Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1 |
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引用本文: | 刘斌,蔡道章,戎利民,董健文,曾春,史德海,张非.Study on chondrogenic differentiation of canine mesenchymal stem cells induced by Type 2 recombinant adeno-associated viral mediated transfer of hTGF-β1[J].中华显微外科杂志,2008,32(1):213-216,插7. |
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作者姓名: | 刘斌 蔡道章 戎利民 董健文 曾春 史德海 张非 |
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作者单位: | 中山大学附属第三医院骨科,广州,510630; |
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基金项目: | 广东省科技计划项目中山大学医科青年教师科研启动基金 |
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摘 要: | Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.
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关 键 词: | 间充质干细胞 骨髓 腺相关病毒 转化生长因子β1 软骨细胞 组织工程 Transforminggrowth factor β1 |
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