自噬在巨噬細胞介導的炎癥中的作用及其機制研究
發(fā)布時間：2019-02-19 17:28
【摘要】：目的： 研究自噬在脂多糖(lipopolysaccharide LPS)誘導的RAW264.7巨噬細胞介導的炎癥中的作用,并初步探討其相關機制。 方法： 第一部分：以Raw264.7巨噬細胞為研究對象,通過LPS建立細胞炎癥模型。采用Western blot法檢測TNF-α(tumor necrosis factor-a)的表達,檢驗模型是否建立成功；免疫熒光方法觀察LC3的熒光聚集；Western blot法測定不同時間點自噬相關蛋白LC3(microtubule-associated protein light chain3)、p62及Beclinl的表達水平。第二部分：分別采用自噬誘導劑雷帕霉素(Rapamycin)以及自噬抑制劑3-甲基腺嘌呤(3-methyladenine3-MA)預處理LPS誘導的巨噬細胞,Western blot法檢測自噬相關蛋白的表達；ELISA檢測炎癥相關因子TNF-α、ICAM-1(intercellular adhesion molecule)及Griess Reagen檢測NO (nitric oxide)的生成和釋放：采用RT-PCR方法檢測p62轉錄水平變化;利用siRNA干擾技術構建敲減自噬底物蛋白p62的細胞模型,同時ELISA檢測炎癥因子釋放水平。 結果： 第一部分：成功建立LPS誘導Raw264.7巨噬細胞炎癥模型,細胞免疫熒光結果顯示LPS刺激12h后自噬特異性蛋白LC3呈點顆粒狀聚集增多。同樣,Western blo結果顯示,與對照組相比,不同時間點(2h、4h、8h、12h、24h)自噬相關蛋白LC3,p62及Beclinl的表達增加,且此作用呈時間依賴性(P0.05)；去除刺激兇素LPS后,自噬相關蛋白表達并未下降(P0.05)。第二部分：自噬誘導劑Rapamycin預處理后自噬相關蛋白表達增加,炎癥因子(TNF-α、ICAM-1、NO)釋放也增加(P0.05)；相反,自噬抑制劑3-MA抑制了自噬相關蛋白的表達,同時下調了炎癥因子的水平(P0.05)。RT-PCR結果顯示,干預后,p62mRNA表達與蛋白水平趨勢相一致。利用siRNA的方法敲減p62蛋白,炎癥因子釋放減少(P0.05),提示p62在LPS誘導的Raw264.7巨噬細胞介導的炎癥反應中起著重要的作用。 結論： 自噬參與了LPS激活的巨噬細胞炎癥反應。自噬應激狀態(tài)下,自噬誘導劑促進自噬相關蛋白的聚集,p62蛋白轉錄水平上升,同時上調炎癥因子的生成和釋放；相反,自噬抑制劑抑制自噬相關蛋白,p62轉錄及炎癥因子的產生；siRNA敲減p62,炎癥因子釋放減少。這些結果說明,在自噬應激反應中,p62蛋白在LPS誘導的Raw264.7巨噬細胞介導的炎癥反應中起著重要的作用。
[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- 偽 (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- 偽, 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- 偽 (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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363
本文編號：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- 偽 (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- 偽, 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- 偽 (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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363
【參考文獻】
相關期刊論文 前1條
1 石際俊;劉康永;楊亞萍;劉春風;;自噬在帕金森病發(fā)病中的作用[J];生理科學進展;2009年01期
，本文編號：2426705
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