结核分枝杆菌感染巨噬细胞后细胞凋亡/坏死相关LncRNA分子的筛选及其调控机制研究

As target cell of mycobacterium tuberculosis, Apoptosis and necrosis of Macrophages after infection with Mtb play an important role in the development and prognosis of the disease, therefore, to clarify the regulatory mechanism of the process has important scientific significance. In recent years, studies have shown that LncRNAs are involved in the development of a variety of disease, but the regulatory role of LncRNAs in apoptosis and necrosis of Macrophages after infection with Mtb remains unclear.. Based on the research background, the present study aims to identify LncRNAs related to apoptosis and necrosis of macrophage infection with Mtb through LncPath ™ disease/signaling pathways microarray. In order to clarify regulatory role of LncRNAs in apoptosis and necrosis of Macrophages infection with Mtb. overexpression vector and siRNA vector will be constructed to transfect RAW264.7 cells respectively, and then the transfected macrophage will be infected with Mtb, Furthermore, the caspase inhibitor and RNAi technology will be used to inhibit and silence apoptosis and necrosis pathway genes respectively. The research results will provide further insight into the pathogenesis of tuberculosis (TB) and theory basis for development of targeted drugs.

巨噬细胞作为结核分枝杆菌的靶细胞，它与结核分枝杆菌相互作用后发生的凋亡与坏死对结核病的发生、发展及预后具有重要影响，因此，阐明 该过程的调控机制具有十分重要的科学意义。近年研究表明，LncRNA在多种疾病的发生发展过程中发挥了重要的调控作用，但是其在结核分枝杆菌与巨噬细胞的相互作用过程中对细胞凋亡与坏死的调控作用尚未见相关报道。. 基于以上研究背景，本项目拟采用LncPath™ 疾病/信号通路特异性LncRNA芯片筛选结核分枝杆菌感染巨噬细胞后与宿主细胞凋亡/坏死信号通路相关的LncRNA分子，通过构建其过表达载体和干扰载体并转染RAW264.7细胞后并用结核分枝杆菌感染，进而用caspase抑制剂和RNAi技术分别抑制和沉默凋亡与坏死通路相关基因，以期阐明LncRNA对巨噬细胞凋亡和坏死的调控作用及其分子机制。研究结果将为深入理解结核病的发病机制及靶向药物研制提供理论依据。

结核病（Tuberculosis，TB）是由结核分枝杆菌（Mycobacterium tuberculosis，Mtb）感染引起的人畜共患传染病。巨噬细胞作为宿主细胞和结核分枝杆菌的靶细胞，在抗结核分枝杆菌感染中发挥着极其重要的作用。一方面，巨噬细胞在被Mtb感染后，会通过自身凋亡来清除结核分枝杆菌；另一方面，Mtb感染巨噬细胞后会诱导其发生坏死，从而逃避对它的清除作用，实现扩散和繁殖。凋亡和坏死过程十分复杂、涉及多个环节，并受到多种因素调控。在众多调控因素中，长链非编码RNA（Long non-coding RNA，LncRNA）近年来受到了广泛关注。本项目在成功建立BCG感染诱导巨噬细胞凋亡/坏死模型的基础上，通过转录组学分析，筛选到了与细胞凋亡/坏死相关的两个LncRNA分子：LncRNA-Cox2和LncRNA-GAS5。进一步研究了所筛选的LncRNA分子对结核分枝杆菌感染巨噬细胞诱导其发生凋亡与坏死的作用机器分子机制，取得了如下研究结果：.（1）基于转录组学对结核分枝杆菌感染巨噬细胞后宿主细胞发生凋亡/坏死相关的LncRNA分子以及相应的靶基因进行了筛选和鉴定。筛选到了两个候选的LncRNA分子：LncRNA-Cox2和LncRNA-GAS5。 .（2）设计并筛选了LncRNA-Cox2的小干扰RNA(siRNA-LncRNA-Cox2)，研究了下调LncRNA-Cox2对BCG诱导的巨噬细胞凋亡相关指标的影响。结果表明，下调LncRNA-Cox2后可显著提高细胞的凋亡率，促进凋亡相关因子的表达；显著升高细胞内Ca2+浓度和内质网应激相关因子的表达；而阻断内质网应激会抑制巨噬细胞凋亡的发生。表明下调LncRNA-Cox2会通过激活内质网应激途径促进BCG诱导的巨噬细胞凋亡。.（3）研究了下调LncRNA-Cox2对BCG诱导的巨噬细胞坏死的调控作用及机制。结果发现，下调LncRNA-Cox2可显著降低细胞坏死率以及细胞坏死关键因子RIP1、RIP3和MLKL的表达，表明下调LncRNA-Cox2通过抑制RIP1-RIP3-MLKL途径调控BCG诱导的巨噬细胞坏死。.（4）初步探究了LncRNA-GAS5对BCG诱导巨噬细胞坏死调控作用及机制。结果表明，LncRNA-GAS5通过激活RIP1-RIP3-MLKL途径正调控BCG诱导的巨噬细胞坏死。