miR-207调控结核分枝杆菌在巨噬细胞内存活的机制研究  

Mechanistic study of miR-207 regulation of Mycobacterium tuberculosis survival in macrophages

作　　者：杜文雅 代禹美 乐林芝 马涛 吴利先 DU Wenya;DAI Yumei;YUE Linzhi;MA Tao;WU Lixian(Department of Microbiology and Immunology,School of Basic Medical Sciences,Dali University,Dali 671000,China)

机构地区：[1]大理大学基础医学院微生物学与免疫学教研室,云南大理671000

出　　处：《中国比较医学杂志》2024年第12期41-49,共9页Chinese Journal of Comparative Medicine

基　　金：云南省地方本科高校基础研究重点项目(202101BA070001-038)。

摘　　要：目的miR-207在许多疾病中存在差异性表达,探究miR-207过表达对调控结核分枝杆菌(H37Ra)在巨噬细胞内存活的机制研究,为结核病的靶向治疗提供理论基础。方法将实验分为4组:空白组(Ana-1细胞)、对照组(感染H37Ra的细胞)、mi组(感染H37Ra并转染miRNA-207 mimics)和mi-NC组(感染H37Ra并转染mimics NC)。使用H37Ra感染Ana-1细胞建立结核感染模型,使用脂质体转染法将miRNA-207 mimics和mimics NC转入Ana-1细胞。结核菌落形成单位计数评估miR-207对胞内分枝杆菌负荷的影响以及对胞外残留分枝杆菌的清除作用。采用流式细胞术检测细胞总凋亡率。实时荧光定量聚合酶链反应(qPCR)检测miR-207、细胞凋亡基因、焦亡基因、炎症基因及自噬基因相对表达量。Western blot检测细胞凋亡蛋白、焦亡蛋白、自噬蛋白的相对表达量。采用荧光显微镜及多功能酶标仪检测细胞内ROS的荧光强度,以及检测细胞内LDH的含量。结果显微镜下观察成功建立感染模型,qPCR检测发现对照组miR-207表达量低于空白组(P<0.01),表明miR-207在空白组和对照组中差异性表达,mi组miR-207表达量显著高于mi-NC组(P<0.0001),表明成功建立转染模型。菌落形成单位计数发现mi组的菌落数高于mi-NC组和对照组(P<0.001)。流式细胞术检测发现mi组总凋亡高于mi-NC组和对照组(P<0.05)。qPCR及Western blot检测发现对照组凋亡基因和凋亡蛋白相对表达量高于空白组(P<0.05),mi组高于mi-NC组(P<0.05)。对照组炎症基因相对表达量高于空白组(P<0.001),mi组高于mi-NC组(P<0.05),对照组焦亡基因和焦亡蛋白相对表达量高于空白组(P<0.01),mi组高于mi-NC组(P<0.05)。自噬正调节基因LC3和Beclin1在对照组的相对表达量高于空白组(P<0.0001),mi组低于mi-NC组(P<0.05)。负调节自噬基因相对表达量与自噬正调节基因趋势相反。自噬相关蛋白相对表达量与自噬基因趋势一致。ROS荧光强度检测发现�Objective miR-207 is differentially expressed in many diseases.We investigated the mechanism by which miR-207 overexpression regulates the survival of Mycobacterium tuberculosis(H37Ra)in macrophages,to provide a theoretical basis for the targeted therapy of tuberculosis.Methods Macrophages were divided into four groups:blank(Ana-1 cells),control(cells infected with H37Ra),mi(infected with H37Ra and transfected with miRNA-207 mimics),and mi-NC(infected with H37Ra and transfected with NC mimics)groups.A model of tuberculosis infection was established using H37Ra-infected Ana-1 cells,and miRNA-207 and NC mimics were transfected into Ana-1 cells using the liposome transfection method.Tuberculosis colony-forming units were counted to assess the effect of miR-207 on intracellular mycobacterial load and clearance of extracellular residual mycobacteria.The total apoptosis rate was detected by flow cytometry.The relative expression levels of miR-207 and apoptosis,pyroptosis,inflammation,and autophagy genes were measured by quantitative real-time polymerase chain reaction(qPCR).Relative expression levels of apoptosis,pyroptosis,and autophagy proteins were detected by Western blot.Fluorescence microscopy and multifunctional enzyme labeling were used to detect the fluorescence intensity of intracellular reactive oxygen species(ROS)and lactate dehydrogenase(LDH).Results Successful establishment of the infection model was observed under the microscope.qPCR showed that miR-207 expression was lower in the control compared with the blank group(P<0.01),indicating differential expression between these two groups.miR-207 expression was significantly higher in the mi compared with the mi-NC group(P<0.0001),indicating successful establishment of the transfection model.The number of colonies and total apoptosis were both higher in the mi group compared with the mi-NC and control groups(P<0.001).qPCR and Western blot showed that the relative expression levels of apoptotic genes and proteins were higher in the control group than in the b

关 键 词：结核分枝杆菌 H37RA miR-207 自噬 巨噬细胞 凋亡 焦亡 

分 类 号：R-33[医药卫生]