miRNAs对肺泡巨噬细胞自噬溶酶体成熟及抗结核分枝杆菌感染的调控机制

项目名称： miRNAs对肺泡巨噬细胞自噬溶酶体成熟及抗结核分枝杆菌感染的调控机制

项目编号： No.31472168

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 徐广贤

作者单位： 宁夏医科大学

项目金额： 83万元

中文摘要： 微小RNA是已被广泛认可的基因调控子，通过调控细胞自噬基因的转录，参与调控自噬过程。结核分枝杆菌（MTB）在宿主细胞内的存活与自噬溶酶体的成熟有着密切的关系，miRNAs对巨噬细胞自噬的调控机制，尚不明晰。本研究拟用雷帕霉素与3-MA处理RAW264.7细胞，建立自噬与抑制自噬细胞模型，基于miRNA免疫共沉淀方法，筛选感染MTB后，参与调控自噬与自噬溶酶体成熟的特异性miRNAs与mRNAs，利用双荧光素酶报告系统，验证miRNAs与Atg1、Beclin1、ATG8和mTOR等自噬基因的靶向调控，绘制出miRNAs与自噬基因之间的调控网络；构建miRNA过表达与抑制表达的慢病毒载体，建立相应的MTB感染RAW264.7细胞自噬模型，利用透射电镜、Western blot、Real-Time PCR和激光共聚焦技术，揭示miRNAs对巨噬细胞自噬及MTB抑制自噬溶酶体成熟的调控机制。

中文关键词： miRNAs；自噬；自噬溶酶体；结核分枝杆菌；肺泡巨噬细胞

英文摘要： microRNAs(miRNAs), well-known gene regulators, regulate the autophagy process by regulating the transcription of autophagy genes. The survival of Mycobacterium tuberculosis (MTB) in the host cells is related with the mature of autophagy lysosome. And the regulatory mechanism of miRNAs on the macrophage autophagy is still unclear. In this study, we firstly establish the model of autophagy and autophagy lysosome mature of RAW264.7 cells after treated with rapamycin and 3-MA. Then we infected the cell model with MTB, and screen the specific miRNAs and mRNAs involved in the regulatory process of autophagy and autophagy lysosome mature by the method of miRNA co-immunoprecipitation. Next, we verify the relationship of miRNAs and the key molecules (Atg1, Beclin1, ATG8, mTOR et al.) by the dual-luciferase reporter system, making the regulatory network between the miRNAs and the autophagy genes. Further, we construct the miRNA over-expressed and lost-expressed lentiviral vectors and establish the relative MTB-effected autophagy model of RAW264.7 cells. At last, we use the methods of transmission electron microscope, Western blot, Real-Time PCR and laser scanning confocal microscope to reveal the regulatory mechanism of miRNAs on the effect of the macrophage autophagy and the inhibition effect of Mycobacterium tuberculosis on autophagy lysosomal mature.

英文关键词： miRNAs;autophagy;Autolysosomes;Mycobacterium tuberculosis;Macrophages Alveolar