慢病毒介导骨形态发生蛋白2基因转染大鼠骨髓内皮祖细胞

背景：基因治疗已成为疾病治疗的新趋势,为某些难治性疾病带来了曙光,合适的细胞、基因及载体的选择是治疗的关键。目的：探讨慢病毒介导骨形态发生蛋白2基因转染大鼠骨髓内皮祖细胞应用于基因治疗的有效性和可行性。方法：从SD大鼠骨髓分离、培养、鉴定内皮祖细胞,采用构建的慢病毒载体（LV）将空载体（LV-eGFP）或骨形态发生蛋白2基因（LV-eGFP-BMP2）转染至内皮祖细胞,通过eGFP荧光检测转染效率。ELISA法检测上清液中骨形态发生蛋白2蛋白的分泌,Western blot法检测细胞骨形态发生蛋白2蛋白的表达,比较转染后细胞的迁移、增殖和抗凋亡能力。结果与结论：慢病毒介导骨形态发生蛋白2载体的转染效率可达90%。与正常内皮祖细胞、转染空载体内皮祖细胞比较,转染骨形态发生蛋白2基因的内皮祖细胞分泌和表达骨形态发生蛋白2蛋白增加（P〈0.01）,迁移和增殖能力无明显改变（P〉0.05）,肿瘤坏死因子α损伤后的细胞凋亡率明显降低（P〈0.05）。结果表明慢病毒介导骨形态发生蛋白2载体可成功转染内皮祖细胞,转染后可增加内皮祖细胞分泌和表达骨形态发生蛋白2蛋白,增强对炎性损伤的抗凋亡能力,对迁移能力和增殖活性无明显影响。

Lentiviral vector-mediated transfection of bone morphogenetic protein 2 gene into endothelial progenitor cells from rat bone marrow

BACKGROUND： Gene therapy has become a new trend for disease therapy and brought promise for some refractory diseases. The key point is to choose the proper cell, gene and vector. OBJECTIVE： To investigate the effectiveness and feasibility of bone morphogenetic protein 2（BMP2） gene transfected into endothelial progenitor cells（EPCs） from rat bone marrow for gene therapy. METHODS： The EPCs were isolated, cultured and identified from the bone marrow of Sprague-Dawley rats. Empty vector（LV-eGFP） or BMP2 gene（LV-eGFP-BMP2） was transferred into EPCs by the constructed lentiviral vector（LV）. We examined the transfection efficiency by eGFP fluorescence, BMP2 secretion by ELISA, BMP2 expression by Western blot, and compared the capacities of migration, proliferation and anti-apoptosis after transfection in the three groups of normal EPCs, empty vector-EPCs, and BMP2-EPCs. RESULTS AND CONCLUSION： The transfection efficiency of lentiviral vector was 90%. BMP2 gene-transferred EPCs secreted and expressed more BMP2 proteins（P 〈 0.01）, and showed enhanced anti-apoptotic ability（P 〈 0.05）. The proliferation and migration capacity did not change obviously（P 〉 0.05）. After successful transfection with lentivirus-BMP2 gene, EPCs can secrete and express more BMP2 protein and show enhanced anti-apoptotic ability without obvious influence on the proliferation and migration capacity.