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

1 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【摘要】为了研究低氧诱导因子(hypoxia inducible factor-1alpha , HIF-1 α) 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-1 α质粒至 EPC , 计算转染效率, RT-PCR 方法测定 HIF-1 α、 HIF-1 β及 HIF-1 α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-1 α蛋白在 HIF-1 α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-1 α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-1 α mRNA 在常氧中有表达, 并在 HIF-1 α过表达组含量增加( ); 其下游 2 靶基因 VEGF 在 HIF-1 α过表达组表达上调( ); HIF-1 β表达在各组中无明显差异( ) 。免疫组织化学显示常氧下 HIF-1 α蛋白无表达, 转染 HIF-1 α质粒 12 小时后 HIF-1 α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-1 α质粒使 CD31+ 细胞的百分比增加( )。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-1 α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-1 α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 Overexpression of Hypoxia Inducible Factor-1alpha (HIF-1 α) Promotes the Differentiation of Endothelial Progenitor Cell Ex Vivo Abstract To investigate the influence of HIF-1 α overexpression on the differentiation of endothelial progenitor 3 cells (EPCs) ex vivo , EPCs were isolated from human peripheral blood by density gradient centrifugation , overexpressed HIF-1 α was transfected to EPCs by electroporation; HIF1 α, HIF1 β, vascular endothelial growth factor (VEGF) mRNA level were measured with RT-PCR; HIF-1 α protEin was detected with immunohistochemistry ina time course. CD31+ cells were measured with flow cytometry. Cell morphology was observed after transfection. The results showed that the transfection efficiency of HIF-1 α to EPCs was about 20%. HIF-1 α and its controlled target gene VEGF were markedly induced by HIF-1 α vector (). HIF1 β had its same level as it before interference (). HIF-1 α protEIn was induced by HIF-