【摘要】 目的构建骨形态发生蛋白-2(BMP-2)真核表达质粒,使其在人骨髓基质干细胞(hBMSCs)中表达,并观察表达产物的诱导成骨活性。方法在脂质体介导下将BMP-2基因导入hBMSCs,流式细胞仪、ALP检测和VEGF探针原位杂交分析其对细胞增殖、ALP活性和VEGF表达的影响。并利用转染后的培养液上清诱导小鼠成纤维细胞(L929),检测其骨钙素、Ⅱ型胶原表达。结果转染后细胞稳定表达BMP-2基因,S期细胞比例增多,ALP活性和VEGF的表达明显增加。且诱导L929细胞骨钙素、Ⅱ型胶原阳性表达。结论BMP-2重组质粒转染hBMSCs后表达目的蛋白,促进自身增殖、分化和上调VEGF的表达,并诱导成纤维细胞向成骨细胞转化,为BMP-2基因治疗奠定了实验基础。更多还原

【Abstract】 Objective To construct an eukaryotic expression plasmid carr ying hBMP-2 gene and have it expressed in hBMSC and to study induced activities of the expressed protein. Methods With the help of lipofectamine, BMP-2 gene w as transduced into hBMSC, and the changes of proliferation in these cells were o bserved with a flow cytometry. In situ hybridization by VEGF cDNA probe and ALP analysis were applied to make sure the effects of BMP-2 gene transfection on th e expressions of VEGF and ALP in these cells. The supplement secreted by transfe cted hBMSC was used to induce L929 cells and the expression of osteocalcin and t ype Ⅱ collagen were detected. Results Stable expression of hBMP-2 in PcDNA3.1-hBMP-2 transfected hBMSC was confirmed. Cellular proportion in S period and t he expression of VEGF and ALP obviously increased in these cells. After inductio n, positive staining of osteocalcin and type Ⅱ collagen was oserved. Conclusio n BMP-2 expressed by PcDNA3.1-hBMP-2 transfected hBMSC can promote cellular p roliferation, differentiation and up-grade VEGF expression and induce fibroblas ts to osteoblasts.更多还原