SOX-9和GDF-5共同转染骨髓间充质干细胞向类髓核细胞的分化

背景：移植间充质干细胞预防和治疗椎间盘退变是一种可行的方法，将SOX-9和GDF-5共同转染骨髓间充质干细胞，使其向髓核细胞转化，以期获得更大的髓核诱导和促增殖效应。 目的：探讨SOX-9和GDF-5基因共同诱导兔骨髓间充质干细胞向类髓核细胞分化的效果。 方法：提取、分离、纯化4周龄新西兰大白兔骨髓间充质干细胞，取第3代骨髓间充质干细胞分为5组体外诱导其向类髓核细胞分化，分别为未转染组、空载体转染组、SOX-9转染组、GDF-5转染组、共转染组。转染后第14 天采用RT-PCR检测SOX-9，GDF-5和Ⅱ型胶原的mRNA表达，免疫组化染色法检测髓核细胞标记物KRT19表达。 结果与结论：共转染组SOX-9 mRNA表达高于转染SOX-9组，差异有显著性意义(P < 0.05)；共转染组GDF-5 mRNA表达高于转染GDF-5组，差异有显著性意义(P < 0.05)。共转染组Ⅱ型胶原表达高于转染SOX-9组、转染GDF-5组，差异有显著性意义(P < 0.05)。SOX-9转染组及GDF-5转染组KRT19呈阳性表达，共转染组呈强阳性表达，可见被转染的骨髓间充质干细胞向类髓核细胞分化，且双基因转染诱导骨髓间充质干细胞向类髓核细胞分化的能力和分泌细胞外基质的能力明显高于单基因转染。

Differentiation of SOX-9 and GDF-5 co-transfected bone marrow mesenchymal stem cells into nucleus pulposus cells

BACKGROUND: Transplantation of mesenchymal stem cells to prevent and treat degeneration of the intervertebral disc is a feasible method. Mesenchymal stem cells co-transfected by SRY-related high mobility group-box gene 9 (SOX-9) and growth differentiation factor-5 (GDF-5) can differentiate into nucleus pulposus cells, in order to obtain greater effect of induction and proliferation of nucleus pulposus cells. OBJECTIVE: To investigate the effect of SOX-9 and GDF-5 co-transfection on the differentiation of rabbit bone marrow mesenchymal stem cells into nucleus pulposus cells. METHODS: We separated and cultured bone marrow mesenchymal stem cells from the bone marrow of rabbit aged 4 months. Passage 3 cells were divided into five groups and in vitro induced to differentiate into nucleus pulposus cells: non-transfected group, empty vector transfection group, SOX-9 transfection group, GDF-5 transfection group, SOX-9 and GDF-5 co-transfection group. At 14 days after transfection, RT-PCR was employed to assay SOX-9, GDF-5 and collagen type II mRNA expressions in bone marrow mesenchymal stem cells. The marker of nucleus pulposus cells-KRT19 expression was also detected by immunohistochemical staining.   RESULTS AND CONCLUSION: In the co-transfection group, the mRNA expressions of SOX-9, GDF-5, and collagen type II were significantly higher than those in the SOX-9 transfection group, GDF-5 transfection group, and both these two groups, respectively (P < 0.05). Cells were positive for KRT19 in the SOX-9 and GDF-5 groups, and strongly positive for KRT19 in the co-transfection group. These findings indicate that double gene-transfected bone marrow mesenchymal stem cells are better than single gene-transfected cells with regard to differentiation into nucleus pulposus cells and secretion of extracellular matrix.