心磷脂激活NLRP3炎症小体促进非酒精性脂肪肝的机制研究

发布时间：2018-07-10 05:49

本文选题：非酒精性脂肪肝 + NLRP3　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的构建非酒精性脂肪肝小鼠模型,探讨心磷脂激活NLRP3炎症小体而促进非酒精性脂肪肝病变的机制。方法1.分别使用高脂饲料和低脂饲料喂养C57BL/6J小鼠各10只,16周后取肝脏组织,采用油红O染色、HE染色、免疫组化染色及血清肝功能测定,评估非酒精性脂肪肝模型建立状况。2.分别提取非酒精性脂肪肝模型组和对照组小鼠肝组织的总蛋白,采用Western Blot技术比较两组标本NLRP3的表达水平。3.分离培养C57BL/6J小鼠Kupffer细胞。分别用棕榈酸(模拟体内游离脂肪酸在Kupffer细胞沉积)和生理盐水(对照)刺激两组Kupffe细胞后,采用Western Blot比较两组的NLRP3表达水平;ELISA测定细胞培养液上清液的IL-1β水平;用带His标签的NLRP3过表达质粒转染Kupffer细胞,将细胞培养48h后提取蛋白,用protein-lipid overlay技术检测心磷脂与NLRP3蛋白结合情况。结果1.高脂饮食组较低脂饮食组小鼠的肝脏体积增大、肝细胞脂肪变性显著,Kupffer细胞明显增生,灶性炎症细胞浸润,外周血ALT显著增高(高脂组ALT为134.3U/L,低脂组为26U/L)。2.非酒精性脂肪肝模型组小鼠NLRP3炎症小体表达水平较对照组增高了42.16%。3.棕榈酸刺激的Kupffer细胞上清较对照组Kupffer细胞的上清含有更多的IL-1β,分别是169.3±6.8 pg/ml and134.6±2.4 pg/ml respectively(p0.01)。棕榈酸刺激组NLRP3蛋白表达水平较对照组高103.68%;c-IL-1β蛋白水平也较对照组高38.78%。将Kupffer细胞总蛋白孵育PIP strip膜,再用抗His抗体杂交,含心磷脂的孔出现免疫印迹。结论1.非酒精性脂肪肝模型构建成功。2.NLRP3炎症小体在非酒精性脂肪肝组织中表达上调,提示其可能参与非酒精性脂肪肝的病变进程。3.棕榈酸刺激的Kupffer细胞NLRP3和c-IL-1β表达水平均明显增高。心磷脂能与Kupffer细胞中NLRP3炎症小体结合并将其激活,可能促进非酒精性脂肪肝的病理进程。
[Abstract]:Objective to construct a nonalcoholic fatty liver mouse model and explore the mechanism of cardiolipin activation of NLRP3 inflammatory body to promote nonalcoholic fatty liver disease. Methods 1. 10 C57BL/6J mice were fed with high fat diet and low fat diet respectively. After 16 weeks, liver tissue was taken by oil red O staining, HE staining, immunohistochemical staining and serum liver function test. To evaluate the establishment of non-alcoholic fatty liver model (.2.), the total protein of the liver tissues of the nonalcoholic fatty liver model group and the control group was extracted respectively. The expression level of NLRP3 in the two groups was compared with the C57BL/6J Blot technique by using the Western Blot technique to isolate the C57BL/6J mice Kupffer cells. After two groups of Kupffe cells were stimulated by cell deposition and normal saline (control), the expression level of NLRP3 in two groups was compared with Western Blot; IL-1 beta level in supernatant of cell culture liquid was measured by ELISA; Kupffer cells were transfected with NLRP3 overexpressed plasmid with His label, the cells were cultured for 48H and extracted protein, and the cardiac phosphorus was detected by protein-lipid overlay technique. Results in 1. high fat diet group, the liver volume increased, the liver cell fatty degeneration was significant, the Kupffer cells were obviously proliferated, the focal inflammatory cells were infiltrated, the peripheral blood ALT was significantly increased (high fat group ALT was 134.3U/L, the low fat group was 26U/ L).2. non-alcoholic fatty liver model group mice NLRP3 inflammation small Compared with the control group, the level of 42.16%.3. palmitic acid stimulated Kupffer cell supernatant was more IL-1 beta than that of the control group, which was 169.3 + 6.8 pg/ml and134.6 + 2.4 pg/ml respectively (P0.01). The expression level of NLRP3 protein in the palmitic acid stimulation group was 103.68% higher than that in the control group, and the c-IL-1 beta protein level was also higher than that of the control group. The total protein of Kupffer cell was incubated with the total protein of PIP strip membrane in the group of high 38.78%., and then hybridized with anti His antibody, and the pore of cardiac phospholipid appeared to be immunoblotting. Conclusion 1. non-alcoholic fatty liver model was successfully constructed and the expression of.2.NLRP3 inflammatory body was up-regulated in non-alcoholic fatty liver tissue, suggesting that it could be involved in the process of non-alcoholic fatty liver disease,.3. brown. The expression level of NLRP3 and c-IL-1 beta in Kupffer cells stimulated by acid was significantly increased. Cardiolipin could be combined with NLRP3 inflammatory bodies in Kupffer cells and activated it, which may promote the pathological process of non-alcoholic fatty liver.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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