| Objective: To find a way of using TGF-β3 in clinical treatment of injury of cartilage by foundation and application research on chondrogenesis induced by TGF-β3, and it would be a beginning of using biological cytokines to repair cartilage in all kinds of orthopedic diseases. Method: Firstly, primary marrow cells were cultured with low density. When there were monoclones of marrow cells, the cells were picked out from one monoclone by micromanipulative technique. Then these cells from a monoclone were co-culture with the mouse embryonic fibroblasts which were cultured as feeder cells. When the cells coming from a monoclone generated to passage 20, these MSCs were identified by means of cell surface markers such as STRO-1,CD34,CD45,OCT-4,CD105 and Nestin. Then the identified MSCs were induced to differentiate to osteoblasts, chondrocyts, tendon cells and neurons. Secondly, the phosphorylation of Erk1/2 was checked by western blot during chondrogenesis induced by TGF-β3. Then U206 which was the specific inhibitor of phosphorylation of Erk1/2 was used during the chondrogenesis, and the expression of sox9, COLⅡand Agc was detected by RealTime-PCR. The expression vector of Smad4 with point mutation at Thr277 was constructed by overlap PCR technique, and the Smad4 with mutation was transfected into MSCs during the chondrogenesis induced by TGF-β3. Then the expression of sox9, COLⅡand Agc were detected by RealTime-PCR as same as above. At the same time the accumulation of proteoglycan was confirmed by TB staining respectly. Thirdly, the total length cDNA of rattus TGF-β3 was obtained by RT-PCR from rattus embryo(embryonic bud), then the cDNA was cloned into eukaryotic vector pIRES-EGFP for constucting the expression vector pIRES2–EGFP-TGF-β3. Then pIRES2– EGFP- TGF-β3 was transfected into MSCs, and the effect of transfection on chondrogensis was analyzed by measured the expression of COLⅡ, COLХ, Agc and the accumulation of proteoglycan. In vivo experiment, these genetically modified MSCs were co-cultrued with decalcifies bone matrixs for 7days, then the mixtures were transplanted to huge cartilage defect in animal model. The healing of cartilage defect was checked by macroscopic observations and histomorphological observations. Fourthly, engineering TGF-β3 was obtained by genetic engineering technique. The recombinant eukaryotic plasmid pIRES-EGFP-LAP-MMP-mTGF-β3 was constructed by inserting gene region of matrix metalloproteinase cutting site between latent associated protein(LAP) and mature TGF-β3(mTGF-β3). Then the pIRES-EGFP-LAP-MMP-mTGF-β3 was transfected into MSCs and chondrogenic progenitor cells(CPCs), and the expression of COLⅡ, Agc and tissue inhibitor of metalloproteinase(TIMP) was checked by RT-PCR when the both genetically modified cells above culture in medium with or without MMP. At last, the antisense oligodeoxyribonucleotide of GADD45-βwas synthetized, and it was used at terminal of chondrogenesis induces by TGF-β3. The expression of MMP-13, COLХand VEGF were checked by RealTime-PCR, and the accumulation of proteoglycan was evaluated by TB staining. Result: Firstly, We got plenty monocloned MSCs successfully by amplifying cells which came from one monclone in primary culture of marrow cells, and the monocloned MSCs could be induced differentiating to osteocytes, chondrocytes, tendon cells and neurons successfully. Secondly, western blot showed that phosphorylation of Erk1/2 increased during the chondrogensis induced by TGF-β3, and U206 could weaken the chondrogenesis. Furthermore, the mutation of Smad4 on Thr277 could take the same effect on chondrogenesis as U206 and decrease the expression of Sox9, COLⅡ, Agc and the accumulation of proteoglycan during the chondrogenesis induced by TGF-β3. Thirdly, the embryonic plasmid pIRES2–EGFP-TGF-β3 was constructed successfully, and plenty TGF-β3 was detected in the medium after MSCs were transfected by pIRES2– EGFP- TGF-β3. The genetically modified MSCs differentiated to chondrocyts and turned to chondral pellets by pellet culture successfully in vitro. In vivo, the genetically modified MSCs with DBM as carrier could repair huge cartilage defect more efficiently than nature MSCs with DBM as carrier or DBM without MSCs. Fourthly, eukaryotic expression vector of engineering TGF-β3 was constructed successfully, and the engineering protein was gotten by transfecting pIRES-EGFP-LAP-MMP-mTGF-β3 into CHO cells, and the expression of engineering TGF-β3 also had been detected after CPCs and MSCs was transfected with pIRES-EGFP-LAP-MMP-mTGF-β3. During the pellet culturing of CPCs and MSCs which were transfected with pIRES-EGFP-LAP-MMP-mTGF-β3, CPCs and MSCs could differentiate to chondrocytes only in the medium with MMP. At last, the antisense oligodeoxyribonucleotide of GADD45-βcould inhibit the expression of GADD45-βand blocked the terminal differentiation of chondrogenesis induced by TGF-β3. Intervention of antisense oligodeoxyribonucleotide of GADD45-βcould decrease expression of MMP-13, VEGF and COLХefficiently which were the representative cytokines of terminal differentiation in chondrogenesis. Conclusion: Monocloned MSCs came from one marrow cell could transdifferentiate to the mature cells of endoderm and ectoderm. Phosphorylation of Erk1/2 on Thr277 was the key point of MAPK signal pathway which was the other important signal pathway except Smad signal pathway in chondrogenesis, and Smad4 is the junction point of Smad pathway and MAPK pathway. Genetically modified MSCs with TGF-β3 could be used as seed cells in cartilaginous tissue engineering more efficiently. And the engineering TGF-β3 protein could repair the injury of cartilage in all kinds of orthopedic diseases more specifically. The GADD45-βwas the key point of terminal differentiation of chondrogenesis, and the inhibitor of GADD45-βcould block the terminal differentiation more efficiently. |
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