TLR4基因小干扰RNA对缺氧复氧诱导BV-2细胞炎症因子分泌的影响

Inhibitory effects of special siRNA targeting TLR4 gene on the TNF-α expression of BV-2 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BV-2细胞分泌TNF-α的抑制作用。方法小鼠小胶质细胞株BV-2置于六孔培养板培养，随机分为N组（正常培养组）、H组（缺氧复氧组）、T组（缺氧复氧＋TLR4-siRNA转染组，转染质粒pEGFP-H1／TLR4-siRNA）、C组（缺氧复氧＋对照siRNA转染组，转染含对照序列的质粒pEGFP-H1／对照-siRNA）和B组（缺氧复氧＋空白质粒转染组，转染pEGFP-H1空质粒）共5组。其中H组、T组、C组和B组细胞缺氧培养3h后复氧培养24h，运用脂质体转染技术介导质粒转染，流式细胞术检测转染后BV-2细胞EGFP的表达率，RT-PCR方法检测各组BV-2细胞TLR4 mRNA和NF-κB p65 mRNA的表达水平，Western blot方法检测各组BV-2细胞TLR4蛋白表达变化，ELISA方法检测各组上清液中TNF-α含量。采用方差分析进行统计分析。结果转染后BV-2细胞EGFP的表达率为（67．58-＋27．16）％；经缺氧复氧处理后，H组、T组、C组和B组的TLR4 mRNA、NF-κB p65 mRNA、TLR4蛋白水平较N组均明显上调（P〈0．01），各组上清液TNF-α含量较N组也明显升高（P〈0．01）：而T组TLR4 mRNA、NF-κB p65 mRNA、TLR4蛋白表达量和上清液TNF-α含量较H组、C组和B组均明显下调（P〈0．01）；C组和B组分别与H组相比，各项检测指标表达均无明显变化（P〉0．05）。结论针对TLR4 mRNA的小干扰RNA可以有效的抑制缺氧复氧诱导的BV-2细胞的炎症反廊。

Objective To study the inhibition of tumor necrosis factor-alpha （TNF-α） cytokine expression of BV-2 ceils induced by hypoxia-reoxygenation injury by siRNA targeting TLR4 gene via the RNAi mechanisms. Method BV-2 mouse microglial cell line was cultured in six-well plates and randomly divided into group N（normal group）, group H （hypoxia-reoxygenation）, group T （ hypoxia-reoxygenation ＋ TLR4 - siRNA transfected group） ,group C （hypoxia-reoxygenation ＋ pEGFP- HI/control- siRNA transfected group） and group B （hypox- ia-reoxygenation ＋ pEGFP - H1 transfected group）. Group H, group T, group C and group B were cultured in hypoxia condition for 3 h followed by reoxygenation for 24 h. The plasma was transfected into BV-2 ceils mediated by lipofectamine 2000. The efficiency of transfection were detected by flow cytometry to observe the expression of EGFP. RT-PCR method was used to detect the level of mRNA of TLR4 or NF-κB p65. Western blot method was used to test the expression of TLR4 protein, and ELISA was used to test the level of TNF-α in the supematants. Analysis of variances was used for statistical analysis. Results The expression of EGFP gene was （67.58 ± 7.16） % after transfectiou by flow cytometry analysis. Compared to group N, the TLR4 mRNA, NF-κB 1365 mRNA, TLR4 protein level and the TNF-α quantity in group H, group T, group C and group B increased after the hypoxia-reoxygenafion treatment （ P 〈 0.01）. While the expression of the TLR4 mRNA, NF-κB 1365 mRNA, TLR4 protein level and the TNF-α quantity in the group T down-regulated compared to group H, group C and group B （ P 〈 0.01）. And there were no changes in group C, group B and group H about observation index （P 〉 0.05）. Conclusions The siRNA targeting TLR4 mRNA could inhibit the inflammatory reaction released by BV-2 cells induced by hypoxia-reoxygenation stimulation.