siRNA沉默EPAS1基因对低氧条件下大鼠PASMCs增殖的影响

摘要

目的 探讨应用siRNA技术沉默EPAS1 (HIF-2α)基因表达对低氧条件下培养的大鼠肺动脉平滑肌细胞(PASMCs)增殖的影响.方法 原代培养大鼠PASMCs共采用免疫荧光法进行鉴定.构建特异性EPAS1 siRNA脂质体,转染PASMCs,从3个干扰靶点中选取效果最好的靶点进行干扰;在常氧(37℃、5％CO2、20％O2)和低氧(37℃、5％CO2、2％O2)条件下分别培养24、48、72h,采用Western blotting检测PASMCs中EPAS1、VEGF蛋白的表达水平,CCK-8法检测细胞增殖情况.结果 成功分离培养原代大鼠PASMCs并经免疫荧光鉴定证实.将特异性的EPAS1 siRNA脂质体转染到PASMCs,选择干扰效果最好的靶点2进行干扰.与常氧条件比较,低氧条件下PASMCs中VEGF蛋白的表达及细胞增殖水平均明显升高;沉默EPAS1后,无论低氧还是常氧条件下,PASMCs中VEGF蛋白的表达及细胞增殖水平均明显降低.结论 EPAS1基因参与了低氧条件下大鼠PASMCs增殖的调控,其调节可能是通过VEGF介导完成的.%Objective This study explored whether EPAS1 gene is involved in the proliferation of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EPAS1 gene expression was interfered by small interfering RNA (siRNA).Methods The rat primary pulmonary artery smooth muscle cells were cultured and identified by immunofluorescence.The specific lipidosomes of EPAS1 siRNA were constructed and transfected into PASMCs,and the best targets were selected from the three interfering targets.The transfected PASMCs were cultured in hypoxia (37℃,5％ CO2,2％ O2) or normoxia (37℃,5％ CO2,20％ O2) for 24h,48h and 72h,respectively.The PASMCs proliferation was detected by CCK-8 assay.The protein expression of EPAS 1 and vascular endothelial growth factor (VEGF) were determined by Western blotting to investigate the effect of silencing EPAS1 gene expression on the proliferation of PASMCs under hypoxic condition.Results The specific liposomes ofEPAS1 siRNA were successfully constructed and transfected into PASMCs,and the best interfering target were selected from the three interference targets.The proliferation of PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition.Under hypoxic or normoxic condition,PASMCs proliferation and VEGF protein expression of PASMCs were suppressed by EPAS 1 siRNA.Conclusion EPAS 1 gene might be involved in the proliferation of rat PASMCs by regulating VEGF protein level under hypoxic condition.