CN103146755A - Method for inducing synovium mesenchymal stem cells to be differentiated to chondrocytes by in-vitro lentivirus mediated BMP-2 (Bone Morphogenetic Protein) genes - Google Patents

Abstract

The invention relates to a method for inducing synovium mesenchymal stem cells (SMSCs) to be differentiated to chondrocytes by in-vitro lentivirus mediated BMP-2 (Bone Morphogenetic Protein) genes. The method comprises the following steps of: separating and culturing synovium mesenchymal stem cells; constructing a recombinant plasmid pFUGW-oBMP-2; preparing morbus virosus of transfected lentivirus; and transfecting synovium mesenchymal stem cells by the morbus virosus of transfected lentivirus, wherein in the step of preparing morbus virosus of transfected lentivirus, the transfected lentivirus is jointly formed by the recombinant plasmid pFUGW-oBMP-2 and a packaging plasmid; and in the step of transfecting synovium mesenchymal stem cells by the morbus virosus of transfected lentivirus, synovium mesenchymal stem cells over third generation is taken to be mixed with the morbus virosus of transfected lentivirus, and then added into incomplete chondroblast inducing culture liquid to induce so as to obtain chondrocytes. SMSCs transfected by the transfected lentivirus provided by the invention are safe enough and can be spontaneously differentiated to cartilage in vitro.

Description

The gene induced Synovial Mesenchymal Stem Cells of external lentivirus mediated BMP-2 is to the method for Chondrocyte Differentiation

Technical field:

The present invention relates to biomedical sector, relate in particular to a kind of method of cartilage differentiation, particularly the gene induced Synovial Mesenchymal Stem Cells of a kind of external lentivirus mediated BMP-2 is to the method for Chondrocyte Differentiation.

Background technology:

In the last few years, used Synovial Mesenchymal Stem Cells to carry out the hot issue that the meniscal cartilage reparation is joint surgery.Classical organizational engineering method is that the in-vitro multiplication induced dry-cell is formed to seed cell, and the plantation seed cell, on biological support, is retracted organism by support and adds various cytokines and repaired.

Previously many investigators select mesenchymal stem cells MSCs (bone marrow-dervied mesenchymal stem cells, BMSCs) as seed cell.But, after De Barii has found Synovial Mesenchymal Stem Cells (synovium-dervied mesenchymal stem cells, SMSCs), it has replaced rapidly BMSCs.Reason is: although SMSCs is similar with BMSCs aspect cellular form, immunophenotype, colony formation ability and differentiation potential, it has stronger cartilage differentiation potential; In addition, when physiology is taken in meniscus appearance damage, synovial membrane is as the holder of SMSCs, and the field planting of can dividing a word with a hyphen at the end of a line, to injury region, together with local cells, completes repair process.So select SMSCs, more meet physiological process, and more single-minded cartilage differentiation potential is arranged.

So far, have case report investigator to adopt transforming growth factor 3(TGF-β 3) and bone morphogenetic protein 2(BMP-2) as mediating the inducible factor of SMSCs to cartilage differentiation.Sakaguchiii, Shirasawaiii and Horie Miv etc. utilize TGF-β 3, BMP-2 and dexamethasone (Dex), successfully induce SMSCs to break up to fibrous cartilage.But still there are problems in this process: induce required BMP-2 concentration to need very high (50～200g/L is even higher), and chronic, this just causes the possibility of the oriented bone differentiation of SMSCs.And there is the possibility of degraded inactivation in the external factor of application cell repeatedly, and greatly increase cultivation fee use.So in experiment of the present invention, adopt lentivirus mediated rabbit BMP-2 gene transfection SMSCs, and add the EGFP gene as reporter gene, thereby carry out repair of cartilage for application organizational engineering method, provide new approaches and novel method.

Summary of the invention:

The object of the present invention is to provide the method for the gene induced Synovial Mesenchymal Stem Cells of a kind of external lentivirus mediated BMP-2 to Chondrocyte Differentiation, the gene induced Synovial Mesenchymal Stem Cells of described this external lentivirus mediated BMP-2 to the method for Chondrocyte Differentiation to solve external evoked Synovial Mesenchymal Stem Cells in prior art to the method success ratio of cartilage differentiation low and time and the high technical problem of cost.

The invention provides the method for the gene induced Synovial Mesenchymal Stem Cells of a kind of external lentivirus mediated BMP-2 to Chondrocyte Differentiation, comprise that one is separated Synovial Mesenchymal Stem Cells, the step of cultivating, the step that also comprises a construction recombination plasmid pFUGW-oBMP-2, the step of the virus liquid of a preparation transfection slow virus, one by the step of transfection slow-virus transfection Synovial Mesenchymal Stem Cells, in the step of described construction recombination plasmid pFUGW-oBMP-2, at first according to BMP-2 gene design primer, by the reverse transcription reaction BMP-2 gene that increases, pFUGW plasmid and the BMP-2 gene recombination of EGFP reporter gene will be comprised, construction recombination plasmid pFUGW-oBMP-2, in the step of the virus liquid of described preparation transfection slow virus, utilize described recombinant plasmid pFUGW-oBMP-2 to add packaging plasmid, common structure transfection slow virus, and then add damping fluid to cultivate, hatch, remove supernatant, obtain the virus liquid of transfection slow virus after filtration, described by the step of transfection slow-virus transfection Synovial Mesenchymal Stem Cells, the virus liquid of the transfection slow virus of getting the above Synovial Mesenchymal Stem Cells of the third generation and thawing mixes, mixture is centrifugal, cultivate 40-52 hour, then above-mentioned culture is added in incomplete one-tenth chondrocyte induction nutrient solution inducing culture liquid and induce, induce more than 14 days and can obtain the chondrocyte.

Further, in the step of the virus liquid of described preparation transfection slow virus, at first increase recombinant plasmid pFUGW-oBMP-2, packaging plasmid psPAX2 and pMD2.G, purify concentrated, secondly the cell density of 293T cell is adjusted to 10 ⁷, putting into culturing bottle cultivates, described culturing bottle contains the DMEM substratum, also include penicillin G in described substratum, Streptomycin sulphate, calf serum, polylysine, the concentration of described penicillin G in the DMEM substratum is 100U/ml, the concentration of described Streptomycin sulphate in the DMEM substratum is 100 μ g/ml, the mass percent concentration of described calf serum in the DMEM substratum is 10%, concentration in described polylysine DMEM substratum is 5%, the 293T cell is added and contains pFUGW-oBMP-2 after culturing bottle is hatched, psPAX2, the damping fluid of pMD2.G, hatch, remove supernatant, obtain the virus liquid of transfection slow virus after filtration.

Further, in the step of described construction recombination plasmid pFUGW-oBMP-2, by Age1 and Nhe1 double digestion for the cDNA of oBMP-2, simultaneously, to comprise Age1 and the Nhe1 double digestion for pFUGW of EGFP reporter gene, then add the T4DNA reaction to obtain the pFUGW-oBMP-2 recombinant plasmid.

Further, in the process of amplification BMP-2 gene, the primer sequence of described 5 ' end is as shown in SEQ IDNO:1, and the primer sequence of described 3 ' end is as shown in SEQ ID NO:2.

Further, described incomplete one-tenth chondrocyte induction nutrient solution nutrient solution be one containing TGF-β ₃, dexamethasone, 2-phosphoric acid-xitix, proline(Pro), L-Ala, 1:100 dilution the H-DMEM nutrient solution of ITS+Premix solution; Wherein, in described H-DMEM nutrient solution, TGF-β ₃concentration be 10ng/mL, dexamethasone concentration is 1 * 10 ^-7contain Regular Insulin, Transferrins,iron complexes, selenous acid, bovine serum albumin, linolic acid in the ITS+Premix solution that the concentration that the concentration that the concentration of mol/L, 2-phosphoric acid-xitix is 50 μ g/mL, proline(Pro) is 40 μ g/mL, L-Ala is 100 μ g/mL, described 1:100 dilution, in the ITS+Premix solution of described 1:100 dilution, the concentration that the concentration that the concentration that the concentration of Regular Insulin is 6.25 μ g/mL, Transferrins,iron complexes is 6.25 μ g/mL, selenous acid is 6.25ng/mL, bovine serum albumin is that 1.25mg/mL, linoleic concentration are 5.35mg/mL.

Although SMSCs and BMSCs are all similar aspect cellular form, immunophenotype, colony formation, differentiation potential, SMSCs has stronger cartilage differentiation potential, so the present invention selects the seed cell of SMSCs as repair of cartilage.

Be identified at present and can promote SMSCs to comprise to the cytokine of cartilage differentiation: TGF-β 1, BMP-2, IGF-1, DEX.TGF-β 1 can strengthen the amplification of SMSCs, and suppresses its differentiation to bone and fat, and the mechanism of action of TGF-β 1 realizes by the ERK1/2MAPK signal path.

BMP-2 is acknowledged as the strongest osteogenic factor.But research in recent years think its can also inducing mesenchymal stem cell to cartilage differentiation.Although at present its mechanism is known little about it, it applies but day by day augmentation.But in its application process, still exist the manufacturing process of problems: BMP-2 very complicated, output is very low; And metabolism is very fast, and induction time is shorter; And, as foreign protein, when widely applying, can produce deleterious effect to body.Some investigators attempt utilizing support to carry slowly-releasing BMP-2, but BMP-2 is to the material height sensitivity, when being placed on support, and its active can reduction.Institute thinks and addresses the above problem, adopts the BMP-2 gene imported to slow virus, and transfection SMSCs, thereby make BMP-2 albumen generate with SMSCs propagation.What is interesting is, In the view of experimental result, BMP-2 induces the key factor of SMSCs to cartilage differentiation.

Than other virus vector, lentiviral vectors not only can the transfection division cells, can also the non-division cells of transfection.And the expression of goal gene is stable, long-acting.

EGFP, as reporter gene, can directly be observed.It absorbs visible ray at the 470nm place, and sends green fluorescence at the 510nm place.As long as express enough strongly, it can be directly in somatoscopy.Be different from other fluorescent markers, for example LacZ, Luc, β-galactosidase, in its application without adding again other factors.

So, in experiment of the present invention, used the pFUGW that comprises the EGFP gene to carry out transfection SMSCs, become simple and clear thereby make to observe.After transfection, immunofluorescence and Western blot result have all confirmed that BMP-2 albumen generates.SMSCs after transfected, its security is particularly important, because it has determined whether can be used as seed cell.Relevant Growth chart clear-cells quantity tended towards stability at the 7th day, showed that its differentiation capability is non-unconfined.Flow cytometer is used to measure DNA content, and results suggest is without the pseudodiploid cell, and transfected SMSCs is normal diploid cell.In addition, karyotyping shows in SMSCs containing 44 karyomit(e)s.The tumorigenicity test shows that it is without tumorigenicity.These results have all confirmed that SMSCs is safe, can be used as seed cell.

Safe SMSCs is placed on incomplete one-tenth Cartilage culture base afterwards.Find that its cellular form transfers the Polygons cell to from spindle cell.1 Collagen Type VI, 2 Collagen Type VIs and aggrecan mrna expression can be detected by RT PCR.The indirect immunofluorescence result also shows to have 1 Collagen Type VI and 2 Collagen Type VIs to generate.

In normal fetal rickets case, visible MicroRNA-130a expression deletion.It expresses gradually and reduces in the cartilage forming process.Its target gene Runx3 also expression deletion in the fetal rickets case.Micro-145, as the direct attemperator of Sox9 in the natural joint cartilage, it has lowered the expression of Sox9 by the particular combination site in conjunction with 3'-UTR.In the human cartilage phenotype, Micro-145 is extremely important.When its level raises, can reduce specificity cartilage cell epimatrix gene (COL2A1 and aggrecan) and the little RNA(miR-675and miR-140 of organizing specific) expression, simultaneously the raise expression of RUNX2 and MMP13, both are common in the osteoarthritis case afterwards.

All above-mentioned results all show that BMP-2 can induce SMSCs to cartilage differentiation.The invention provides a kind of new seed cell for meniscal repairs.Through the SMSCs of plentiV6-oBMP-2 transfection safe enough, for normal diploid cell, without tumorigenicity, and can certainly be sent to cartilage differentiation in vitro.

The accompanying drawing explanation:

Fig. 1 is the morphology photo of (100 *) SMSCs under first adherent latter 1 day light microscopic of transfection, and light microscopic is shown as spindle cell.

Fig. 2 is the morphology photo of (100 *) SMSCs under first adherent latter 14 days light microscopics of transfection, and light microscopic is shown as spindle cell.

Fig. 3 is the morphology photo of first adherent latter 14 days Electronic Speculum SMSCs of transfection, and Electronic Speculum shows that nuclear-cytoplasmic ratio is large.

Fig. 4 is immunofluorescent test.A, B, C, D are followed successively by as the generation of green fluorescence display waveform albumen; Green fluorescence shows that α-SMA generates; Red fluorescence shows Oct4 and expresses; Red fluorescence shows Sox9 and expresses.

Fig. 5 is the flow cytometer result.A, B, C, D, E are followed successively by CD44 (98.25%), CD90 (98.75%), CD29 (99.14%), CD34 (almost without), CD45 (almost without).

Fig. 6 is the Multidirectional Differentiation experimental result.A, B, C are followed successively by osteogenic induction, become fat to induce, become chondrocyte induction.Visible AKP dyeing (+), oil red dyeing (+), alkaline Toluidine blue staining show extracellular matrix GAG and generate.

Fig. 7 shows the figure after slow-virus transfection; Wherein A is that the FCM results show transfection efficiency surpasses 80%; B is corresponding fluorogram (100 *).

Fig. 8 shows the photo of the rear cellular form (200 *) of transfection cultivation.A is not exclusively becoming on the Cartilage culture base, and cell becomes the Polygons cell by spindle cell; B is that in the experiment contrast group, cellular form is almost unchanged.

Fig. 9 shows the RT-PCR result.Experimental group is expressed 1 Collagen Type VI, 2 Collagen Type VIs etc., and 2 Collagen Type VIs are more; A small amount of 1 Collagen Type VI and 2 Collagen Type VIs etc. of expressing of experiment contrast group; The blank group is not expressed.

Figure 10 shows the photo of immunofluorescence dyeing and Toluidine blue staining experiment (200 *).Red fluorescence shows 1 Collagen Type VI and generates, and green fluorescence shows 2 Collagen Type VIs and generates, and experimental group is by Toluidine blue staining, experiment contrast group and the equal dye-free of blank group.

Figure 11 shows that MicroRNA analyzes.The expression that A is Mi-130a in experimental group, experiment contrast group, complete control group, baseline group, the expression that B is Mi-145 in experimental group, experiment contrast group, complete control group, baseline group.Embodiment:

Embodiment 1:SMSCs obtains, separates and identifies

1.1 obtain SMSCs

Prepared 63 monthly ages new zealand white rabbit (the SPF level, the Slack Experimental Animal Center, the Chinese Academy of Sciences, http:// www.slaccas.com) (mean body weight 2.1 ± 0.3kg, male and female are not limit).After fixing anesthesia, get the other straight cut of kneecap, successively open to joint capsule, with organizing clip suprapatellar bursa synovial tissue.The synovial membrane of obtaining is shredded, under 37 ℃ with 2 Collagenase Types (0.2%, Sigma-Aldrich company, http:// www.sigmaaldrich.com) digest 3 hours, through the 200-lm filter screen (Jia Bao company, http:// www.caigou.com.cn/c20931) obtain tissue block after filtration, and be incubated at the company containing 20%FBS(Hyclone http:// www.hyclone.com) DMEM(GibcolBRL company, http:// www.gelcompany.com/products/gibco-brl) in.Above-mentioned all processes all super clean bench (SW-CJ-1F, Jia Bao company, http:// www.caigou.com.cn/c20931) in complete.Be stored in subsequently 5%CO ₂in incubator (CCL-170A-8, ESCO company, http:// escoglobal.com/home.php) in hatch 14 hours.After again shredding subsequently, filtering, be stored in the 60mm culture dish containing 10%PBS.Within every 24 hours, change liquid to remove not adherent cell.

1.2SMSCs separation and purifying

Comprise various kinds of cell in culture dish.Separate SMSCs based on negative partition method principle.To added in cell mass contain CD14 wear the promise magnetic bead (111-49D, Dynabeads CD14, Zhuo Kang biotechnology company, http:// www.caigou.com.cn/c42082), pass through subsequently the magnetic bead sorting instrument (KingFisher, peace promise biotechnology company, http:// ww.hbzhan.com/st31204/product_1832042.html) obtain the SMSCs of purifying.By these cells according to 100cells/cm ²density cultivate after 14 days frozen under-80 ℃ as P1 generation.

1.3 Morphological Identification

All observe the upgrowth situation of P1 for cell by inverted phase contrast microscope every day.After cell dissociation is become to suspension, choose at random 100 visuals field, measure the diameter of finding cell.

Simultaneously, also carried out electron microscopic observation.By P2 for cell 0.25%Trypsin/EDTA(YW003, should Teng biotech firm, http:// www.yitbio.com) after digestion, use 2.5% glutaraldehyde and osmic acid (chemical company of Supreme Being section, http:// www.whdico.com/index.htm) fix 90 minutes.With 70% alcoholization acetate solution uranium dyeing, be embedded in (rich big chemical company, http://cqtnbw.qjy168.com) in epoxy resin 618 after alcohol and acetone decolouring again.Be cut into subsequently the 90nm ultrathin section(ing), then after the two dyeing of uranyl acetate and lead citrate, in transmission electron microscope (H-7650, HITACHI company, http:// www.hitachi.co.jp) lower microscopy.

Result: adherent latter 2 days, some spindle cells have been arranged; After 14 days, cell almost is spindle cell.In addition, transmission electron microscope shows that nuclear-cytoplasmic ratio is larger, and the large and tenuigenin undeveloped (Fig. 1,2,3) of kernel meets the morphological feature of stem cell.

1.4 immunofluorescence analysis

By P3 for cell seeding on slide, with 4% paraformaldehyde, fix, then immerse in 2%BSA/PBS 10 minutes.Primary antibodie comprises the Oct4(goat-anti, 1:50, and Sigma-Aldrich company, http:// www.sigmaaldrich.com), vimentin and α-SMA(all derive from mouse-anti, 1:200, Sigma-Aldrich company, http:// www.sigmaaldrich.com) and the Sox-2(mouse-anti, 1:400, Sigma-Aldrich company, http:// www.sigmaaldrich.com).Cultivate after 24 hours, add two anti-, two anti-comprise the FITC-sheep anti-mouse igg (1:100, Sigma-Aldrich company, http:// www.sigmaaldrich.com), Cy3-sheep anti-mouse igg and the anti-sheep IgG(of Cy3-donkey be 1:150, KPL company, http:// www.kpl.com).DAPI dyeing is subsequently observed.

Result: immunofluorescence analysis show P3 for cell expressing vimentin and α-SMA, this two cell surface marker that is SMSCs.In addition, Oct4 and Sox2 express also very strong (Fig. 4), especially Oct4, show that cell has multi-lineage potential.So can think that P3 substantially has been mescenchymal stem cell for cell, and multi-lineage potential is arranged.

1.5 flow cytometer detects

P3 is used to 0.25%Trypsin/EDTA digestion for cell.Choose subsequently 1 * 10 ⁵cell, add multiple fluorescent-labeled antibody, has CD45-FITC, CD29-FITC, CD34-FITC, CD90-FITC, CD44-FITC(all to derive from brilliant U.S. biotech firm, http:// www.jingmei.com) and corresponding homotype control antibodies, mix rear lucifuge room temperature standing 20 minutes.Add subsequently 2mlPBS, centrifugal 5 minutes, remove supernatant, add 300 μ lPBS, recentrifuge, upper machine testing (FACS Calibur type, Becton Dickinson company, http:// www.bd.com) the counting positive cell percentage, all data are by supporting CellQuest software analysis (Becton Dickinson).

Result: the flow cytometer result the has shown above-mentioned cell expressing molecular marked compound of SMSCs, for example CD44(98.25%), CD29(99.14%), CD90(98.75%).And molecular marked compound CD34, the CD4 of hemopoietic stem cell almost do not express (Fig. 5).The above results has confirmed that the cell of amplification is SMSCs.

1.6 Multidirectional Differentiation of Cells potential is measured

In order to become fat, cell is cultured on the one-tenth fat substratum containing 0.5 μ M dexamethasone, 0.5mM isobutyl methylxanthine, 50 μ M indomethacins.After 4 days, 0.3% oil red dyeing is observed.

For skeletonization, cell is cultured on the skeletonization substratum containing 100nM dexamethasone, 10mM beta-glycerophosphate, 50 μ M xitix.After 21 days, use NBT/BCIP method (grind brilliant company, http:// www.yjbio.com) analyze the expression of alkaline phosphatase.

In order to become cartilage, cell is cultured in BMP-2, the 10ng/ml TGF-β 3,10 containing 500ng/ml ^-7m dexamethasone, 50ug/ml2-phosphoric acid-xitix, 40ug/ml proline(Pro), 100ug/ml L-Ala, and the ITS+Premix(of 1 to 100 dilution is containing 6.25ug/ml Regular Insulin, 6.25ug/ml Transferrins,iron complexes, 6.25ng/ml selenous acid, 1.25mg/ml BSA, and 5.35mg/ml linolic acid) the DMEM substratum on.Use Toluidine blue staining after the particle embedded section.

The AKP dyeing of osteogenic induction shows that rabbit SMSCs is positive, the dyed particles of visible brown color in positive cell.Become fat to induce, have as seen the large fat that drips to be dyeed by oil red.Become chondrocyte induction visible cell form to transfer polygon to, alkaline Toluidine blue staining shows extracellular matrix GAG composition (Fig. 6).

The above results confirms that rabbit SMSCs has multi-lineage potential.

Embodiment 2 vector constructions and transfection

2.1 design of primers

The GenBank of rabbit BMP-2 is numbered NM_0010826507, and reading frame is 70-1257bp.Utilize accordingly SGD software ( http:// www.yeastgenome.org/cgi-bin/web-primer) the design primer.Design of primers is 5 ' end: 5 '-CTAGCTAGCTGGAGGCTCTTTCAATGG-3 ' (SEQ ID NO:1).The restriction enzyme site that GCTAGC is restriction enzyme Nhe1, ATG is terminator codon; 3 ' end:

5’-GGGTTTACCGGTAAAGCAAAAACAAACCAA-3’（SEQ?ID?NO:2）。The restriction enzyme site that ACCGGT is restriction enzyme Age1, AAG is terminator codon.

2.2BMP-2 amplification

Use Trizol(Gibco company, http:// zh.invitrogen.com) carry out total RNA of extracting rabbit liver, use 5IU MMLV(industry power company, http:// www. industry power bio.cn) carry out reverse transcription reaction.Under 70 ℃, reaction is 5 minutes, and reaction is 60 minutes under 37 ℃, and after the reaction circulation of 5 minutes, cDNA is synthesized under 95 ℃.Use PCR(TC9600-G-230V, Labnet company, http://www.labnet.net) oBMP-2 is expanded to 1.2kb.Reaction conditions is: 94 ℃ lower 2 minutes; 94 ℃ lower 5 seconds, 58 ℃ lower 45 seconds, 72 ℃ once circulations after lower 45 seconds, every 30 seconds; 72 ℃ lower 5 minutes.

2.3PlentiV6-oBMP2 packing

By Age1 and Nhe1(TakaRa company, http:// www.takara.com.cn) be diluted to 1unit/ μ L from 10units/ μ L.Again by the cDNA double digestion of oBMP-2, and in 70 ℃ of lower deactivations 10 minutes.After the 100V electrophoresis from the rubber tapping of the 1.2kb band on 1% agarose gel.Simultaneously, Age1 and the Nhe1 double digestion for pFUGW of EGFP reporter gene, standing 2 hours will be comprised.By double digestion, good carrier and the adhesive tape cut off react 1 hour under 16 ℃ according to the ratio of 1:3, during add T4DNA(industry power company, http:// www.yelibio.cn).Obtain the pFUGW-oBMP-2 recombinant plasmid.

Subsequently, amplification recombinant plasmid pFUGW-oBMP-2, packaging plasmid psPAX2 and pMD2.G(Didier Tro laboratory, http:// www.lentiweb.com); It is standby that purification is concentrated into 1 μ g/ μ L.First 1 day of transfection, first by 293T cell (preserve in this laboratory) cell density modulation 10 ⁷, then put into the 75ml culturing bottle, bottle includes the DMEM substratum, includes penicillin G (100U/ml), Streptomycin sulphate (100 μ g/ml), 10%FBS, polylysine.Culturing bottle is stored in to the 5%CO under 37 ℃ ₂in incubator 24 hours.Separately carefully add pFUGW-oBMP-231 μ g, psPAX29 μ g, pMD2.G10 μ g to heat up in a steamer in water to two, total volume is adjusted to 1600 μ L, then add 150 μ LCaCl ₂(2.5mol/L) He 1750 μ LBES(industry power companies, http:// www.yelibio.cn), mix under rear room temperature and place 20 minutes.Then dropwise said mixture is added in the culturing bottle containing the 293T cell.Culturing bottle is stored in to the 5%CO under 37 ℃ again ₂in incubator 16 hours. then remove supernatant, filter with 0.45 μ m filter paper.The virus liquid of gained is frozen standby under-80 ℃.

Done equally control group, removed and do not comprise oBMP-2, all the other the institute in steps the same state identical.

2.4plentiV6-oBMP-2 transfection SMSCs and transfection efficiency analysis thereof

Get P3 for SMSCs, after digestion and counting, the virus liquid mixing of choosing 5 * 104 cells and thawing.Mixture centrifugal (2500r/min), after 3 hours, is heavily planted back on 12 orifice plates.Every hole adds 1ml to contain the L-DMEM of 10%FBS, within every 2 days, changes substratum.During each replacing substratum, all use the expression of fluorescence microscope EGFP.

After mixture is cultivated 24 hours, by the fluorescence phase microscope, observe whether green glow is arranged.During each replacing substratum, all use the fluorescence phase microscope to observe green fluorescence, the fluorescence phase of getting same field of view compares mutually with natural light, thereby determines transfection efficiency.Also used the flow cytometry analysis positive rate simultaneously.

In addition, also having carried out Western blot detects.Get to cultivate and turn the then supernatant liquor of SMSCs, with the 3%PBS fixed cell, after 3 hours, add oBMP-2 antibody and FITC-goat anti-rabbit igg, after reaction hour, add 10mlNBT/BCIP, the lucifuge reaction after 15 minutes row SDS-PAGE detect.

Result: because plentiV6-oBMP-2 includes the EGFP reporter gene, so can be directly at the fluorescence microscopy Microscopic observation.SMSCs cell by successful transfection can send green fluorescence, not successful transfection without fluorescence.Real Time Observation shows that the green fluorescence that after 48 hours, EGFP expresses reaches climax, and the flow cytometer result shows that now expression rate is about 80%(Fig. 7).

Choose the supernatant liquor of cultivating SMSCs and be SDS-PAGE, turn subsequently glue and be Western blot.Experimental group has band to show at 40000 places, control group without.But SMSCs expressed BMP-2 albumen of explanation after by the plentiV6-oBMP-2 transfection.

After embodiment 3 transfections, the SMSCs security is identified

3.1 dynamics of cell proliferation analysis

SMSCs after transfection is pressed to 50cells/cm ²density plant after 24 orifice plates, put into CO ₂in incubator.Choose 3 holes every day, with after 200 μ L0.25%Trypsin/EDTA digestion, with liquid-transfering gun, repeatedly blow and beat into single cell suspension.Until it is porose to take institute, substratum is not all changed before.After all being counted, cell quantity in every day every hole gets equal value record.According to numerical value, according to equation, draw growth curve and calculate the doubling time successively.The equation used is TD=tlog2/log (N/N0) (t: from starting to cultivate the time to being counted day, unit hour; N: cell counting during time T; N0: total cellular score).

Result: counted the cell count of 7 days, then drawn accordingly cell growth curve.According to correlation formula, calculated cell doubling time, be about 30.2 ± 2.4 hours.The curve display cell quantity is the capable distribution of S, and during by the 7th day, it is stable that cell quantity almost keeps.The growth of presentation of results SMSCs is not unrestrictedly doubled.

3.2 karyotyping

At first prepared the karyomit(e) sample of SMSCs after the transfection.SMSCs is added into containing cultivating 4 hours in the nutritive medium of 0.1 μ g/ml colchicine, with 0.25%Trypsin/EDTA digestion, with liquid-transfering gun, repeatedly blows and beats into single cell suspension subsequently.With after PBS washing 2 times, centrifugal mixed solution is also got its throw out again.After using subsequently 0.075M Repone K hypotonic medium resuspended, be placed on 37 ℃ lower 30 minutes, recentrifuge (1000rpm), get its precipitation, with methyl alcohol/Glacial acetic acid (3:1) is resuspended, fixes 15 minutes.Cell suspension is dropwise dropped in the ice slide from the 20cm eminence, overdo 3 times, then dye 10 minutes with Giemsa.Finally use om observation counting karyomit(e).

Then use the flow cytometry analysis DNA content.With PBS washing SMSCs3 time, with 0.25%Trypsin/EDTA digestion, with liquid-transfering gun, repeatedly blow and beat into single cell suspension subsequently.After centrifugation, get its cell precipitation resuspended fixing with 75% alcohol, be placed on lower 24 hours of-20 ℃ of environment.Use afterwards 75% alcohol and PBS washed cell 3 times, then add 100100 μ g/ml rnases.37 ℃ after lower 30 minutes, with 40 μ g/ml propidium iodides dyeing.Simultaneously, chosen the rabbit femoral medullary cell, contrasted as homotype after the removal red corpuscle.All the other all operations are all identical.Use the flow cytometry analysis DNA content.

Result: use flow cytometer to determine DNA content.The curve of visible G2-M phase, G0-G1 phase is comparatively level and smooth, and showing does not have pseudodiploid.SMSCs is distributed as 87.41%, 0.00%, 12.59% G0-G1 phase, G2-M phase, S phase, shows that SMSCs is normal stem cell.

3.3 tumorigenicity analysis

By 2 * 10 of P3 generation ⁷sMSCs be expelled to the left side inguinal region of NOD/SCID mouse (n=3) subcutaneous (the SPF level, the Slack Experimental Animal Center, the Chinese Academy of Sciences, http:// www.slaccas.com).Observe and within 3 months, see if there is the tumour generation.

Result: observe weekly the NOD/SCID mouse be injected, amount to 3 months, do not find that any tumour generates.Show that transfected SMSCs is without tumorigenicity.The above results is common confirms that transfected SMSCs is normal diploid cell, non-infinite multiplication, and, without the tumorigenesis type, be safe, thereby can be used as seed cell for follow-up experiment.

SMSCs after embodiment 4 transfections is to the cartilage directed differentiation

4.1RT pcr analysis genetic expression

In follow-up all experiments, all be provided with following 3 groups, be respectively experimental group: through the SMSCs of plentiV6-oBMP-2 transfection, be designated as plentiV6-oBMP-2 (+); Experiment contrast group: through the plentiV6 transfection, but do not comprise oBMP-2, be designated as plentiV6-oBMP-2 (-); Blank group: without the SMSCs of transfection.

The experimental group cell is placed on to the upper cultivation of DMEM, containing 5mlFBS, 5 μ L dexamethasone (1mol/L), 50 μ LTGF-β 3(10 μ g/ml).After 14 days, observation of cell form under light microscopic.In addition, utilize the expression of mRNA etc. of the aggrecan of RT pcr analysis 1 Collagen Type VI, 2 Collagen Type VIs and extracellular matrix.

Result: SMSCs non-cultivate 14 days in becoming the Cartilage culture base fully after, can find that very large change has occurred cellular form, spindle cell has originally become similar chondrocyte's Polygons gradually, and experiment contrast group shape does not change (Fig. 8) (the blank group is without observing).

Frozen under-70 ℃ after use Trizol extracted total RNA.Oligo dT-Adaptor primer (the Bio-Rad laboratory, http:// www.biorad.com), Rase Free dH ₂the great biotech firm of O(, http://www.haoranbio.com), the dNTP mixture (Promega company, http:// www.promega.com.cn), RNase inhibitor (great biotech firm, http://www.haoranbio.com), M-MMLV reversed transcriptive enzyme (Promega company, http:// www.promega.com.cn) etc. be used to carry out reverse transcription reaction.

Design of primers is as follows:

1 Collagen Type VI (positive-sense strand: 5 '-AGGTCCACATGGCCCCGTGG-3 ' (SEQ ID NO:3)), antisense strand: 5 '-AAAGGGGCCCAGAGGTCTTCCT-3 ' (SEQ ID NO:4));

2 Collagen Type VIs (positive-sense strand: 5'-AACACTGCCAACGTCCAGAT-3'(SEQ ID NO:5)), antisense strand: 5'-AGTGGATATCGGCTA GACG-3'(SEQ ID NO:6));

Rabbit BMP-2(positive-sense strand: 5 '-TTGCTGGACACCCGGCTGAT-3 ' (SEQ ID NO:7), antisense strand: 5 '-AGACCCTTACTGGTCACCTT-3 ' (SEQ ID NO:8));

RhoA(positive-sense strand: 5 '-AAAAGGAACAGATTTAAGAAGT-3 ' (SEQ ID NO:9), antisense strand: 5 '-TTTTGGAGTTCCCCTCACAGTC-3 ' (SEQ ID NO:10));

Sox9(positive-sense strand: 5 '-TTCCGTGACGTGGACATCGGC-3 ' (SEQ ID NO:11), antisense strand: 5 '-AGCCTGACCCCCCTGGGGACCAG-3 ' (SEQ ID NO:12));

GAPDH(positive-sense strand: 5 '-AGCCACATCGCTCAGACAC-3 ' (SEQ ID NO:13), antisense strand: 5 '-GAGGCATTGCTGATGATCTTG-3 ' (SEQ ID NO:14)).

Building-up process is in FASMAC(Fasmac company, http:// www.fasmac.co.jp) in complete.

Use the rich photo bio of 10 * RNAPCRBuffer(company, http:// shiading06545.11467.com), dNTP mixture, Taq enzyme (TakaRa company, http:// www.takara.com.cn), and the above-mentioned primer of mentioning is together, carries out reverse transcription reaction in RT PCR.

Result: after cultivating 14 days, by RT PCR result, visible experimental group is obviously expressed distinctive 1 Collagen Type VI of chondrocyte, 2 Collagen Type VIs, and the RhoA of cartilage differentiation signaling molecule and Sox-9, in addition obvious expressed BMP-2 albumen.And experiment contrast group and blank group are expressed not obvious (Fig. 9).Show that the experimental group cell is to cartilage differentiation.

4.2 indirect immunofluorescence experiment and Toluidine blue staining

After transfection 14 days, get cell with after PBS washing 3 times, fix 30 minutes with 4% paraformaldehyde.Then add primary antibodie (1 Collagen Type VI and 2 Collagen Type VIs, all come from Sigma-Aldrich company, http:// www.sigmaaldrich.com) cultivate 24 hours, then add two anti-(FITC-sheep anti-mouse igg (1:100), Cy3-sheep anti-mouse iggs (1:500)) to observe.Cell only adds two to resist as a control group.

In addition, also carried out Toluidine blue staining.Cell, with after PBS washing 3 times, is fixed to 15 minutes with acetone.And then washing, and use 0.1% Toluidine blue staining.Use under alcohol and the rear light microscopic of p-Xylol gradient dehydration and observe.

Result: experimental group is expressed I, II Collagen Type VI through the SMSCs of slow-virus transfection after in incomplete Cartilage culture base, inducing 14 days, and the II Collagen Type VI is more, more is partial to fibrous cartilage, and the RT-PCR result is similar; The experiment contrast group is owing to still having part to become the cartilage factor in substratum, thereby expresses part I, II Collagen Type VI, but expression amount is obviously few than experimental group; The blank group is not expressed I, II Collagen Type VI.

And alkaline toluidine blue cytochemical staining result shows that experimental group has produced distinctive GAG in the cartilage differentiation process, also more trend towards the Polygons of cartilage like cell on cellular form, the experiment contrast group does not have GAG to express, and the blank group is also without expressing.Illustrate that the cell of inducing differentiation possesses the special secretion characteristic of chondrocyte.Do not secrete GAG(Figure 10 without the SMSCs induced).

4.3Real-Time pcr analysis MicroRNA

With the Trizol extracting after total RNA of 3 experimental group, cultivate 10 days.The reverse transcription system comprises: the total RNA of 1 μ g, dNTP mixture 2 μ l, RT10 * Buffer2 μ l, AMV reversed transcriptive enzyme 0.5 μ l and RNasin0.5 μ l(all derive from Promege company, http://www.promega.com.cn).Design of primers is as follows:

Mi130a：

5’-GTCGTATCCAGTGCGCUCUUUUACAUUCUCUAATTGCACTGGATACGACCGCATT-3’（SEQ?ID?NO:15）,

Mi145：

5 '-GTCGTATCCAGTGCGCAGUUUCCAGGAAUCCCUUAATTGCACTGGATACGACCGCA TT-3 ' (SEQ ID NO:16). respond is in Gene Amp PCR System9700(Applied Biosysytem company, http://www.appliedbiosystems.com) in, complete, U6 is as internal reference.

The reactive system of 29 μ L Real-Time PCR comprises: 25 μ L2 * Real qPCR MasterMix(comprises Modified archaeal dna polymerase, SYBR Green I, Optimized PCR buffer, 5mM MgCI ₂, the dNTP mixture that comprises dUTP.All come from Genencor company, http://www.genencor.com), the contrary primer of the positive primer of 1 μ L, 1 μ L (Mi130a:F5 '-CGGGGCAGTGCAAATGTTAAAA-3 ' (SEQ ID NO:17), Mi145:F5 '-GUCCAGUUUUCCCAGGAAUCCC-3 ' (SEQ IDNO:18), common:R5 '-AGTGCGAACTGTGGCGAT-3 ' (SEQ ID NO:19) and 2 μ L cDNA templates.Use ABIStepone Plus Real-Time PCR(ABI company, http:// www.appliedbiosystems.com) calculate Ct value (cycle threshold).

Above-mentioned experiment repeats 3 times, to obtain Ct mean value.The variance analysis of bilateral for difference.Relatively adopt the Bonferroni method between group, when P<0.05, think that significant difference is arranged.Above-mentioned statistic processes utilizes SAS8.02 software to complete.

Result: Real-Time PCR results suggest by the plentiV6-oBMP-2 transfection after, the MicroRNA relevant to cartilage differentiation in SMSCs, the content of Mi-130a and Mi-145 all significantly raise (Figure 11).For Mi-130a, using the SMSCs of blank group as baseline, experimental group is plentiV6-oBMP-2 (+), and negative control group is plentiV6-oBMP-2 (-).Control group is taken from the normal rabbit meniscal tissue fully.From figure as a result, experimental group is baseline 7.8 times, the experiment contrast group is 1.2 times, control group is 15.5 times fully.Statistical analysis shows that the F value is for 5822.03(P<0.001), show between each group to have difference, further Bonferroni decomposes demonstration, and experimental group and baseline group difference are less than 0.05, and experiment contrast group and baseline group difference are greater than 0.05.

For Mi-145, experimental group is 4 times of baseline group, and the experiment contrast group is 1.2 times, and control group is 4.7 times fully.The F value is 135.52(P<0.001), show to there are differences between group, further Bonferroni decomposes demonstration, and experimental group and baseline group difference are less than 0.05, and experiment contrast group and baseline group difference are greater than 0.05.

Above-mentioned two results demonstrations, after the plentiV6-oBMP-2 transfection, the MicroRNA content in SMSCs approaches the normal meniscus tissue, and untransfected BMP-2 group and baseline group be indifference nearly all.Prompting BMP-2 may play key effect in the process to cartilage differentiation.

Claims (5)

1. the gene induced Synovial Mesenchymal Stem Cells of external lentivirus mediated BMP-2, to the method for Chondrocyte Differentiation, comprises a step that Synovial Mesenchymal Stem Cells is separated, cultivated, it is characterized in that: the step that also comprises a construction recombination plasmid pFUGW-oBMP-2, the step of the virus liquid of a preparation transfection slow virus, one by the step of transfection slow-virus transfection Synovial Mesenchymal Stem Cells, in the step of described construction recombination plasmid pFUGW-oBMP-2, at first according to BMP-2 gene design primer, by the reverse transcription reaction BMP-2 gene that increases, pFUGW plasmid and the BMP-2 gene recombination of EGFP reporter gene will be comprised, construction recombination plasmid pFUGW-oBMP-2, in the step of the virus liquid of described preparation transfection slow virus, utilize described recombinant plasmid pFUGW-oBMP-2 to add packaging plasmid, common structure transfection slow virus, and then add damping fluid to cultivate, hatch, remove supernatant, obtain the virus liquid of transfection slow virus after filtration, described by the step of transfection slow-virus transfection Synovial Mesenchymal Stem Cells, the virus liquid of the transfection slow virus of getting the above Synovial Mesenchymal Stem Cells of the third generation and thawing mixes, mixture is centrifugal, cultivate 40-52 hour, then above-mentioned culture is added in incomplete one-tenth chondrocyte induction nutrient solution inducing culture liquid and induce, induce more than 14 days and can obtain the chondrocyte.

2. the gene induced Synovial Mesenchymal Stem Cells of a kind of external lentivirus mediated BMP-2 as claimed in claim 1 is to the method for Chondrocyte Differentiation, it is characterized in that: in the step of the virus liquid of described preparation transfection slow virus, at first increase recombinant plasmid pFUGW-oBMP-2, packaging plasmid psPAX2 and pMD2.G, purify concentrated, secondly the cell density of 293T cell is adjusted to 10 ⁷, putting into culturing bottle cultivates, described culturing bottle contains the DMEM substratum, also include penicillin G in described substratum, Streptomycin sulphate, calf serum, polylysine, the concentration of described penicillin G in the DMEM substratum is 100U/ml, the concentration of described Streptomycin sulphate in the DMEM substratum is 100 μ g/ml, the mass percent concentration of described calf serum in the DMEM substratum is 10%, concentration in described polylysine DMEM substratum is 5%, the 293T cell is added and contains pFUGW-oBMP-2 after culturing bottle is hatched, psPAX2, the damping fluid of pMD2.G, hatch, remove supernatant, obtain the virus liquid of transfection slow virus after filtration.

3. the gene induced Synovial Mesenchymal Stem Cells of a kind of external lentivirus mediated BMP-2 as claimed in claim 1 is to the method for Chondrocyte Differentiation, it is characterized in that: in the step of described construction recombination plasmid pFUGW-oBMP-2, by Age1 and Nhe1 double digestion for the cDNA of oBMP-2, simultaneously, to comprise Age1 and the Nhe1 double digestion for pFUGW of EGFP reporter gene, then add T4 DNA reaction to obtain the pFUGW-oBMP-2 recombinant plasmid.

4. the gene induced Synovial Mesenchymal Stem Cells of a kind of external lentivirus mediated BMP-2 as claimed in claim 1 is to the method for Chondrocyte Differentiation, it is characterized in that: in the process of amplification BMP-2 gene, the primer sequence of described 5 ' end is as SEQ ID NO: as shown in the of 1, the primer sequence of described 3 ' end is as SEQ ID NO: as shown in the of 2.

5. the gene induced Synovial Mesenchymal Stem Cells of a kind of external lentivirus mediated BMP-2 as claimed in claim 1, to the method for Chondrocyte Differentiation, is characterized in that: described incomplete one-tenth chondrocyte induction nutrient solution nutrient solution is one and contains TGF-β ₃, dexamethasone, 2-phosphoric acid-xitix, proline(Pro), L-Ala, 1:100 dilution the H-DMEM nutrient solution of ITS+Premix solution, wherein, in described H-DMEM nutrient solution, TGF-β ₃concentration be 10 ng/mL, dexamethasone concentration is 1 * 10 ^-7mol/L, the concentration of 2-phosphoric acid-xitix is 50 μ g/mL, the concentration of proline(Pro) is 40 μ g/mL, the concentration of L-Ala is 100 μ g/mL, contain Regular Insulin in the ITS+Premix solution of described 1:100 dilution, Transferrins,iron complexes, selenous acid, bovine serum albumin, linolic acid, in the ITS+Premix solution of described 1:100 dilution, the concentration of Regular Insulin is 6.25 μ g/mL, the concentration of Transferrins,iron complexes is 6.25 μ g/mL, the concentration of selenous acid is 6.25 ng/mL, the concentration of bovine serum albumin is 1.25 mg/mL, linoleic concentration is 5.35 mg/mL.

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