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目的探讨结核分枝杆菌(MTB)小分子热休克蛋白Hsp16.3表达与感染小鼠肺泡巨噬细胞凋亡的关系。方法分别将MTB国际标准强毒株H37Rv株Hsp16.3基因缺失突变菌株(△H37Rv)、结核杆菌国际标准强毒株H37Rv菌株(H37Rv)以及无菌生理盐水溶液(空白对照)通过小鼠尾静脉注入小鼠体内以建立小鼠的感染模型,并在感染后的1、3、5、7、9、11、13、15d分离及鉴定小鼠肺泡巨噬细胞,用激光共聚焦显微镜技术检测及鉴定MTB感染小鼠肺泡巨噬细胞;流式细胞术检测不同时间点感染小鼠肺泡巨噬细胞的凋亡率;Western blot技术检测小鼠肺泡巨噬细胞内半胱天冬酶-3(Caspase-3)和B细胞淋巴瘤/白血病-2(Bcl-2)蛋白的表达。结果△H37Rv菌株感染组和H37Rv菌株感染组凋亡率均高于空白对照组,△H37Rv组的巨噬细胞凋亡率在感染1~7d内逐渐升高,至感染7d时达到高峰,随后呈下降趋势,与H37Rv组相比,1~7d及13~15d,△H37Rv组的巨噬细胞凋亡率高于H37Rv组,差异有统计学意义(P<0.05);H37Rv组和△H37Rv组的巨噬细胞Caspase-3和Bcl-2蛋白表达水平均高于空白对照组;1~7d内,H37Rv感染组和△H37Rv感染组的巨噬细胞Caspase-3表达水平逐渐升高,至第7天达到高峰,且△H37Rv感染组在第13天时出现第二次高峰,但Caspase-3表达水平均高于H37Rv感染组,差异有统计学意义(P<0.05);在感染的早期(1~7d),△H37Rv感染组Bcl-2的表达水平无明显变化(P<0.05),但在9d后逐渐升高;H37Rv感染组巨噬细胞内Bcl-2的表达水平1~7d内无明显变化(P<0.05),7d后逐渐升高,但△H37Rv感染组Bcl-2的表达水平始终低于H37Rv组且7d后表现更为显著。结论在MTB感染的早期和晚期,MTB小分子热休克蛋白Hsp16.3的表达能够有效抑制小鼠肺泡巨噬细胞的凋亡,而这种抑制作用可能是通过抑制凋亡蛋白酶Caspase-3的表达,同时促进Bcl-2蛋白的表达来实现的。
Objective To investigate the relationship between the expression of heat shock protein Hsp16.3 of Mycobacterium tuberculosis (MTB) and the apoptosis of alveolar macrophages in infected mice. Methods H37Rv strain H37Rv strain (H37Rv), H37Rv strain (H37Rv) and sterile saline solution (blank control) of MTB international standard strain H37Rv were respectively injected into mouse tail vein The mice were injected into the body to establish a mouse model of infection. The mouse alveolar macrophages were isolated and identified at 1, 3, 5, 7, 9, 11, 13 and 15 days after infection and detected by laser confocal microscopy MTT was used to detect the alveolar macrophages in mice. Flow cytometry was used to detect the apoptosis rate of alveolar macrophages in mice infected by MTT. The expression of caspase-3 (Caspase-3) in mouse alveolar macrophages -3) and B cell lymphoma / leukemia-2 (Bcl-2) protein expression. Results The apoptosis rate in infected group of H37Rv strain and H37Rv strain was higher than that of blank control group. The apoptosis rate of macrophage in △ H37Rv group increased gradually from 1 to 7 days after infection and peaked at 7 days after infection, (P <0.05). Compared with H37Rv group, the apoptotic rates of macrophages in △ H37Rv group were higher than those in H37Rv group at 1 ~ 7d and 13 ~ 15d (P <0.05), while those in H37Rv group and △ H37Rv group The expression of Caspase-3 and Bcl-2 in macrophages were higher than those in blank control group. Caspase-3 expression in macrophages of H37Rv infected group and △ H37Rv infected group gradually increased from 1 to 7 days. By day 7, (P <0.05). In the early stage of infection (1-7 days), the expression of Caspase-3 in the H37Rv group was higher than that in the H37Rv group (P <0.05) ), While the expression of Bcl-2 in △ H37Rv infection group did not change significantly (P <0.05), but gradually increased after 9 days. The expression of Bcl-2 in H37Rv infection group did not change within 1 ~ 7d P <0.05), and gradually increased after 7 days. However, the expression of Bcl-2 in △ H37Rv infection group was always lower than that in H37Rv group and was more significant after 7 days. Conclusion The expression of heat shock protein Hsp16.3, a small molecule of MTB, can effectively inhibit the apoptosis of alveolar macrophages in the early and late stages of MTB infection, and this inhibition may be through inhibiting the expression of Caspase-3 , While promoting the expression of Bcl-2 protein to achieve.