Necrostatin-1对BCG感染后小鼠巨噬细胞RAW264.7凋亡的调控

发布时间：2018-11-17 09:20

【摘要】：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。
[Abstract]:5- (1H-indole-3-methylmethyl) -3-methyl-2-thione-4-imidazolone (Necrostatin-1,Nec-1) is a small molecule that can specifically inhibit programmed cell apoptosis. To investigate the effect of Nec-1 on RAW264.7 apoptosis induced by Bacillus Calmette-Gu 茅 rin,BCG in mouse (Mus musculus) macrophages. In this study, the cell survival rate of, Annexin V and (propidine iodide, were determined by 3- (4- (4-) -dimethylthiazole-2) -2- diphenyltetrazolium bromide (3- (4t5)-dimethylthiahiazo (- z-y1) -3- di-phenytetrazoliumromide) colorimetric assay. The cell apoptosis rate was detected by PI, the mitochondrial membrane potential level was detected by JC-1 staining, and the enzyme activity of Caspase-3 was detected by spectrophotometry. The expression levels of m RNA and protein of apoptosis-related genes were detected by Q RT-PCR and Western blot methods. The results showed that Nec-1 could increase the survival rate and decrease the apoptosis rate of macrophages infected with BCG, and down-regulate the expression of RIP1,RIP3 and BAX genes by decreasing the level of mitochondrial membrane potential and up-regulating the expression of Bcl-2 gene. Thus, the protein expression and enzyme activity of Caspase-3 were reduced effectively. This study suggests 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 protein. This will be helpful to further study the interaction between (Mycobacterium tuberculosis) and macrophages of Mycobacterium tuberculosis, and it is of great significance to reveal the pathogenesis of tuberculosis.
【作者单位】：西部特色生物资源保护与利用教育部重点实验室;宁夏大学生命科学学院;
【基金】：国家自然科学基金(No.31560322;No.31460301) 宁夏高等学校科学研究项目(No.NGY2015029)
【分类号】：R52

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