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目的:利用构建的人骨形成蛋白BMP2真核表达载体pcDNA3/BMP2,检测其转染人骨髓基质细胞后的表达及对其成骨分化 的影响.方法:酶切鉴定构建的真核表达载体pcDNA3/BMP2,利用脂质体介导的转染技术,将所构建的载体导入骨髓基质细胞 中,体外单层培养.分别于转染后48h和4wk,采用原位杂交、免疫组化和碱性磷酸酶、钙化学染色方法检测BMP2的基因蛋白表 达以及对骨髓基质细胞成骨分化的影响.结果:pcDNA3/BMP2酶切片段的大小与理论相符.转染后细胞能检测到BMP2基因和 BMP2蛋白表达,并促进成骨转化.结论:pcDNA3/BMP2转染骨髓基质干细胞中可获得短暂和长期表达,并加强骨髓基质细胞的 成骨分化能力.
OBJECTIVE: To detect the expression of human bone morphogenetic protein 2 (BMP2) eukaryotic expression vector pcDNA3 / BMP2 and its effect on the osteogenic differentiation of human bone marrow stromal cells.Methods: The eukaryotic expression vector pcDNA3 / BMP2 was transfected into bone marrow stromal cells by lipofectamine mediated transfection technique and cultured monolayer in vitro.After 48h and 4wk transfection respectively, in situ hybridization, immunohistochemistry and alkaline phosphatase Enzyme and Ca2 + staining were used to detect the expression of BMP2 gene and its effect on the osteogenic differentiation of bone marrow stromal cells.Results: The size of pcDNA3 / BMP2 fragment was consistent with the theory.The expression of BMP2 and BMP2 were detected in transfected cells , And promote osteogenic transformation.Conclusion: Transient and long-term expression of pcDNA3 / BMP2 transfected bone marrow stromal stem cells can be obtained and the osteogenic differentiation ability of bone marrow stromal cells can be enhanced.