摘要
结核病(tuberculosis,TB)是一种由结核分枝杆菌(Mycobacterium Tuberculosis,Mtb)所致的人畜共患的慢性、消耗性传染病。先天免疫细胞与Mtb的相互作用通过自噬、细胞凋亡及炎症反应等途径影响结核病的发生与发展。旨在探究减毒牛分枝杆菌卡介苗(Bacillus Calmette-Guérin,BCG)感染小鼠巨噬细胞系RAW264.7后,能否发生自噬与细胞凋亡。试验以BCG不同感染复数与感染时间感染小鼠巨噬细胞系RAW264.7,通过Western blot法检测自噬及凋亡相关蛋白LC3B-II、Beclin1、Bcl-2和Caspase3的表达水平,MTT法检测细胞存活率,透射电镜观察感染BCG的巨噬细胞形态学变化,探究BCG感染RAW264.7是否会发生自噬和凋亡,进而确定BCG感染巨噬细胞的最佳浓度与时间。通过对巨噬细胞的分子水平和形态学检测发现,BCG以感染复数为10,感染小鼠巨噬细胞系RAW264.7时间为12 h时,感染组自噬有关蛋白Beclin1、LC3B-II,促凋亡蛋白Caspase3的相对表达水平极显著高于对照组(P<0.01),抑凋亡蛋白Bcl-2的相对表达水平极显著低于对照组(P<0.01);透射电镜观察结果显示,小鼠巨噬细胞系RAW264.7内出现明显的自噬体、自噬溶酶体和细胞核固缩等现象。研究结果表明,BCG感染巨噬细胞系RAW264.7能够发生自噬与细胞凋亡,且具有一定的感染复数和感染时间依赖性,最佳感染复数为10,最佳感染时间为12 h。
Tuberculosis(TB)is a zoonotic chronic wasting and infectious disease caused by mycobacterium tuberculosis(Mtb),and the interaction between innate immune cells and Mtb determines the occurrence and development of tuberculosis mainly through autophagy,apoptosis and inflammatory response.The purpose of this research was to determine whether autophagy and apoptosis occur in mice infected with Bacillus Calmette-Guérin(BCG)with the mouse macrophage line RAW264.7.Mouse macrophage line RAW264.7 infected with BCG of different infection complex numbers and at infection time points was used.The expression levels of autophagy and apoptotic-related proteins LC3 B-II,Beclin1,Bcl-2 and Caspase3 were detected by Western blot.The survival rate of the cells was detected using the MTT method.The morphological changes of macrophages infected with BCG were observed by transmission electron microscopy.Autophagy and apoptosis in BCG-infected macrophage line RAW264.7 were detected.The optimal concentration and timing of BCG-infected macrophages were determined.The results showed that,when BCG infected macrophages with a complex number of 10 of the mouse macrophage line RAW264.7 RAW264.7 for 12 h,the relative expression levels of autophagy-related proteins Beclin1 and LC3 B-II,and the pro-apoptotic protein Caspase3,were significantly higher than those of the control group(P<0.01),but the relative expression levels of the apoptosis protein Bcl-2 were significantly lower(P<0.01).The obvious occurrence of autophagosomes and autophagy lysosomals and nuclear consolidation in macrophages were observed under the transmission electron microscopy(TEM).BCG-infected macrophage RAW264.7 produced autophagy and apoptosis,and depend,to a degree,on the times and timing of infections.The present results suggested that the optimal complex number of infections of BCG-induced macrophage RAW264.7 for autophagy and apoptosis was 10,and the optimal infection timing was 12 h.
作者
李瑞乾
宋阿北
缪西鹏
梁晓珊
许立华
LI Ruiqian;SONG Abei;MIAO Xipeng;LIANG Xiaoshan;XU Lihua(College of Agriculture,Ningxia University,Yinchuan 750021,China)
出处
《畜牧与兽医》
CAS
北大核心
2022年第12期115-121,共7页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金项目(31760722)。
关键词
结核分枝杆菌
巨噬细胞系
自噬
凋亡
卡介苗
Mycobacterium tuberculosis
macrophage line
autophagy
apoptosis
BCG
