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目的利用构建的人骨形成蛋白-2(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4周采用原位杂交、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 after transfection into human bone marrow stromal cells and its effect on osteogenic differentiation. Methods The constructed eukaryotic expression vector pcDNA3 / BMP2 was digested by restriction endonuclease. The constructed vector was introduced into bone marrow stromal cells by liposome-mediated transfection technique and cultured in vitro monolayer. The expression of BMP2 gene and the osteogenic differentiation of bone marrow stromal cells were detected by in situ hybridization, immunohistochemistry, alkaline phosphatase and calcium staining 48 h and 4 weeks after transfection. Results The size of pcDNA3 / BMP2 fragment was consistent with the theory. Transfection cells can detect BMP2 gene and BMP2 protein expression, and promote osteogenesis. Conclusion The transient and long-term expression of pcDNA3 / BMP2 transfected bone marrow stromal cells can be obtained and the osteogenic differentiation ability of bone marrow stromal cells can be enhanced.