TLR4基因小干扰RNA对缺氧复氧诱导肾小管上皮细胞凋亡的影响

Inhibitory effects of special siRNA targeting TLR4 gene on the apoptosis of renal proximal tubule epithelial cells induced by hypoxia-reoxygenation

目的研究Toll样受体4(TLR4)基因小干扰RNA(siRNA)对体外缺氧复氧条件下诱导肾小管上皮细胞分泌TNF-α的抑制作用。方法 Wistar大鼠原代肾小管上皮细胞(PTECs)置于六孔培养板培养,随机分为A组(正常培养组)、B组(缺氧复氧+TLR4-siRNA转染组)、C组(缺氧复氧组)、D组(缺氧复氧+对照siRNA转染组)共4组。其中B组、C组和D组细胞缺氧3h,随后复氧24h,RT-PCR方法和Westernb lot检测TLR4 mRNA和蛋白表达情况;流式细胞仪检测细胞凋亡情况,ELISA方法检测各组上清液中TNF-α含量。结果经缺氧复氧处理后,B组、C组和D组的TLR4 mRNA、蛋白水平较A组均显著上调(P<0.01),各组上清液TNF-α含量较A组也显著升高(P<0.01):而B组TLR4 mRNA、TLR4蛋白表达量和上清液TNF-α含量较C组和D组均显著下调(P<0.01);D组与C组相比,各项检测指标表达无显著变化(P>0.05)。结论针对TLR4 mRNA的siR-NA可以有效地抑制缺氧复氧诱导的肾小管上皮细胞的凋亡和炎症反应。

Objective To study the inhibition of apoptosis of proximal tubule epithelial cells(PTECs) induced by hypoxia-reoxygenation injury by siRNA targeting TLR4 gene via the RNAi mechanisms.MethodsPTECs of Wistar rats were cultured in six-well plates and randomly divided into group A(normal group),group B(hypoxia-reoxygenation+TLR4-siRNA transfected group),group C(hypoxia-reoxygenation),group D(hypoxia-reoxygenation+con-siRNA transfected group).Group B,C,D were cultured in hypoxia condition for 3 h followed by reoxygenation for 24 h.RT-PCR and western blot were used to detect the expression of mRNA and protein of TLR4.Flow cytometry(FCM)was used to detect apoptosis of PTECs.ELISA was used to test the level of TNF-α in the supernatants.ResultsAfter hypoxia-reoxygenation,the expression of TLR4 mRNA and protein increased significantly in B,C and D group compared with A group(P<0.01),the levels of TNF-α in the three groups increased significantly compared with A group(P<0.01).The apoptosis cells in B,C and D increased significantly compared with A(P<0.05).While the expression of TLR4 mRNA,TLR4 protein,the level of TNF-α in the supernatants and the apoptosis cells in B group decreased significantly compared with C and D group(P<0.01);every measurement indicator was similar in D and C group(P>0.05).ConclusionsSpecial siRNA targeting TLR4 gene could effectively prevent the apoptosis of PTECs and inflammatory reaction induced by hypoxia-reoxygenation.