靶向沉默Mcl-1基因对感染不同毒力结核杆菌小鼠腹腔巨噬细胞凋亡的影响

 目的: 通过RNA干扰技术特异性沉默 Mcl-1 基因,探讨下调 Mcl-1 基因对感染不同毒力结核杆菌小鼠腹腔巨噬细胞凋亡的影响。方法: 分别用制备好的新疆地区流行的优势强毒结核分枝杆菌临床分离株(简称强毒株)、结核分枝杆菌国际标准强毒株H37Rv(简称H37Rv)、结核分枝杆菌国际标准无毒株H37Ra(简称H37Ra)和卡介苗(BCG)菌悬液感染BALB/c小鼠,再以筛选并构建好的Mcl-1-shRNA作用于感染的小鼠模型,同时设立相应对照组,于作用后1 d、3 d、5 d和7 d提取小鼠腹腔巨噬细胞,应用实时荧光定量PCR和Western blot检测各组小鼠腹腔巨噬细胞中Mcl-1 mRNA和蛋白的表达;应用流式细胞术检测各组巨噬细胞的凋亡水平。结果: 小鼠被不同毒力的结核杆菌感染后其腹腔巨噬细胞中Mcl-1 mRNA和蛋白的表达水平均有不同程度的升高,其中以感染了强毒株和H37Rv的腹腔巨噬细胞升高最为明显(P<0.05);应用RNA干扰技术沉默 Mcl-1 基因后,Mcl-1 mRNA和蛋白的表达水平明显低于对照组(P<0.05);流式细胞术分析显示,下调 Mcl-1 基因的表达可诱导小鼠腹腔巨噬细胞凋亡。结论: 应用Mcl-1-shRNA可有效沉默 Mcl-1 在感染了不同毒力结核杆菌小鼠腹腔巨噬细胞中的表达,并能上调巨噬细胞的凋亡水平。

Effects of Mcl-1 silencing on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis

AIM: To investigate the effect of inhibiting Mcl-1 gene expression on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis using a technique of RNA interference. METHODS: The BALB/c mice were infected with prepared bacterium of the virulence strains of Xinjiang, H37Rv, H37Ra and BCG. Mcl-1-shRNA was applied to the mouse model of infection, and the control groups were set up. On 1 d, 3 d, 5 d and 7 d, the mouse peritoneal macrophages were collected. The expression of Mcl-1 at mRNA and protein levels was determined by real-time PCR and Western blot. The apoptotic rate of peritoneal macrophages was analyzed by flow cytometry. RESULTS: The expression of Mcl-1 at mRNA and protein levels was up-regulated in the peritoneal macrophages from the mice infected with different virulence of Mycobacterium tuberculosis, and the cells from the mice infected with virulence strains of Xinjiang and H37Rv expressed higher level of Mcl-1 than the uninfected control cells (P<0.05). The expression of Mcl-1 at mRNA and protein levels was reduced by RNA interference as compared with control group (P<0.05). Inhibition of Mcl-1 expression induced apoptosis of peritoneal macrophages in the mice. CONCLUSION: The Mcl-1 expression at mRNA and protein levels in mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis was effectively suppressed by Mcl-1-shRNA, which can induce macrophage apoptosis.