论文部分内容阅读
背景:利用重组腺病毒载体转染外源性基因到组织工程骨的种子细胞是骨缺损基因治疗研究的热点。目的:用人骨形态发生蛋白2和人成纤维细胞生长因子2双基因共表达腺病毒载体转染人骨髓基质干细胞,以探讨基因转染对人骨髓基质干细胞增殖的影响。方法:将Ad-hBMP2-IRES-hFGF2转染至人骨髓基质干细胞中,荧光显微镜观察转染效果,RT-PCR方法观察人骨形态发生蛋白2cNDA和人成纤维细胞生长因子2cNDA在人骨髓基质干细胞中的转录情况,Westerblot方法检测人骨形态发生蛋白2和人成纤维细胞生长因子2蛋白表达情况,锥虫蓝测定细胞活力,流式细胞仪分析其对细胞增殖的影响。结果与结论:转染后人骨形态发生蛋白2和人成纤维细胞生长因子2基因在mRNA水平和蛋白水平均有表达,细胞活力无明显变化,流式细胞仪分析细胞周期中增殖细胞比例明显增加。说明该双基因可高效转染人骨髓基质干细胞,且促进细胞增殖。
BACKGROUND: The use of recombinant adenovirus vector to transfect exogenous genes into seed cells of tissue engineered bone is a hotspot in gene therapy of bone defects. OBJECTIVE: To investigate the effect of gene transfection on the proliferation of human bone marrow stromal cells (BMSCs) by transfecting human bone marrow stromal stem cells with human bone morphogenetic protein-2 and human fibroblast growth factor 2 co-expression adenovirus vector. Methods: Ad-hBMP2-IRES-hFGF2 was transfected into human bone marrow stromal cells, and the transfection efficiency was observed by fluorescence microscopy. The expression of human bone morphogenetic protein 2cNDA and human fibroblast growth factor 2cNDA in human bone marrow stromal cells Westerblot method was used to detect the expression of human bone morphogenetic protein 2 and human fibroblast growth factor 2 protein. Trypan blue assayed the cell viability and the effect of cell proliferation by flow cytometry. RESULTS AND CONCLUSION: Human bone morphogenetic protein 2 and human fibroblast growth factor 2 gene were expressed at both mRNA and protein levels after transfection, with no significant changes in cell viability. The proportion of proliferating cells in the cell cycle was significantly increased by flow cytometry . This double gene can efficiently transfect human bone marrow stromal cells and promote cell proliferation.