| Abstract [objective] The target gene TGF-P3 (transforming growth factorβ-3)and TIMP-1 (tissue inhibitor of metalloproteinase -1) transfected into the rabbit's mesenchymal stem cells cells through the adeno-associated virus, and induce the transformation into cartilage phenotype in vitro. And then MSCs, which successfully transfected and embedded in alginate hydrogel, were implanted into rabbit's articular cartilage defects to repair the acute defect of articular cartilage.[Method] Bone marrow fluid, collected from the rabbit tibia and femur under sterile conditions, were applied for the primary culture and subculture。The third generation MSCs in logarithmic growth time were selected and implanted on the six pole's platelet at the density of 2.5x105/ml, and then were transfected by AAV with target genes. After transfection, total protein was extracted from cells at 3,6,9,12day, and TGF-β3 was detected through ELISA (enzyme-linked immunosorbent assay). After two week's induction, mRNA were extracted by TRIZOL, expressions of typeⅡcollagen were determined by RT-PCR (Reverse transcription Polymerase chain reaction), WESTERN BLOT was used to detect collagenⅡ, The expression of proteoglycan was shown by toluidine blue stain. After MSCs were successfully transformed into cartilage phenotype through endogenous induction in vitro, MSCs, which successfully transfected,were embedded by alginate hydrogel and were implanted into rabbit's defected articular cartilage.Indication of defected cartilage repair were generally observated and assessed by HE stain till the rabbits were sacrificed two months later.[Result] BMSCs could stably express TGF-β3 and TIMP-1 after recombinant adeno-associated virus transfection. Channels of group II collagen genes could be expressed by RT-PCR from the first week after transfection. We could see that aizen was obvious within the cytoplasm and nucleus from the result of armor alcine blue stain at the first week, which was more obvious at the second week. Small amounts of groupⅡcollagen expression could be detected by western-blot at the first week, but expression of that was more obvious at the second week. All these results were consistent from both gene and protein levels. Amounts of every index- in joint transfection group were better than experimental group. White chondroid substances could be produced in rabbit articular cartilage defects of all experimental groups. And cartilage of joint transfection group was closer to transparent cartilage. There were statistically differences in grades of joint transfection group and TGF-β3 group. It suggested from the results of HE stain that joint transfection group was better than TGF-β3 group.[Conclusion] TGF-β3 can induce stem cells to cartilage phenotypic differentiation by recombinant adeno-associated virus transinfection of BMSCs. The better differentiation of joint transfection group can suggest that TIMP-1 and TGF-β3 have synergistic effects. BMSCs compounded with Alginate gel transfected by TIMP-1 and TGF-β3 have better effects in rabbit cartilage defects repair in animal experiment. With the improvement of transgenic carrier and explanation of cytokines biological effects, effective and reasonable transgenic strengthening tissue engineering to cure cartilage defects will be a better treatment in future work.
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