骨形态发生蛋白-2基因转染骨髓基质干细胞复合壳聚糖体外构建组织工程软骨的实验研究

In vitro cartilage tissue engineering with BMP-2 transfected BMSCs and chitosan scaffold

目的观察转人骨形态发生蛋白-2(BMP-2)基因的骨髓基质干细胞(BMSCs)向软骨细胞方向分化的能力,并种植于壳聚糖体外构建组织工程软骨。方法应用慢病毒介导把人BMP-2基因和增强型绿色荧光蛋白(eGFP)基因转入大鼠BMSCs,通过荧光观察、反转录-聚合酶链反应(RT-PCR)、Westernblot、免疫组织化学等方法检测BMP-2、Ⅱ型胶原表达及软骨基质分泌情况;噻唑蓝(MTT)检测转基因后细胞的增殖活力。结果BMP-2基因和eGFP基因成功转染BMSCs,Ⅱ型胶原及蛋白聚糖的表达均显著增强,细胞种植壳聚糖支架后生长、粘附良好。结论BMP-2可在BMSCs内表达,并诱导其向软骨方向分化;与壳聚糖复合培养可构建组织工程骨。

Objective To observe cartilaginous differentiation capacity of bone marrow stromal cells （BMSCs） modified with bone morphogenetic protein-2 （BMP-2） gene, then to construct tissue engineered cartilage using the transgenic BMSCs planted into chitosan scaffold. Methods SD rat BMSCs were transferred by lentiviral vector with BMP-2 and eGFP genes. The expression of BMP-2 and the secretion of collagenlI were confirmed by fluorescence microscopy, RT-PCR, Western blotting, and immunocytochemistry. MTT was used to test the muhiplication activities of the transgenic cells. Result BMSCs were modified by BMP-2 and eGFP genes successfully. The secretion of collagen Ⅱ and proteoglycan expression were significantly enhanced as compared to the controls. The adhesion and growth of transgenic BMSCs were satisfactory on the chitosan scaffold. Conclusion Exogenous BMP-2 gene could be stably expressed in BMSCs, and may be used to construct tissue engineered cartilage when cultured with chitosan in vitro.