COM晶体刺激巨噬细胞TLR2-NADPH-NLRP3炎症体系的实验研究

发布时间：2018-06-28 15:49

本文选题：巨噬细胞 + TLR2受体　；参考：《广西医科大学》2017年博士论文

【摘要】：第一部分TLR2/NADPH氧化酶通路在COM晶体-巨噬细胞反应中的作用目的:观察COM晶体-巨噬细胞反应中TLRs受体的表达情况,初步探讨TLRs-NADPH通路在COM晶体-巨噬细胞反应中的作用。方法:根据前期实验,选用500ug/ml COM晶体刺激巨噬细胞,利用Real-time PCR法检测细胞TLR2m RNA、TLR4m RNA、p47phoxm RNA与p91phoxm RNA基因相对表达量;利用流式细胞仪检测细胞膜蛋白TLR2表面受体的表达;利用Wester-blot法检测p47phox与p91phox蛋白表达情况;利用荧光酶标仪观察细胞内ROS表达情况。在巨噬细胞与COM晶体培养体系中给予NADPH氧化酶阻断剂Apocynin后,观察细胞内ROS表达情况。给予TLR2受体抑制剂后,利用Wester-blot法检测细胞p47phox蛋白的表达情况。结果:COM晶体刺激巨噬细胞后,TLR2m RNA表达明显升高(*P0.01);TLR4m RNA表达未见明显变化(P0.05);TLR2蛋白表达明显升高(*P0.01);细胞内ROS明显升高(*P0.01)。给予NADPH氧化酶阻断剂Apocynin干预后,细胞内ROS明显降低(*P0.01)。给予TLR2受体抑制剂(FSL-1)后,p47phox蛋白的表达明显降低(P0.05)。结论:巨噬细胞可能通过TLR2受体识别COM晶体或晶体结合的基质,进而激活NADPH氧化酶产生大量ROS。TLR2/NADPH氧化酶通路在草酸钙结石形成中可能发挥了重要作用。第二部分COM晶体通过NADPH氧化酶依赖性ROS生成诱导巨噬细胞NLRP3炎症小体活化的研究目的:观察COM晶体-巨噬细胞反应中NLRP3炎症小体表达情况,探讨NLRP3炎症小体的活化机制。评估阿托伐他汀对COM晶体-巨噬细胞反应中NLRP3炎症小体活化的影响。方法:在巨噬细胞与COM晶体共培养体系中,利用Real-time PCR法检测细胞NLRP3m RNA和Caspase-1m RNA基因相对表达量。利用Wester-blot法检测细胞NLRP3和Caspase-1蛋白的表达。酶联免疫吸附法(ELISA)测定细胞上清液中IL-1β、IL-18的蛋白表达情况。分别用NADPH氧化酶抑制剂(Apocynin)、TLR2受体抑制剂(FSL-1)及药物阿托伐他汀预处理巨噬细胞后,利用Wester-blot法检测细胞NLRP3和Caspase-1蛋白的表达。结果:NLRP3和Caspase-1蛋白水平与m RNA表达量在COM晶体刺激巨噬细胞后均明显升高(*P0.01);细胞上清液中IL-1β、IL-18表达明显升高(*P0.05)。给予NADPH氧化酶抑制剂、TLR2受体阻滞剂或阿托伐他汀干预后,NLRP3及Caspase-1的活性蛋白P20的表达明显降低(*P0.05)。结论:COM晶体刺激巨噬细胞NLRP3炎症小体激活。COM晶体通过NADPH氧化酶依赖性ROS生成诱导巨噬细胞内NLRP3炎症小体活化。阿托伐他汀能够抑制COM晶体诱导巨噬细胞NLRP3炎症小体的激活。第三部分COM晶体刺激巨噬细胞NLRP3-HMGB1炎症信号通路作用的研究目的:观察细胞培养液中HMGB1的表达情况,探讨NLRP3-HMGB1炎症信号通路在晶体-巨噬细胞炎症反应过程中的作用。方法:利用Real-time PCR法检测细胞HMGB1m RNA基因的相对表达量。利用Wester-blot法检测细胞培养液上清中HMGB1蛋白的表达情况。使用RNA干扰技术,将NLRP3-Si RNA转染巨噬细胞后,利用Wester-blot法检测细胞培养液上清中HMGB1蛋白的表达。结果:COM晶体刺激巨噬细胞后,细胞培养液中HMGB1的表达明显升高(*P0.05)。NLRP3-Si RNA转染巨噬细胞后,HMGB1的表达明显降低(*P0.01)。结论:COM晶体刺激巨噬细胞HMGB1的释放,NLRP3-HMGB1炎症信号通路在COM晶体-巨噬细胞炎症反应中发挥了重要作用。
[Abstract]:Part 1: the role of TLR2/NADPH oxidase pathway in the COM crystal macrophage reaction: To observe the expression of TLRs receptor in the COM crystal macrophage reaction, and to explore the role of TLRs-NADPH pathway in the COM crystal macrophage reaction. Method: according to the earlier experiment, 500ug/ml COM crystal was selected to stimulate macrophage, and Re was used in Re. Al-time PCR method was used to detect the relative expression of TLR2m RNA, TLR4m RNA, p47phoxm RNA and p91phoxm RNA gene, and the expression of cell membrane protein TLR2 surface receptor was detected by flow cytometry, and the expression of the protein was detected by Wester-blot method. After giving the NADPH oxidase blocking agent Apocynin in the crystal culture system, the expression of ROS in the cell was observed. After the TLR2 receptor inhibitor was given, the expression of p47phox protein was detected by Wester-blot. The results showed that the RNA expression of TLR2m increased significantly (*P0.01) after the COM crystal stimulated the macrophage (*P0.01), and the expression of TLR4m RNA was not obviously changed. The expression of LR2 protein increased significantly (*P0.01), and the intracellular ROS increased significantly (*P0.01). ROS in cells decreased significantly (*P0.01) after the NADPH oxidase blocker Apocynin. The expression of p47phox protein was significantly reduced after the TLR2 receptor inhibitor (FSL-1). Conclusion: macrophages may identify crystals or crystal binding by the receptor. The matrix, and then activation of NADPH oxidase to produce a large number of ROS.TLR2/NADPH oxidase pathway may play an important role in the formation of calcium oxalate stones. The second part of COM crystal can induce the activation of macrophage NLRP3 inflammatory corpuscle through NADPH oxidase dependent ROS: observe the NLRP3 inflammation in the COM crystal macrophage reaction The effect of atorvastatin on the activation of NLRP3 inflammatory corpuscles in COM crystal macrophage reaction was evaluated by the expression of NLRP3. Methods: in the co culture system of macrophages and COM crystals, the relative expression of NLRP3m RNA and Caspase-1m RNA genes was detected by Real-time PCR method. Wester-blot method was used to detect the relative expression of NLRP3m RNA and Caspase-1m RNA genes. The expression of NLRP3 and Caspase-1 protein in cells was detected. Enzyme linked immunosorbent assay (ELISA) was used to determine the protein expression of IL-1 beta and IL-18 in the supernatant. NADPH oxidase inhibitor (Apocynin), TLR2 receptor inhibitor (FSL-1) and drug atorvastatin were used to pretreat macrophage cells, and NLRP3 and Caspase-1 were detected by Wester-blot. Results: the level of NLRP3 and Caspase-1 protein and the expression of M RNA increased significantly after the COM crystal stimulated macrophages (*P0.01), and the expression of IL-1 beta and IL-18 in the cell supernatant was significantly increased (*P0.05). The prognosis of NADPH oxidase inhibitor, TLR2 receptor blocker or Atorvastatin The expression was significantly reduced (*P0.05). Conclusion: COM crystal stimulated macrophage NLRP3 inflammatory body to activate.COM crystal through NADPH oxidase dependent ROS generation to induce the activation of NLRP3 inflammatory corpuscle in macrophage. Atorvastatin could inhibit the activation of NLRP3 inflammatory corpuscle in macrophage induced by COM crystal. Third part COM stimulates NL macrophage NL. Study on the role of RP3-HMGB1 inflammatory signaling pathway: To observe the expression of HMGB1 in cell culture fluid and to explore the role of NLRP3-HMGB1 inflammatory signaling pathway in the process of inflammatory reaction of crystal macrophage. Methods: the relative expression of HMGB1m RNA gene was detected by Real-time PCR method. Cell culture fluid was detected by Wester-blot method. The expression of HMGB1 protein in the supernatant. After transfecting NLRP3-Si RNA into macrophages using RNA interference technique, the expression of HMGB1 protein in the supernatant of cell culture liquid was detected by Wester-blot. Results: after the stimulation of the macrophage by COM crystal, the expression of HMGB1 in the cell culture fluid increased significantly (*P0.05).NLRP3-Si RNA transfection of macrophage, HMGB1. The expression was significantly reduced (*P0.01). Conclusion: the COM crystal stimulates the release of HMGB1 in macrophages, and the NLRP3-HMGB1 signaling pathway plays an important role in the inflammatory response of COM crystal macrophage.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R692.4

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