FADS1基因在高甘油三酯血症大鼠模型中表达及甲基化状态研究

发布时间：2018-04-20 22:38

本文选题：FADS1 + 高甘油三酯血症　；参考：《华中科技大学》2016年硕士论文

【摘要】：目的:通过观察FADS1在高甘油三酯血症大鼠肝组织、BRL-3A肝细胞中表达水平及其Cp G岛甲基化状态,探讨FADS1在高甘油三酯血症发生、发展中的作用。方法:1、36只雄性SD大鼠,普通饲料适应性喂养1周后随机分为对照组和高血脂组,分别给予普通饲料及高脂饲料喂养,喂养3-5周后测血清中总胆固醇、甘油三酯、低密度脂蛋白和高密度脂蛋白,建立高甘油三酯血症大鼠模型。再选择合适浓度的医用脂肪乳诱导大鼠肝细胞BRL-3A细胞脂变,检测细胞中总胆固醇、甘油三酯含量,建立高脂细胞模型,并利用5-Aza-Cd R处理高脂组细胞。2、利用荧光实时定量PCR检测大鼠肝组织及BRL-3A细胞FADS1 m RNA的表达,酶联免疫吸附试验检测大鼠血清及BRL-3A细胞上清液中FADS1蛋白的表达情况。3、提取大鼠肝脏组织中DNA后,经亚硫酸转化后行PCR扩增目的基因,并用克隆测序检测FADS1基因甲基化水平。4、将含有目的基因的质粒使用甲基化试剂Sss I处理及未处理后,分别转染至BRL-3A细胞中,利用荧光素酶报告基因载体系统明确甲基化对FADS1表达的影响。结果:1、高甘油三酯血症组大鼠血清及高脂BRL-3A细胞中甘油三酯浓度均高于对照组,差异具有统计学意义。2、高甘油三酯血症组大鼠肝组织及高脂BRL-3A细胞中FADS1 m RNA及蛋白表达均低于对照组,而5-Aza-Cd R处理高脂BRL-3A细胞后,FADS1 m RNA及蛋白表达高于高脂组,差异均有统计学意义。3、FADS1启动子区16个甲基化岛共6个检测位点中,高脂组甲基化水平高于对照组,差异均有统计学意义,并且甲基化水平与甘油三酯浓度呈正相关性。4、与甲基化试剂Sss I未处理组相比,处理组荧光酶活性显著降低,差异具有统计学意义,表明FADS1启动子区甲基化抑制了其基因的表达。结论:1、本研究利用高脂饲料喂养雄性SD大鼠,成功建立高甘油三酯血症动物模型;利用医用脂肪乳成功诱导BRL-3A细胞脂变,建立体外模拟高甘油三酯血症肝细胞模型,且浓度为6%的医用脂肪乳为最佳诱导浓度。2、在高甘油三酯血症大鼠肝组织及脂变BRL-3A细胞中,FADS1基因表达量降低,且甲基化水平高于对照组,FADS1基因启动子区甲基化可能参与了高甘油三酯血症发病过程。
[Abstract]:Aim: to investigate the role of FADS1 in the pathogenesis and development of hypertriglyceridemia by observing the expression of FADS1 in the liver tissue of hypertriglyceridemia rats and the methylation status of CP G island. Methods Thirty-six male Sprague-Dawley rats were randomly divided into two groups: control group and hyperlipidemia group after one week of adaptive feeding with normal diet. Serum total cholesterol and triglyceride were measured after 3-5 weeks of feeding. The rat model of hypertriglyceridemia was established by low density lipoprotein (LDL) and high density lipoprotein (HDL). Then select the appropriate concentration of medical fat emulsion to induce rat hepatocyte BRL-3A cell lipid change, detect the total cholesterol, triglyceride content in the cells, establish a hyperlipidemic cell model. The expression of FADS1 m RNA in rat liver tissues and BRL-3A cells was detected by real-time fluorescence quantitative PCR. Enzyme linked immunosorbent assay (Elisa) was used to detect the expression of FADS1 protein in rat serum and supernatant of BRL-3A cells. After extracting DNA from rat liver, the target gene was amplified by PCR after sulfite transformation. The methylation level of FADS1 gene was detected by clone sequencing. The plasmid containing the target gene was treated with methylation reagent Sss I and then transfected into BRL-3A cells. Luciferase reporter gene vector system was used to determine the effect of methylation on FADS1 expression. Results the concentration of triglyceride in serum and hyperlipidemic BRL-3A cells in hypertriglyceridemia group was higher than that in control group. The expression of FADS1 m RNA and protein in hypertriglyceridemia group was lower than that in control group, while the expression of FADS1 m RNA and protein in hypertriglyceridemia group was higher than that in hyperlipidemia group. The methylation level in hyperlipidemia group was higher than that in control group, and the difference was statistically significant. There was a positive correlation between methylation level and triglyceride concentration. Compared with the untreated group of methylation reagent Sss I, the activity of fluorescence enzyme in the treatment group was significantly lower than that in the untreated group, and the difference was statistically significant. The results showed that methylation of FADS1 promoter inhibited its gene expression. Conclusion in this study, hypertriglyceridemia animal model was established by feeding male Sprague-Dawley rats with high fat diet, and hypertriglyceridemia hepatocyte model was established by using medical fat milk to induce lipid change of BRL-3A cells. The optimal induction concentration of medical fat milk was 6%, and the expression of FADS1 gene was decreased in hypertriglyceridemia rats' liver tissue and BRL-3A cells, and the expression of FADS1 gene was decreased in hypertriglyceridemia rats, and the expression of FADS1 gene was decreased in hypertriglyceridemia rats. The methylation level of FADS1 gene promoter was higher than that of control group, which may be involved in the pathogenesis of hypertriglyceridemia.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R589.2;R-332

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