HIF-1αsiRNA重组腺病毒对缺氧肺微血管内皮细胞HIF-1α和ET-1表达的影响

The effect of HIF-1αsiRNA recombinant adenovirus on the expression of HIF-1αand ET-1 in hypoxia-cultured rat lung microvascular endothelial cells

目的研究缺氧诱导因子-1αsiRNA重组腺病毒(Ad/siHIF-1α)对缺氧肺微血管内皮细胞(RPMVECs)HIF-1α及其下游基因ET-1表达的影响。方法原代培养肺微血管内皮细胞(RPMVECs);应用HEK293细胞大量扩增Ad/siHIF-1α。将RPMVECs分6组:A组(正常对照:常氧下5%CO2、95%空气、37℃培养);B组(含100μmol/L COCl2培养基培养4h);C组(Ad/siHIF-1α感染24h后,加入COCl2培养4h);D组(Ad/siHIF-1α感染48h后,加入COCl2培养4h);E组(Ad/siHIF-1α感染72h后,加入COCl2继续培养4h);F组(单纯腺病毒感染72h后,加入COCl2培养4h)。采用荧光定量PCR法检测HIF-1α和ET-1mRNA表达;Western blotting法检测HIF-1α蛋白表达;ELISA法检测ET-1蛋白表达。结果与B组比较,C、D及E组HIF-1αmRNA、HIF-1α蛋白表达显著下调,差异有显著性(均P<0.05);与B组比较,C、D及E组ET-1mRNA、ET-1蛋白也相应下调,差异有显著性(均P<0.05)。结论Ad/siHIF-1α能够有效沉默缺氧诱导的大鼠肺血管内皮细胞HIF-1α基因,继而影响其下游ET-1基因的表达,为下一步在动物体内研究HIF-1α及其下游基因ET-1在新生儿肺出血发病机制中发挥作用。

Objective To study the expression of HIF -1α and ET - 1 in hypoxia -cultured rat lung mierovaseufar endothelial cells transfeeted with HIF - 1α siRNA recombinant adenovirns. Methods Primary rat pulmonary microvascular endothelial cells （PMVECs） and 293 cell were cultured in the medium containing the primary adenovirus to gain more HIF - 1α siRNA recombinant adenovirus. All RPMVECs were divided into six groups： A （control group： cultured in normal conditions, 5% CO2,95% atmosphere, 37℃）. B （CoCl2 stimulatation group： cultured in medium containing CoCl2 for 4 h）. C （infected with Ad/siHIF- 1α for 24 h, then cultured for 4 h in CoCl2 -treated culture fluid）. D （infected with Ad/siHIF- 1α for 48 h, then cultured for 4 h in CoCl2 -treated culture fluid）. E （infected with Ad/HIF- 1α siRNA for 72 h, then cultured for 4 h in CoCl2 -treated culture fluid）. F（ infected with Ad/null for 72 h, then cultured for 4 h in COCl2 - treated culture fluid）. The expression of HIF - 1α mRNA and ET - 1 gene mRNA was detected by quantitation PCR. The expression of HIF - 1α protein were detected by western Blotting. ET - 1 in cell culture supernatant was detected by ELISA. Results In compared with group B, the expression of HIF - lotmRNA and HIF - 1α protein significantly decreased in group C, D and E, with statistical difference （ P 〈 0.05 ）. In compared with group B, the expression of ET - 1 mRNA and ET - 1 protein significantly decreased in group C, D, E, with statistical difference （ P 〈 0. 05 ）. Conclusion HIF - 1α siRNA recombinant adenovirus efficiently silences the hypoxia inducible HIF - 1α and ET - 1 gene expression in RPMVECs. Ad/siHIF -1α can be used as powerful tool for investigating the effect of HIF - 1α and ET - 1 I in the pathogenesis of NPH.