone obtained confirms still have stem cell properties to differentiation periodontal ligament stem cell through experiment, and has reduced immunogenicity and immunosuppression feature; If PDLSCs is divided into osseous tissue in vivo, then can form new bone, repair and destroyed by periodontitis and the Cranial defect that formed, thus become a kind of novel method of effectively treatment periodontitis.
(2) periodontitis leads higher late adult onset at present, but the autologous PDLSCs ten points suffering from the elderly of periodontitis is limited, we can not pull out a healthy tooth of patient, separation and Culture PDLSCs treats periodontitis, will lose more than gain like this, the present invention adopts teenager to need when carrying out orthodontic treatments to pull out and is raw material as Biohazard Waste by the premolar teeth of health thrown away and third molar, separation and Culture PDLSCs, carry out bone to induction method, not only effectively achieve the recycling of Biohazard Waste, if and PDLSCs can apply by allosome, just obviously expand the source of PDLSCs, greatly can advance the development of periodontitis treatment.
Accompanying drawing explanation
Shown in Fig. 1 is the experimental result of embodiment 3 (PDLSCs can carry out bone to differentiation), wherein A: bone is to induction 1 day PDLSCs and do not induce the alkaline phosphatase activities of PDLSCs close, and bone obviously raises to the alkaline phosphatase activities of induction 3 days PDLSCs, bone then has remarkable rising to the alkaline phosphatase activities of induction 7 days PDLSCs; B:RT-PCR finds, bone to induction after 2 weeks, the mark of these three osseous tissues of PDLSCs high expression level OCN, BSP and Msx1; C:PDLSCs is at bone to induction after 4 weeks, and the calcareous dyeing of Von-kossa presents more brown positive reaction region; D: do not break up when PDLSCs carries out Von-kossa calcareous dyeing and do not occur positive reaction.White post: do not break up PDLSCs; Black post: bone is to differentiation PDLSCs; ALP: alkaline phosphatase; BSP: resorption lacunae; OCN: Bone Gla protein; Msx1: homeobox protein; NS: there was no significant difference;
*p<0.05,
*p<0.01; The multiple of figure C, D: 400 times.
Shown in Fig. 2 is the experimental result of embodiment 4, wherein flow cytometry finds, PDLSCs does not express the mark CD14 (A) of hematopoietic lineage cell, CD34 (B) and CD45 (C), still express the mark STRO-1 (D) of mescenchymal stem cell, CD90 (E) and CD146 (F), positive rate is respectively 14.9%, 72.73% and 86.24%.
Shown in Fig. 3 is the experimental result of embodiment 5, wherein A: bone does not cause the lymphocytic propagation of T to the differentiation PDLSCs of 2 weeks, shows that it has reduced immunogenicity; B: bone significantly suppress to the differentiation PDLSCs of 2 weeks the allogeneic T lymphocytes propagation that mitogen PHA causes; NS: there was no significant difference; PBMCs: peripheral blood mononuclear cell; PHA: phytohaemagglutinin; MLR: mixed lymphocyte reacion;
*p<0.01.
Shown in Fig. 4 is the schematic diagram that in embodiment 5, prostaglandin E2 plays a significant role in the immunosuppressive properties of bone to differentiation PDLSCs: set up the co-cultivation system of bone to the differentiation PDLSCs+ equivalent allosome PBMCs+PHA of 2 weeks, then anti-TGF-beta antibody is added respectively, anti-pHGF antibody, anti-IL-10 antibody, isotype control Ab, L-NAME (inhibitor of induction type NO synthetic enzyme), 1-methyl-L-tryptophan (indoles amine-2, the inhibitor of 3-dioxygenase), indomethacin (inhibitor of prostaglandin E2), found that, only have in the system of row parathyrine E2 inhibitor before addition, again propagation is there occurs by the T lymphocyte that bone suppresses to differentiation 2 weeks PDLSCs, PBMCs: peripheral blood mononuclear cell, PHA: phytohaemagglutinin, 1-MT:1-methyl-L-tryptophan, L-NAME:N-nitro-L-arginine methyl esters, TGF-beta: Peritoneal fibrosis beta, HGF: pHGF, IL-10: interleukin 10, NS: there was no significant difference.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail.Iting is noted that following illustrating is all exemplary, being intended to the invention provides further instruction.Except as otherwise noted, all Science and Technology terms that the present invention uses have the identical meanings usually understood with the technical field of the invention personnel.
Embodiment 1: the separation of people PDLSCs, cultivation:
Choose normal third molar or the premolar teeth (Weifang Yidu Central Hospital's Stomatology Clinic provides) of the healthy patients pulled out, be separated according to the method for bibliographical information and cultivate PDLSCs.Be summarized as follows: the tooth pulled out is put into the aseptic centrifuge tube that precooling PBS is housed immediately, transfers into Cytology Lab, separation and Culture PDLSCs in 24 hours.Peel off periodontal periodontal tissue gently, get the periodontal tissue in stage casing, repeatedly clean with PBS, shred, be placed in the Digestive system containing NTx enzyme (3g/L) and neutral protease (4g/L), digest 1 hour at 37 DEG C, cross 70 μm of cell sieve collecting cells, 1000 revs/min centrifugal 10 minutes, becomes single cell suspension with nutrient solution Eddy diffusion.Single cell suspension is inoculated in 25cm
2in Tissue Culture Flask, 37 DEG C, 5%CO in α-MEM substratum (containing the L-AA 2-phosphoric acid of 15% foetal calf serum, 100 μm of ol/L, 2mmol/L glutamine, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates)
2cultivate, within every 2 ~ 3 days, change liquid 1 time.Every day is observation of cell upgrowth situation under inverted microscope.When Growth of Cells to 80% converging state, press 1:2 had digestive transfer culture with 0.25% trypsinase, then carry out the bone of PDLSCs to differentiation-inducing.
The bone of embodiment 2:PDLSCs is to differentiation-inducing
The PDLSCs that embodiment 1 obtains is inoculated in bone in inductive differentiation medium, inoculum size is 2.0 × 10
4/ cm
2, be then placed in 37 DEG C of cell culture incubators and carry out differentiation-inducing 2 weeks, wherein bone adds 10 to substratum based on the formula of inductive differentiation medium
– 3mol/L icarin, 10
-6mol/L Schuttgelb, 10
-5mol/L puerarin, 10
-6mol/L Berberine, 10
-5mol/L ulmoprenol extract, 2mg/L Process of Total Flavonoids in Drynaria Fortunei, 4mg/mL psoralene, 4mg/mL Oleanolic Acid and 10
-6mol/L asperosaponin VI.
Above-mentioned bone is α-MEM to the formula of basic medium in inductive differentiation medium.
Embodiment 3:PDLSCs bone is to differentiation-inducing detection
Following three kinds of methods are adopted to detect embodiment 2 bone to induction PDLSCs:
1, alkaline phosphatase activities detects:
PDLSCs bone detects with alkaline phosphatase activities test kit to induction for latter 1 day, 3 days, 7 days.Concrete operations illustrate according to test kit to be carried out.
Result: detect its alkaline phosphatase activities to induction after 1 day, 3 days, 7 days at PDLSCs bone, discovery bone is to induction 1 day PDLSCs and do not induce the alkaline phosphatase activities of PDLSCs close, both do not have significant difference, and bone obviously raises to the alkaline phosphatase activities of induction 3 days, 7 days PDLSCs, be significantly higher than and do not induce PDLSCs, show PDLSCs obviously bone to differentiation.
2, RT-PCR experiment:
PDLSCs bone detects the expression of Bone Gla protein (osteocalcin, OCN), resorption lacunae (bone sialoprotein, BSP) and homeobox protein (Msx1) for 2 weeks after induction with RT-PCR.Primer sequence is respectively:
OCN, upstream: 5 '-CATGAGAGCCCTCACA-3 '
Downstream: 5 '-AGAGCGACACCCTAGAC-3 '
BSP, upstream: 5 '-CTATGGAGAGGACGCCACGCCTGG-3 '
Downstream: 5 '-CATAGCCATCGTAGCCTTGTCCT-3 '
Msx1, upstream: 5 '-TAAACCCCTTGCTACACACTTCC-3 '
Downstream: 5 '-AAAAACCTGAACATCTTCCGACC-3 '
GAPDH (internal reference) upstream: 5 '-GGGCATGAACCATGAGAAGT-3 '
Downstream: 5 '-AAGCAGGGATGATGTTCTGG-3 '
Concrete operation method is as follows: extract cell total rna, synthesize the first chain cDNA.Get 0.5mlPCR pipe, add following reagent successively: the first chain cDNA2 μ l; Upstream primer (20mM) 1 μ l; Downstream primer (20mM) 1 μ l; DNTP (5mM) 4 μ l; 10 × PCRbuffer5 μ l; EXTaq enzyme 1 μ l.Add appropriate ddH2O, make cumulative volume reach 50 μ l.Mix gently, centrifugal.Setting PCR program: 94 DEG C of denaturation 5min, 94 DEG C of sex change 20s, 57 DEG C of renaturation 25s, 72 DEG C of extensions, carry out 35 circulations, extend 5min 72 DEG C of ends subsequently.Row agarose gel electrophoresis, observations under ultraviolet lamp.
Result: PDLSCs bone after 2 weeks, detects the expression of OCN, BSP and Msx1 to induction with RT-PCR, finds the mark of these three osseous tissues of PDLSCs high expression level.
3, Von-kossa dyeing:
PDLSCs bone detects calcium deposition situation by Von-kossa staining in 4 weeks after induction.Concrete grammar is as follows: the cell PBS after induction is washed 3 times; The alcohol fixation of 95% 30 minutes; Distilled water flushing 5 minutes; With 2% silver nitrate solution reductase 12 0 minute under UV-light; Fully rinse with distilled water; With 5% Sulfothiorine effect 3 minutes, termination reaction; The calcareous coloration result of basis of microscopic observation Von-kossa.
Result: PDLSCs is under the induction of bone to induction broth, and cell proliferation capacity weakens, and form changes gradually, becomes cube, polygon gradually, then become irregular type by spindle-type, cultivates and occurs cladding in about 3 weeks, form similar tuberculiform structure.After 4 weeks, the calcareous dyeing of Von-kossa presents more brown positive reaction region.And not there is positive reaction when not breaking up PDLSCs dyeing.
More than test confirmation, PDLSCs can carry out bone to differentiation (Fig. 1).
Embodiment 4: bone is to the expression of induction PDLSCs stem cell markers after 2 weeks
Flow cytometry:
By the CD14 of Flow cytometry bone to differentiation PDLSCs after 2 weeks, the expression of CD34, CD45, STRO-1, CD90, CD146.Concrete steps are as follows:
(1) .PDLSCs 4% paraformaldehyde room temperature fixes 20 minutes.
(2). wash once with PBS, then use PBS suspendible cell, adjustment cell concn is 10
5individual cell/200 μ l.
(3). get 200 μ l cell suspensions, add anti-human CD14 antibody (1:100) respectively, CD34 antibody (1:100), CD45 antibody (1:100), STRO-1 antibody (1:100), CD90 antibody (1:200), CD146 antibody (1:200), react 1 hour under room temperature.
(4) .1000 rev/min centrifugal 5 minutes, wash once with PBS.
(5). abandon supernatant, add 100 μ lPBS suspendible cells.
(6). for CD14, CD34 and CD45 antibody, direct flow cytometer loading, detects expression.
(7). for STRO-1, CD90 and CD146 antibody, then add the anti-mouse IgG antibody (1:200) of FITC mark, room temperature lucifuge 30 minutes.Flow cytometer loading, detects expression.
Result: bone is to induction after 2 weeks, flow cytometry finds, PDLSCs still expresses mark STRO-1, CD90 and CD146 of mescenchymal stem cell, and positive rate is respectively 14.9%, 72.73% and 86.24%, do not express the mark CD14 of hematopoietic lineage cell, CD34 and CD45.
More than test confirmation, bone still has the characteristic (Fig. 2) of mescenchymal stem cell to the differentiation PDLSCs of 2 weeks.
Embodiment 5:
In following immunological experiment, separated by PDLSCs and PBMCs by the Transwell culture systems with 0.4 μm of aperture mocromembrane, PDLSCs is placed in lower floor, and PBMCs is placed in upper strata.
1, bone is to the immunogenicity of differentiation PDLSCs: by bone to the differentiation PDLSCs of 2 weeks (5.0 × 10
4) bed board is adherent, adds the allosome PBMCs of equivalent, at 37 DEG C, 5%CO
2cultivate 5 days under condition.With 5.0 × 10
4simple PBMCs cultivates, from 5.0 × 10 of two Different Individual
4pBMCs reaction in contrast.The proliferative conditions of PBMCs is detected with CCK-8 test kit.
2, bone is to the immunomodulatory properties of differentiation PDLSCs: by the bone of difference amount to differentiation 2 weeks PDLSCs (2.5 × 10
4, 5.0 × 10
4or 2.5 × 10
5) after bed board is adherent, add 5.0 × 10
4pBMCs (PDLSCs:PBMCs is respectively 1:5,1:1,5:1) and final concentration be the phytohaemagglutinin (PHA) of 0.5 μ g/mL, 37 DEG C, cultivate 5 days under 5%CO2 condition.The proliferative conditions of PBMCs is detected with method CCK-8 test kit.
3, bone is to the Exploration of Mechanism of differentiation PDLSCs immunomodulatory properties: by bone to the differentiation PDLSCs of 2 weeks (5.0 × 10
4) bed board is adherent, add the PHA that the allosome PBMCs of equivalent and final concentration are 0.5 μ g/mL, then add anti-transforming growth factor (transforminggrowthfactor-beta, TGF-beta respectively; 10 μ g/ml) antibody, anti-pHGF antibody (10 μ g/ml), anti-IL-10 antibody (10 μ g/ml), isotype control Ab (10 μ g/ml), the L-NAME (inhibitor of induction type NO synthetic enzyme; 1mmol/L), the 1-methyl-L-tryptophan (inhibitor of indoles amine-2,3-dioxygenase; 500 μm of ol/L), the indomethacin (antagonist of prostaglandin E2; 500 μm of ol/L).Within 5 days, detect the proliferative conditions of PBMCs afterwards.
Result:
The lymphocytic propagation of Allogeneic T can be caused to the PDLSCs broken up to observe bone, namely whether bone has immunogenicity to the differentiation PDLSCs of 2 weeks, we by bone to the differentiation PDLSCs of 2 weeks and equivalent allosome PBMCs mixed culture 5 days, within 5 days, detect T lymphopoiesis situation afterwards, found that bone does not cause the lymphocytic propagation of T to the differentiation PDLSCs of 2 weeks, show that bone has reduced immunogenicity to the differentiation PDLSCs of 2 weeks.
We study the impact that bone is bred to the differentiation PDLSCs of 2 weeks the allogeneic T lymphocytes that mitogen PHA causes further.Bone after 5 days, is found that bone significantly suppress the propagation of allosome PBMCs to the differentiation PDLSCs of 2 weeks to the differentiation PDLSCs+ allosome PBMCs+PHA co-cultivation of 2 weeks.
Inquire into the mechanism of bone to the immunosuppressive properties of differentiation 2 weeks PDLSCs subsequently, first the co-cultivation system of bone to the differentiation PDLSCs+ equivalent allosome PBMCs+PHA of 2 weeks is set up, then anti-TGF-beta antibody is added respectively, anti-pHGF antibody, anti-IL-10 antibody, isotype control Ab (10 μ g/ml), L-NAME (inhibitor of induction type NO synthetic enzyme), 1-methyl-L-tryptophan (indoles amine-2, the inhibitor of 3-dioxygenase), indomethacin (inhibitor of prostaglandin E2), found that, only in the system adding indomethacin, again propagation is there occurs by the T lymphocyte that bone suppresses to differentiation 2 weeks PDLSCs, show that prostaglandin E2 has played vital role in the immunosuppressive properties of bone to differentiation PDLSCs.
More than test confirmation, bone has reduced immunogenicity and immunosuppressive properties (Fig. 3, Fig. 4) to the differentiation PDLSCs of 2 weeks.
Attached: human peripheral blood single nucleus cell (peripheral blood mononuclearcells, PBMCs) separation, cultivation: heparin (25u/ml) the anticoagulation 2ml getting healthy volunteer, 3 times are diluted with PBS, join the test tube upper strata containing 5mlFicoll (1.077g/mL), blood slowly adds along tube wall, makes to keep clear interface between two liquid.2000 revs/min centrifugal 30 minutes, is respectively plasma layer, mononuclearcell layer, Ficoll layer, GCL and red blood cell layer in test tube from top to bottom.Mononuclearcell layer between careful absorption blood plasma and Ficoll, adds the PBS of 5 times and mixes, and 1000 revs/min centrifugal 5 minutes, and remove upper asking, precipitation is PBMCs.Precipitation is suspended in containing containing 10% foetal calf serum, 20mol/LHEPES, 2mmol/LL-glutamine, in 100U/mL penicillin and 100 μ g/mL Streptomycin sulphate RPMI-1640 substratum, at 37 DEG C, 5%CO
2cultivate under condition.
The material that the embodiment of the present invention relates to, reagent and experimental installation, if no special instructions, be the commercially available prod meeting biological chemistry and stem cell induction.
The above, be only the preferred embodiments of the present invention, should be understood that; for those skilled in the art; under the prerequisite not departing from core technology of the present invention, can also make improvements and modifications, these improvements and modifications also should belong to scope of patent protection of the present invention.Any change in the implication suitable with claims of the present invention and scope, all should think to be included in the scope of claims.
Claims (2)
1. the bone of periodontal ligament stem cell is to an induction method, it is characterized in that comprising the following steps:
S1: get the third molar pulled out from healthy patients or premolar teeth is raw material, is separated and turns out PDLSCs;
S2: the PDLSCs obtained is inoculated in bone in inductive differentiation medium, inoculum size is 2.0 × 10
4/ cm
2, be then placed in 37 DEG C of cell culture incubators and carry out differentiation-inducing 2 weeks, wherein bone adds 10 to substratum based on the formula of inductive differentiation medium
– 3mol/L icarin, 10
-6mol/L Schuttgelb, 10
-5mol/L puerarin, 10
-6mol/L Berberine, 10
-5mol/L ulmoprenol extract, 2mg/L Process of Total Flavonoids in Drynaria Fortunei, 4mg/mL psoralene, 4mg/mL Oleanolic Acid and 10
-6mol/L asperosaponin VI.
2. the bone of a kind of periodontal ligament stem cell according to claim 1 is to induction method, it is characterized in that: in described S2 step, the formula of basic medium is α-MEM liquid nutrient medium.
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| CN110616190B (en) * | 2019-02-19 | 2023-08-18 | 中国人民解放军第四军医大学 | Method for regulating and controlling periodontal ligament stem cell osteogenic differentiation based on extracellular matrix |
| CN112899225A (en) * | 2021-03-25 | 2021-06-04 | 徐会娟 | Periodontal ligament stem cell culture medium and culture method thereof |
| CN112899225B (en) * | 2021-03-25 | 2023-11-17 | 云南贺尔思细胞生物技术有限公司 | Periodontal ligament stem cell culture medium and culture method thereof |
| CN115607729A (en) * | 2022-11-01 | 2023-01-17 | 吉林大学 | Biological ink, 3D printing hydrogel support, preparation method and application |
| CN115607729B (en) * | 2022-11-01 | 2023-11-17 | 吉林大学 | Biological ink, 3D printing hydrogel bracket and preparation method and application |
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