结核分枝杆菌Hsp16.3蛋白影响小鼠巨噬细胞自噬形成的实验研究

Effects of Mycobacterium tuberculosis Hsp16.3 protein on the autophagy function of mice macrophages

目的:观察热休克蛋白Hsp16.3对感染结核分枝杆菌(MTB)的巨噬细胞自噬体形成的作用。方法:以50 ng/μL雷帕霉素诱导小鼠巨噬细胞RAW264.7自噬体形成后,用结核分枝杆菌毒株H37Rv感染巨噬细胞,再用Hsp16.3蛋白作用于巨噬细胞,电镜观察自噬体相成的变化,抗酸染色观察胞内细菌形态,计数MTB的菌落数。提取巨噬细胞总蛋白,Western blot方法检测自噬相关蛋白LC3表达水平的变化。结果:雷帕霉素诱导巨噬细胞形成自噬后感染结核分枝杆菌可使胞内细菌局限化,加入Hsp16.3蛋白可明显抑制了自噬体的形成,影响了结核分枝杆菌在巨噬细胞内的生存,显著增加了细菌的菌落形成单位,降低了自噬相关蛋白LC3的表达(P<0.05)。结论:Hsp16.3蛋白可能通过调节Atg8的表达水平抑制自噬体的形成。

AIM： To investisate the inhibition of Hsp-16.3 on the autophagosomes formation of macrophages. METHODS： Mouse RAW264.7 macrophages were induced by rapamycin （50 ng/μL） following infection with M. tuberculosis H37Rv strains, thereafter, co-incubated with Hspl6.3 protein （25 μg/mL）. The effects of Hspl6.3 protein on the autophagosomes formation was observed with transmission electron microscope. The expression of autophagy-related genes （atg8） for macrophages was detected by Western blotting. RESULTS： It was found that rapamycin-induced autophagy of macrophages infected with M. tuberculosis H37Rv enhanced localization of mycobacteria with autophagosomes. Hspl6.3 protein inhibits autophagosome formation and affects M. tuberculosis survival inside infected macrophages. Furthermore, Hsp16. 3 protein significantly increased M. tuberculosis colony forming units （CFU）, and decreased the expression of microtubule-associated protein light chain-3 （ LC3 ） expression level （P〈0.05）. CONCLUSION： The results showed that Hsp16.3 protein inhibits the formation of autophagosomes by regulating the expression of LC3 protein.