低氧诱导因子过表达对人外周血内皮祖细胞分化影响的体外研究.doc

低氧诱导因子过表达对人外周血内皮祖细胞分化影响的体外研究 　　作者：姜萌,王长谦,王彬尧,何奔,邵琴,黄定九　　作者单位：上海交通大学医学院附属仁济医院心内科，上海 200001 　　【摘要】为了研究低氧诱导因子(hypoxia inducible factor-1alpha，HIF-1Α)在体外对人外周血内皮祖细胞(endothelial progenitor cells，EPC)向血管内皮细胞(endothelial cells，EC)分化的影响，用密度梯度离心法分离人外周血EPC，电穿孔技术转染HIF-1Α质粒至EPC，计算转染效率，RT-PCR方法测定HIF-1Α、HIF-1Β及HIF-1Α下游靶基因血管内皮生长因子(vascular endothelial growth factor，VEGF) mRNA在质粒转染前后含量变化，免疫细胞化学方法检测在常氧下HIF-1Α蛋白在HIF-1Α质粒转染前后不同时点蛋白表达情况，细胞膜表面抗原决定簇流式细胞术测定FITC-CD31+ EPCs/EC在未转染组、pEGFP空质粒或HIF-1Α质粒转染组转染3-10天后的组间差异，光镜下观察转染前后细胞形态及分化程度。 结果表明： EPC获得后经电穿孔转染，质粒转染效率约20%;RT-PCR示HIF-1Α mRNA在常氧中有表达，并在HIF-1Α过表达组含量增加(P0.05);其下游靶基因VEGF在HIF-1Α过表达组表达上调(P0.05);HIF-1Β表达在各组中无明显差异(P0.05)。免疫组织化学显示常氧下HIF-1Α蛋白无表达，转染HIF-1Α质粒12小时后HIF-1Α蛋白表达阳性，转染24小时后蛋白表达阴性。流式细胞仪检测显示电穿孔转染HIF-1Α质粒使CD31+细胞的百分比增加(P0.05)。细胞形态学观察显示：未转染及pEGFP空质粒转染组细胞在培养6天后呈集落样散在分布，培养14天后贴壁细胞部分呈梭样;穿孔转染的HIF-1Α质粒组细胞于培养6天后集落周边分化出梭样贴壁细胞，培养14天后呈梭样，或铺路石样牢固贴壁。结论： HIF-1Α质粒能有效地用于基因干扰治疗，有助于EPC向EC分化，为体内研究奠定了基础，也为进一步体内诱导血管新生、治疗缺血性心脏病提供了更广阔的治疗选择。 　　【关键词】 低氧诱导因子-1Α 　　Abstract To investigate the influence of HIF-1Α overexpression on the differentiation of endothelial progenitor cells (EPCs) ex vivo，EPCs were isolated from human peripheral blood by density gradient centrifugation，overexpressed HIF-1Α was transfected to EPCs by electroporation; HIF1Α，HIF1Β，vascular endothelial growth factor (VEGF) mRNA level were measured with RT-PCR; HIF-1Α protein was detected with immunohistochemistry in a time course. CD31+ cells were measured with flow cytometry. Cell morphology was observed after transfection. The results showed that the transfection efficiency of HIF-1Α to EPCs was about 20%. HIF-1Α and its controlled target gene VEGF were markedly induced by HIF-1Α vector (P0.05). HIF1Β had its same level as it before interference (P0.05). HIF-1Α protein was induced by HIF-1Α transfection after 12 hours but was undetectable at 24 hours. After 7-14 days cultured in 21% oxygen pressure，fluorescence-trace experiments revealed that CD31+EPCs/EC could be generated more e