中文摘要：

目的建立结核分枝杆菌感染RAW264.7株巨噬细胞模型,研究结核分枝杆菌Hsp16.3与感染巨噬细胞凋亡的相关性。方法分别用结核分枝杆菌国际标准强毒株H37Rv株和卡介苗菌株感染RAW264.7巨噬细胞株,于感染后1、3、6及12h,利用激光共聚焦显微镜鉴定卡介苗及结核分枝杆菌国际标准强毒株H37Rv株感染RAW264.7巨噬细胞模型;同时利用流式细胞技术于上述各时间点检测各组感染巨噬细胞的凋亡率,并分析其时相性变化;利用PCR技术扩增模型目的基因,利用Western blot检测上述各个时间点Hsp16.3蛋白表达水平的时相性变化。结果激光共聚焦显微镜下观察到RAW264.7巨噬细胞内有标记MTB16KD的绿色荧光,证实结核分枝杆菌感染巨噬细胞模型构建成功。巨噬细胞感染结核分枝杆菌后凋亡率升高,其中感染后1、3和12h凋亡率H37Rv感染组与BCG感染组比较差异有统计学意义（P〈0.05）;巨噬细胞感染结核分枝杆菌后的PCR产物大小为435bp;感染细胞Hsp16.3蛋白表达升高,其中感染后1h和3h升高更显著。结论结核分枝杆菌感染可导致巨噬细胞凋亡,感染巨噬细胞的凋亡率与菌株毒力强弱有关,弱毒力结核分枝杆菌感染的巨噬细胞早期凋亡更显著;结核分枝杆菌感染巨噬细胞的凋亡与结核分枝杆菌表达Hsp16.3水平高低有关,在感染早期强毒力结核分枝杆菌Hsp16.3表达水平较高。

英文摘要：

Objective To establish the models of Mycobacterium tuberculosis（MTB） infecting RAW264.7 macrophages,and study the correlation between Hsp16.3 of MTB and the apoptosis of infected macrophages.Methods RAW264.7 cells were infected by H37Rv and BCG strains,after being infected of 1 h,3 h,6 h,and 12 h,the infected RAW264.7 macrophages models were identified by confocal laster scanning microscope.Then flow cytometry was used to detect the macrophage apoptosis rate and analysis infected macrophages group apoptosis rate’s change in different time phases.After PCR amplifying model purpose gene,western blot was used to compare and analysis the Hsp16.3 levels of phase changes.Results The infected RAW264.7 macrophages were identified by Confocal laster scanning microscope： MTB16KD green mark was detected.The mycobacterium tuberculosis infecting macrophages models were established.After being infected by H37Rv and BCG,the apoptosis rate of macrophages increased.The apoptosis rate in 1 、3 and 12 h increased obviously,and the difference was statistically significant（P〈0.05）.With macrophages being infected by MTB,there was a specific band at 435 bp of PCR amplification products.The expression of MTBHsp16.3 increased after infection,which increased obviously in 1 h and 3 h.Conclusion The infection of MTB induced the macrophages’ apoptosis;the apoptosis rate was related to the virulence of MTB;macrophages which were infected by the weak virulent MTB had a much more pronouned early apoptosis.Being infected by different virulence of MTB,the apoptosis rate of macrophages was related to the expression levels of MTB Hsp16.3 of which the expression of the power full virulent MTB was much more significant than that of the less virulent in the early phase of infection.