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5-(1H-吲哚-3-基甲基)-3-甲基-2-硫酮-4-咪唑烷酮(Necrostatin-1,Nec-1)是一种能够特异的、有效抑制细胞程序性凋亡的小分子物质。为了探讨Nec-1对卡介苗(Bacillus Calmette-Guérin,BCG)诱导的小鼠(Mus musculus)巨噬细胞RAW264.7凋亡的调控作用,本研究采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,MTT)比色法检测细胞存活率,Annexin V和碘化丙啶(propidine iodide,PI)双染法检测细胞凋亡率,JC-1染色法检测细胞线粒体膜电位水平,采用分光光度法检测细胞内Caspase-3的酶活性,q RT-PCR和Western blot法检测凋亡相关基因的m RNA和蛋白表达水平。结果表明,Nec-1可提高被BCG感染巨噬细胞的存活率,降低其凋亡率,通过降低线粒体膜电位水平、上调抑凋亡基因Bcl-2的表达同时下调RIP1、RIP3和BAX基因的表达水平,从而有效降低了Caspase-3的蛋白表达量及酶活性。本研究表明Nec-1可通过提高线粒体膜电位水平、并下调促凋亡蛋白的表达量,从而抑制被BCG感染后巨噬细胞的凋亡,这将有助于进一步研究结核分枝杆菌(Mycobacterium tuberculosis)与巨噬细胞间的相互作用,对于揭示结核病的致病机理具有重要意义。
Necrostatin-1 (Nec-1) is a specific and potent inhibitor of cellular processes Apoptotic small molecules. In order to investigate the regulatory effect of Nec-1 on the apoptosis of RAW264.7 induced by Bacillus Calmette-Guérin (BCG) in mice, this study used 3- (4,5-dimethylthiazole- 2) -2,5-di-phenytetrazoliumromide (MTT) assay was used to detect the cell viability by using 2,5-diphenyltetrazolium bromide (3- (4,5) -dimethylthiahiazo (-z- Annexin V and propidium iodide (PI) staining were used to detect the apoptosis rate. JC-1 staining was used to detect the mitochondrial membrane potential. The intracellular Caspase-3 activity was measured by q RT The expression of m RNA and protein in apoptosis-related genes were detected by PCR and Western blot. The results showed that Nec-1 can increase the survival rate of BCG-infected macrophages and decrease the apoptosis rate. By down-regulating mitochondrial membrane potential, up-regulating the expression of Bcl-2, down-regulating RIP1, RIP3 and BAX genes The level of Caspase-3 protein expression and enzyme activity were decreased effectively. This study shows that Nec-1 can inhibit the apoptosis of macrophages after BCG infection by increasing the level of mitochondrial membrane potential and down-regulating the expression of pro-apoptotic proteins, which will be helpful for the further study of Mycobacterium tuberculosis and macrophages are important for revealing the pathogenesis of tuberculosis.