下调Bcl-2家族蛋白促进MTB感染的小鼠巨噬细胞系凋亡

Down-regulation of Bcl-2 family protein promotes apoptosis of mouse macrophages infected with MTB

目的探讨Mcl-1shRNA靶向下调Mcl-1的表达后,Bcl-2家族蛋白对不同毒力结核杆菌感染的小鼠Raw264.7巨噬细胞凋亡的调控作用。方法用XJ-MTB、H37Rv、H37Ra和卡介苗(BCG)感染小鼠Raw264.7巨噬细胞,并用Mcl-1 shRNA作用于感染的巨噬细胞,流式细胞术检测细胞凋亡率;Western blot检测Bcl-2家族蛋白Mcl-1和Bax的表达;实时荧光定量PCR检测Mcl-1、Bcl-2、Bax、caspase-3与细胞色素C mRNA的表达。结果 1)不同毒力的MTB感染可诱导小鼠Raw264.7巨噬细胞的凋亡,靶向下调Mcl-1的表达可显著提高XJ-MTB和H37Rv感染的宿主巨噬细胞的凋亡率。2)XJ-MTB和H37Rv感染可显著上调Bcl-2家族抗凋亡成员Mcl-1与Bcl-2的表达,应用Mcl-1 shRNA沉默Mcl-1基因后Mcl-1与Bcl-2水平显著下降,同时增高Bax的表达。3)不同毒力的MTB感染小鼠Raw264.7巨噬细胞后caspase-3和细胞色素C表达升高,靶向下调Mcl-1可以进一步上调感染组caspase-3和细胞色素C的表达。结论 MTB感染后小鼠Raw264.7巨噬细胞中Bcl-2家族蛋白与基因的表达水平与菌株毒力相关,应用Mcl-1shRNA下调Mcl-1的表达可以促进宿主巨噬细胞的凋亡。

Objective To investigate the regulator role of Bcl-2 family proteins on the apoptosis of mouse Raw264.7 macrophages infected with different virulent of M. tuberculosis strains when target Mcl-1 expression by using RNA interference. Methods Raw264.7 macrophages were infected by XJ-MTB, H37Rv, H37Ra and BCG strains, Mcl-1 shRNA was treated the infection cells. The apoptosis rate of macrophages was detected by flow cytometry. The expression of Mcl- 1 and Bax protein was detected by Western blot. The expression of Bcl- 2 family genes and the expression of caspase-3 and cytochrome-C were detected by real-time PCR. Results 1）Infection with different virulence of M. tuberculosis induced mouse Raw264.7 macrophages apoptosis, down-regulate Mcl- 1 could significantly increase the apoptosis rate of host macrophages infected with XJ-MTB and H37Rv. 2）Infection with XJ-MTB and H37Rv significantly up-regulated the expression of Mcl-1 and Bcl-2 in host macrophages, the level of Mcl-1 and Bcl-2 was significantly decreased after silencing Mcl- 1 expression, while increasing the expression of Bax. 3） M. tuberculosis infection could induce host macrophages caspase- 3 and cytochrome-C expression, down-regulation of Mcl-1 up-regulated the expression of caspase-3 and cytochrome-C in host. Conclusions The expression of Bcl-2 family members in host macrophages induced by M. tuberculosis infection associated with the virulence of M. tuberculosis . Down-regulated Mcl-1 expression by Mcl-1 shRNA may promote host macrophages apoptosis.