重组pcDNA3.1-hBMP-2转染人骨髓基质干细胞和对其增殖及血管内皮生长因子表达的影响

目的构建人骨形成蛋白-2(hBMP-2)真核表达载体pcDNA3.1-hBMP-2,转染人骨髓基质干细胞(MSCs),探讨基因转染对其增殖和血管内皮生长因子(VEGF)表达的影响. 方法利用重组DNA和基因克隆技术构建重组载体pcDNA3.1-hBMP-2;细胞培养和基因转染技术体外转染人MSCs;免疫细胞化学、原位杂交和蛋白印迹法检测细胞BMP-2的表达;通过流式细胞仪和VEGF探针原位杂交分析其对细胞增殖和VEGF表达的影响. 结果转染后细胞在mRNA水平和蛋白质水平均表达BMP-2;转染后S期细胞比例增多,提示细胞DNA的合成增加;BMP-2基因转染上调细胞VEGF的表达. 结论在脂质体介导下,pcDNA3.1-hBMP-2转染MSCs获得成功.基因转染后能促进细胞增殖并将通过使VEGF的表达增加促进血管再生,为进一步骨缺损的基因治疗及构建组织工程骨奠定了实验基础.

EFFECTS OF RECOMBINED pcDNA3.1-hBMP-2 TRANSFECTION ON HUMAN MARROW STROMAL STEM CELLS PROLIFERATION AND VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION

Objective To construct the recombined DNA pcDNA3.1-hBMP-2 and transfect into human marrow stromal stem cells (MSCs) in vitro, and to explore the effects of transfection on cellular proliferation and expression of vascular endothelial growth factor (VEGF). Methods The expression of human bone morphogenetic protein 2(hBMP-2) in these cells after transfection was determined by in situ hybridization and immunohistochemical analysis and Western blot analysis. The changes of cell proliferation were observed by flow cytometry. The effects of BMP-2 gene transfection on expression of VEGF in the cells were analyzed by in situ hybridization of VEGF cDNA probe. Results Stable expression of hBMP-2 in pcDNA3.1-hBMP-2 transfected MSCs was confirmed in the levels of mRNA and protein. Cellular proportion in S period increased, which indicated that the synthesis of cell DNA increased. The expression of VEGF in the cells increased obviously. Conclusion With the help of lipofectamine, the pcDNA3.1-hBMP-2 were transfected into human MSCs successfully. hBMP-2 plays an important role in promoting cellular proliferation and vascular generation during bone repair.