bcl-2基因对血管内皮细胞保护作用的研究

Study on the Protective Role of Endothelial Cell Transinfeeted bcl-2 Gene

目的探讨转染bcl-2基因对血管内皮细胞的保护作用。方法1.重组真核细胞表达载体:将含编码人bcl-2 cDNA完整开放阅读框的bcl-2 cDNA重组至逆转录病毒载体pLXSN的EcoR Ⅰ位点上,用脂质体包裹重组质粒导入PA317细胞株,并经G418筛选阳性克隆;收集含重组病毒的上清液。2.用含高滴度重组病毒的上清液感染内皮细胞;用PCR法检测转染细胞bcl-2基因和Neo基因,用免疫组织化学方法检测转染细胞bcl-2蛋白表达以证实转染成功。设pLXSN／s-bcl-2、pLXSN、未转染三组。3.观察各组血管内皮细胞在正常培养条件下的生长特征情况,以及在无血清、10％CO2以及10-7M的AngⅡ等不同刺激因素下各组培养血管内皮细胞的生存能力的变化。结果1.成功的重组含bcl-2的真核细胞表达质粒,并经碱基自动测序检测未发现重组的bcl-2基因有碱基丢失或错义配对;2.经PCR检测bcl-2片段和Neo基因片段以及免疫组织化学方法检测转染细胞的bcl-2蛋白表达证实转染成功并获得表达。3.在正常培养条件下各组血管内皮细胞间的形态和生存率无显著差异(p>0.05);4.转染bcl-2基因组血管内皮细胞在无血清、10％CO2以及10-7MAngⅡ等刺激因素下的存活率明显高于转染pLXSN组和未转染组(P<0.05)。结论转染bcl-2基因能显著提高血管内皮细胞的抗损伤能力。

Purpose To determine whether the endothelial cell transinfected bcl-2 gene can increase survival ability than those unin-fected cells.Methods 1.Recombinant eukaryotic expression vectors:pLXSN/s-bcl-2 was constructed by cloning human bcl-2 cD-NA containing the full-length open reading frame,and bcl-2 cDNA was orientations into the EcoR I site of retrovirus vector,pLX-SN.PA317 cell lines were transinfected with recombinant expression vectors (pLXSN/s-bcl-2) by using lipsome technique,pLX-SN/s-bcl-2 and pLXSN/Neo,were selected for resistance to G418.And supernant was collected.2.Endothelial cells were infected with supernant containing high titer recombinant viruses produce by pLXSN/s-bcl-2 and pLXSN/Neo,respectively.3.The growth properties and resistance to some apoptotic stimuli were studied in endothelial cells transinfected by human bcl-2 gene.Results Recombinant mammalian cell expression vector by cloning human bcl-2 cDNA in MCS of pLXSN.Cells transinfected by bcl-2 gene can significantly increase their survival with serum-starved,10% CO2 and Ang Ⅱfactors,respectively.Conclusion Transin-fecting bcl-2 gene into endothelial cells can significantly enhance their survival to serum-starved,acidosis and Ang Ⅱ stimulation.