p53信号通路在MTB感染肺泡Ⅱ型上皮细胞系A549中的免疫调控作用

Effect of p53 signaling pathway on AEC Ⅱ line A549 infected with Mycobacterium tuberculosis (MTB)

【目的】探讨p53通路在结核分枝杆菌(MTB)感染肺泡Ⅱ型上皮细胞(AECⅡ)中的免疫调控作用。【方法】将供试的p53基因过表达和干扰载体转染293T细胞,对其作用效果进行验证。利用脂质体分别转染p53基因过表达和干扰载体到AECⅡ细胞系A549中,建立p53基因过表达和干扰A549系模型细胞。用牛结核分枝杆菌减毒株(BCG)分别感染未经任何处理的A549细胞(感染对照组)及模型细胞,以未感染BCG的各类A549细胞为参照。以β-actin为内参基因,通过实时荧光定量PCR和Western blot来分析信号分子p53、p300、NF-κB、TLR-4、TRAF6在mRNA和蛋白水平的表达,以及炎症细胞因子TNF-α、IFN-γ、IL-6和IL-8的mRNA表达情况。【结果】过表达载体plRES2-EGFP-p53 WT和干扰载体TP53-RNAi(2648)作用效果明显,成功建立了p53基因过表达和干扰的A549细胞模型。BCG感染的对照组、p53基因过表达组和干扰组的A549细胞中,p53、p300、NF-κB、TLR-4、TRAF6在mRNA和蛋白水平的表达显著或极显著高于相应的BCG未感染组,其中p300表达与p53基因表达量呈正相关,NF-κB、TLR-4、TRAF6表达与p53基因表达量呈负相关;BCG感染可引起TNF-α、IFN-γ、IL-6和IL-8的显著表达,且与p53基因表达量呈负相关。【结论】BCG感染A549细胞时,p53信号通路中的p53协同p300来抑制NF-κB、TLR-4和TRAF6的活化及负调控TNF-α、IFN-γ、IL-6和IL-8的分泌,从而抵抗MTB的侵染。

【Objective】This study aimed to investigate the immunity regulation of p53 signaling pathway in typeⅡalveolar epithelial cells(AECⅡ)infected with Mycobacterium tuberculosis(MTB).【Method】The overexpression and interference vectors of p53 gene were transfected into 293 T cells and confirmed.The p53 gene overexpression and interference vectors were transfected into the AECⅡcell line A549 separately to establish the AECⅡcell models by liposome.The normol(as control group of infected with BCG)and model A549 cells were infected with the attenuated strain of Mycobacterium tuberculosis(BCG).Using the BCG un-infected A549 cells as blank cells andβ-actin as internal reference gene,the mRNA and protein expressions of p53,p300,NF-κB,TLR-4 and TRAF6 were analyzed by real-time quantitative PCR and Western blot,and the mRNA levels of inflammatory cytokines including TNF-α,IFN-γ,IL-6 and IL-8 were investigated.【Result】The overexpression vector plRES2-EGFP-p53 WT and the interference vector TP53-RNAi(2648)were functional,and the AECⅡcell models of p53 gene overexpression and interference were successfully established.The expressions of p53,p300,NF-κB,TLR-4,and TRAF6 in the BCG-infected A549 cell control group,p53 gene overexpressed group and interfered group were significantly or extremely significantly higher than those of blank group.The expressions of p53 and p300 were positively correlated to the over or interfering expressions of p53 gene.The expressions of NF-κB,TLR-4 and TRAF6 were negatively correlated to the expressions of p53 gene.The BCG infection could increase the expressions of TNF-α,IFN-γ,IL-6 and IL-8 in A549 cells,and the expressions of these inflammatory cytokine were negatively correlated to the expressions of p53.【Conclusion】When A549 cells are infected with BCG,p53 would cooperate with p300 in the p53 signaling pathway to inhibit the activation of NF-κB,TLR-4 and TRAF6,and negatively regulate the secretion of TNF-α,IFN-γ,IL-6 and IL-8 against MTB infection.