论文部分内容阅读
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。
To investigate the function of RhoA protein during the invasion of A549 cells by mycobacterium tuberculosis (MTB). RhoA siRNA was constructed and transfected into A549 cells. After 36 h, the adhesion and invasion assay of MTB was performed. The expression of RhoA protein was detected by Western blot. The expression of RhoA was observed by electron microscopy Ultrastructural changes were observed by confocal laser scanning microscope changes in the actin cytoskeleton and calculated F-actin rearrangement index, quantitative PCR method for measuring cell adhesion and endocytosis MTB number. The results showed that the expression of RhoA protein decreased by 84.7% in A549 cells transfected with si-RhoA, and the adhesion ability of MTB did not change. MTT assay showed that the rearrangement index of F-actin and the number of endocytosed bacteria were (63.0 ± 3.1)% and (3.19 ± 0.26) × 103 copies in the si-RhoA transfected group and (84.5 ± 1.8%) and (4.45 ± 0.29) × 103 copies, the former were less than the latter (P <0.01). Thus, the invasion of A549 cells need Mycobacterium tuberculosis RhoA protein involved, probably through the “zipper” mechanism of Mycobacterium tuberculosis invasion of non-phagocytic cells.