RhoA蛋白对结核杆菌侵袭A549细胞的影响

The Effect of RhoA Protein on Mycobacterium tuberculosis Invasion of A549 Cells

探讨结核杆菌(mycobacterium tuberculosis,MTB)侵袭A549细胞过程中RhoA蛋白功能。构建靶向RhoA基因的小干扰RNA(siRNA)表达载体si-RhoA,转染A549细胞,36 h后进行MTB黏附和侵袭细胞实验,蛋白印迹法(Western blot)检测RhoA蛋白表达水平,电镜观察细胞超微结构改变,激光共聚焦显微镜观察细胞微丝骨架变化并计算F-actin重排指数,定量PCR法测量细胞黏附和内吞的MTB数量。结果显示,si-RhoA转染A549细胞后,RhoA蛋白表达下降84.7%,细胞对MTB的黏附能力未改变。MTB侵袭细胞实验显示,si-RhoA转染组细胞F-actin重排指数和内吞细菌数量分别是(63.0±3.1)%和(3.19±0.26)×103拷贝,无关siRNA组分别是(84.5±1.8)%和(4.45±0.29)×103拷贝,前者均少于后者(P<0.01)。由此可见,结核杆菌侵袭A549细胞需要RhoA蛋白参与,可能通过"拉链"机制介导结核杆菌侵袭非吞噬细胞。

This paper was to investigate the role of RhoA protein in the process of Mycobacterium tuberculo- sis （MTB） invasion ofA549 cell line （human lung adenocarcinoma cells）. Small intefering RNA （siRNA） expressing vector targetting RhoA gene （si-RhoA） was constructed and transfered to A549 cells. MTB adhesion and invasion of A549 cells assays were performed 36 hours post transfection; The level of RhoA protein was determined by Western blot; The ultrastructural changes of cells were observed by electron microscope; Changes of filaments actin （F-actin） cytoskeleton were detected by laser scanning confocal microscope （LSCM）, and the number of MTB adhering to cell and internalized by cell were measured by quantitative polymerase chain reaction （PCR）. These results showed that the level of RhoA protein decreased by 84.7% after A549 cells transfered by si-RhoA, with the capacity of MTB adhering to cell no change. The test of MTB invasion of cells showed that the index of F-actin rearrangement and the number of MTB internalized by cell were （63.0±3.1）% and （3.19±0.26）x 103 copies for A549 cells transfered by si-RhoA, while they were （84.5±1.8）% and （4.45±0.29）x 103 copies for A549 cells transfered by scramble siRNA, and the former was significantly less than the latter （P〈0.01）. All these results indicate that the protein of RhoA has a role on MTB inva- sion ofA549 cells, which may mediate MTB invasion of non phagocytic cells by zipper mechanism.