自噬在巨噬细胞介导的炎症中的作用及其机制研究
发布时间：2019-02-19 17:28
【摘要】：目的： 研究自噬在脂多糖(lipopolysaccharide LPS)诱导的RAW264.7巨噬细胞介导的炎症中的作用,并初步探讨其相关机制。 方法： 第一部分：以Raw264.7巨噬细胞为研究对象,通过LPS建立细胞炎症模型。采用Western blot法检测TNF-α(tumor necrosis factor-a)的表达,检验模型是否建立成功；免疫荧光方法观察LC3的荧光聚集；Western blot法测定不同时间点自噬相关蛋白LC3(microtubule-associated protein light chain3)、p62及Beclinl的表达水平。第二部分：分别采用自噬诱导剂雷帕霉素(Rapamycin)以及自噬抑制剂3-甲基腺嘌呤(3-methyladenine3-MA)预处理LPS诱导的巨噬细胞,Western blot法检测自噬相关蛋白的表达；ELISA检测炎症相关因子TNF-α、ICAM-1(intercellular adhesion molecule)及Griess Reagen检测NO (nitric oxide)的生成和释放：采用RT-PCR方法检测p62转录水平变化;利用siRNA干扰技术构建敲减自噬底物蛋白p62的细胞模型,同时ELISA检测炎症因子释放水平。 结果： 第一部分：成功建立LPS诱导Raw264.7巨噬细胞炎症模型,细胞免疫荧光结果显示LPS刺激12h后自噬特异性蛋白LC3呈点颗粒状聚集增多。同样,Western blo结果显示,与对照组相比,不同时间点(2h、4h、8h、12h、24h)自噬相关蛋白LC3,p62及Beclinl的表达增加,且此作用呈时间依赖性(P0.05)；去除刺激凶素LPS后,自噬相关蛋白表达并未下降(P0.05)。第二部分：自噬诱导剂Rapamycin预处理后自噬相关蛋白表达增加,炎症因子(TNF-α、ICAM-1、NO)释放也增加(P0.05)；相反,自噬抑制剂3-MA抑制了自噬相关蛋白的表达,同时下调了炎症因子的水平(P0.05)。RT-PCR结果显示,干预后,p62mRNA表达与蛋白水平趋势相一致。利用siRNA的方法敲减p62蛋白,炎症因子释放减少(P0.05),提示p62在LPS诱导的Raw264.7巨噬细胞介导的炎症反应中起着重要的作用。 结论： 自噬参与了LPS激活的巨噬细胞炎症反应。自噬应激状态下,自噬诱导剂促进自噬相关蛋白的聚集,p62蛋白转录水平上升,同时上调炎症因子的生成和释放；相反,自噬抑制剂抑制自噬相关蛋白,p62转录及炎症因子的产生；siRNA敲减p62,炎症因子释放减少。这些结果说明,在自噬应激反应中,p62蛋白在LPS诱导的Raw264.7巨噬细胞介导的炎症反应中起着重要的作用。
[Abstract]:Aim: to investigate the role of autophagy in lipopolysaccharide (lipopolysaccharide LPS) -induced RAW264.7 macrophage mediated inflammation and its mechanism. Methods: in the first part, Raw264.7 macrophage was used as the research object, and the inflammatory model was established by LPS. Western blot method was used to detect the expression of TNF- 伪 (tumor necrosis factor-a, and the immunofluorescence method was used to observe the fluorescence aggregation of LC3. The expression levels of LC3 (microtubule-associated protein light chain3), p62 and Beclinl were measured by Western blot. The second part: the expression of autophagy related protein was detected by, Western blot method of macrophage induced by LPS pretreated with autophagy inducer rapamycin (Rapamycin) and autophagy inhibitor 3-methyladenine (3-methyladenine3-MA). ELISA was used to detect TNF- 伪, ICAM-1 (intercellular adhesion molecule) and Griess Reagen were used to detect the production and release of NO (nitric oxide). The transcription level of p62 was detected by RT-PCR method. SiRNA interference technique was used to construct the cell model of knockdown autophagy protein p62, and ELISA was used to detect the level of inflammatory factor release. Results: in the first part, the inflammatory model of Raw264.7 macrophages induced by LPS was successfully established. The results of cellular immunofluorescence showed that the aggregation of autophagy specific protein (LC3) in the macrophage was increased after 12 h of LPS stimulation. The same, Western blo results showed that the expression of autophagy related protein LC3,p62 and Beclinl was increased at different time points (2 h, 4 h, 8 h, 12 h and 24 h) compared with the control group, and the effect was time-dependent (P0.05). After removing LPS, the expression of autophagy related protein did not decrease (P0.05). The second part: after pretreatment with autophagy inducer Rapamycin, the expression of autophagy related protein was increased, and the release of TNF- 伪 (ICAM-1,NO) was also increased (P0.05). On the contrary, autophagy inhibitor 3-MA inhibited the expression of autophagy associated protein and down-regulated the level of inflammatory factor (P0.05). RT-PCR results showed that the expression of p62mRNA was consistent with the trend of protein level after intervention. Using siRNA to knock down p62 protein and reduce the release of inflammatory factors (P0.05), it is suggested that p62 plays an important role in the inflammatory response mediated by Raw264.7 macrophages induced by LPS. Conclusion: autophagy is involved in the inflammatory reaction of macrophages activated by LPS. Under autophagy stress, autophagy inducers promoted the aggregation of autophagy related proteins, increased the transcription level of p62 protein, and upregulated the production and release of inflammatory factors. In contrast, autophagy inhibitors inhibited the production of autophagy associated proteins, p62 transcription and inflammatory factors, and siRNA knocked down p62, which reduced the release of inflammatory factors. These results suggest that p62 protein plays an important role in Raw264.7 macrophage mediated inflammatory response induced by LPS in autophagy stress response.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2426705
[Abstract]:Aim: to investigate the role of autophagy in lipopolysaccharide (lipopolysaccharide LPS) -induced RAW264.7 macrophage mediated inflammation and its mechanism. Methods: in the first part, Raw264.7 macrophage was used as the research object, and the inflammatory model was established by LPS. Western blot method was used to detect the expression of TNF- 伪 (tumor necrosis factor-a, and the immunofluorescence method was used to observe the fluorescence aggregation of LC3. The expression levels of LC3 (microtubule-associated protein light chain3), p62 and Beclinl were measured by Western blot. The second part: the expression of autophagy related protein was detected by, Western blot method of macrophage induced by LPS pretreated with autophagy inducer rapamycin (Rapamycin) and autophagy inhibitor 3-methyladenine (3-methyladenine3-MA). ELISA was used to detect TNF- 伪, ICAM-1 (intercellular adhesion molecule) and Griess Reagen were used to detect the production and release of NO (nitric oxide). The transcription level of p62 was detected by RT-PCR method. SiRNA interference technique was used to construct the cell model of knockdown autophagy protein p62, and ELISA was used to detect the level of inflammatory factor release. Results: in the first part, the inflammatory model of Raw264.7 macrophages induced by LPS was successfully established. The results of cellular immunofluorescence showed that the aggregation of autophagy specific protein (LC3) in the macrophage was increased after 12 h of LPS stimulation. The same, Western blo results showed that the expression of autophagy related protein LC3,p62 and Beclinl was increased at different time points (2 h, 4 h, 8 h, 12 h and 24 h) compared with the control group, and the effect was time-dependent (P0.05). After removing LPS, the expression of autophagy related protein did not decrease (P0.05). The second part: after pretreatment with autophagy inducer Rapamycin, the expression of autophagy related protein was increased, and the release of TNF- 伪 (ICAM-1,NO) was also increased (P0.05). On the contrary, autophagy inhibitor 3-MA inhibited the expression of autophagy associated protein and down-regulated the level of inflammatory factor (P0.05). RT-PCR results showed that the expression of p62mRNA was consistent with the trend of protein level after intervention. Using siRNA to knock down p62 protein and reduce the release of inflammatory factors (P0.05), it is suggested that p62 plays an important role in the inflammatory response mediated by Raw264.7 macrophages induced by LPS. Conclusion: autophagy is involved in the inflammatory reaction of macrophages activated by LPS. Under autophagy stress, autophagy inducers promoted the aggregation of autophagy related proteins, increased the transcription level of p62 protein, and upregulated the production and release of inflammatory factors. In contrast, autophagy inhibitors inhibited the production of autophagy associated proteins, p62 transcription and inflammatory factors, and siRNA knocked down p62, which reduced the release of inflammatory factors. These results suggest that p62 protein plays an important role in Raw264.7 macrophage mediated inflammatory response induced by LPS in autophagy stress response.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
【参考文献】
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1 石际俊;刘康永;杨亚萍;刘春风;;自噬在帕金森病发病中的作用[J];生理科学进展;2009年01期
，本文编号：2426705
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