沉默NF-κB/p65基因抑制C反应蛋白诱导的人气道上皮细胞凋亡

Inhibit C reactive protein induced apoptosis by silencing NF-κB/p65 gene in human airway epithelial cells

目的运用RNA干扰(RNAi)技术沉默人气道上皮细胞株9HTE的核因子κB(NF-κB)/p65基因,探讨NF-κB/p65的活化在C反应蛋白(CRP)诱导的人气道上皮细胞凋亡中的作用及调控机制。方法体外培养人气道上皮细胞株9HTE,利用阳离子脂质体LipofectamineTM2000将化学合成的NF-κB/p65小干扰RNA(siRNA)转染入9HTE细胞,给予终浓度50μg/L的CRP刺激24h,免疫组织化学检测NF-κB/p65的活化,反转录聚合酶链反应(RT-PCR)检测NF-κB/p65、Bcl-2和BaxmRNA的表达,Western blot检测Bcl-2和Bax蛋白的表达,流式细胞术检测细胞凋亡率的变化。结果化学合成的NF-κB/p65 siRNA可以抑制9HTE细胞NF-κB/p65 mRNA的表达及NF-κB/p65的活化,与CRP组和CRP+scramble siRNA组相比,NF-κB/p65 siRNA组Bcl-2 mRNA和蛋白表达明显减少(P﹤0.05),BaxmRNA和蛋白表达明显增加(P﹤0.05),流式细胞仪显示NF-κB/p65 siRNA组细胞凋亡率明显降低(P﹤0.05)。结论利用siRNA干扰沉默NF-κB/p65基因,可以有效抑制CRP介导的气道上皮细胞凋亡,表明NF-κB的活化可以促进气道上皮细胞中的凋亡,其机制之一可能是通过调控Bcl-2和Bax的表达发挥诱导细胞凋亡的作用。

Objective To investigate the influence of C reactive protein(CRP)induced apoptosis through sliencing nuclear factor-kappa B p65(NF-κB)/p65 gene by RNA interference in human airway epithelial cells 9HTE.Methods 9HTE were cultured in vitro and chemically synthesized small interference RNA(siRNA)directed against human NF-κB/p65 was transfected into 9HTE by using cationic liposome LipofectamineTM2000 as the transfecting agent.Then after incubation with CRP for 24 h,the level of NF-κB/p65,Bcl-2 and Bax mRNA was tested by reverse transcription polymerase chain reaction(RT-PC R),the activation of NF-κB/p65 was tested by immunocytochemical stain,the level of Bcl-2 and Bax protein were tested by W estern blot,the rate of apoptosis was tested by flow cytometry.Results Compared with normal group,chemically synthesized small interfering RNA inhibited expression of NF-κB/p65mRNA and the activation of NF-κB/p6.5 in 9HTE.The expression of Bcl-2 mRMA and protein decreased significantly(P﹤0.05),the expression of Bax mRNA and protein increased significantly(P﹤0.05),the rate of apoptosis decreased significantly(P﹤0.05)compared with CRP and scramble siRNA groups.Conclusions Silencing NF-κB/p65 gene by using siRNA interfere could effectively inhibit CRPmediated apoptosis in human airway epithelial cells.lt is indicate that the activation of NF-κB has a role of pro-apoptosis in airway epithelial cells,which one of the mechanisms may be through regulating the expression of Bcl-2 and Bax to induce apoptosis.