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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。
Objective: To observe the effect of heat shock protein Hsp16.3 on autophagosome formation of macrophages infected with Mycobacterium tuberculosis (MTB). Methods: Macrophage RAW264.7 autophagosomes were induced with 50 ng / μL rapamycin, then infected with Mycobacterium tuberculosis strain H37Rv and then stimulated with macrophages using Hsp16.3 protein The changes of autophagosome phase formation were observed under electron microscope. The intracellular bacterial morphology was observed by acid-fast staining and the number of colonies of MTB was counted. The macrophage total protein was extracted and the expression of autophagy-related protein LC3 was detected by Western blot. Results: Rapamycin induced mycobacterium tuberculosis infection after autophagy was induced by macrophages. The addition of Hsp16.3 protein significantly inhibited the formation of autophagosomes, which affected the development of Mycobacterium tuberculosis Survival in macrophages significantly increased bacterial colony forming units and decreased the expression of autophagy-associated protein LC3 (P <0.05). Conclusion: Hsp16.3 protein may inhibit autophagosome formation by regulating the expression of Atg8.