内质网应激对非酒精性脂肪肝的影响及机制研究
发布时间：2018-10-05 21:14
【摘要】：目的:1.通过建立非酒精性脂肪肝(NAFLD)体内外模型,探讨内质网应激(ERS)对NAFLD的影响及机制。2.以雷公藤红素干预NAFLD细胞模型,探讨雷公藤红素对NAFLD的防治作用,以及对其ERS的影响及可能机制。方法:1.NAFLD体内外模型的建立与鉴定:20只SD雄性大鼠以基础饲料适应性饲养1周后,随机分为对照组(Control)和非酒精性脂肪肝组(NAFLD),分别予以基础饲料及高脂饲料喂养。LO2细胞分为control组和NAFLD组,分别用基础培养基和含0.2mmol/L软脂酸培养基培养。HE染色观察肝组织的形态学改变;油红O染色分别观察肝组织和LO2细胞脂肪蓄积情况;胆固醇(TC)和甘油三酯(TG)试剂盒分别检测肝组织和LO2细胞内TC、TG含量。2.免疫组织化学观测肝组织GRP78蛋白的表达水平;PCR和Western-blot分别检测NAFLD细胞模型中ERS相关信号分子ATF6、GRP78、IRE1、SCAP、SREBP-1c和SREBP-2的mRNA及蛋白表达水平。3.雷公藤红素干预实验:将L02细胞分为control组、NAFLD组、雷公藤红素低剂量组(Cel 0.5)、雷公藤红素高剂量组(Cel 1)和辛伐他汀组(SIMVA)。油红O染色观察雷公藤红素干预后LO2细胞内脂质沉积情况;TC和TG试剂盒分别检测LO2细胞内TC、TG含量;PCR和Western-blot分别检测雷公藤红素作用下LO2细胞中ERS相关信号分子ATF6、GRP78、IRE1、SCAP、SREBP-1c和SREBP-2的mRNA以及蛋白表达水平。结果:1.NAFLD体内外模型的建立与鉴定:he染色显示control组大鼠肝小叶结构正常;而nafld组大鼠肝细胞排列紊乱,出现大量脂滴空泡。肝组织和lo2细胞油红o染色均显示control组红染颗粒少,肝细胞结构正常,且排列整齐;而nafld组肝细胞内含有大量红染颗粒且发生脂肪变性。control组肝组织和lo2细胞内的tc和tg含量均低于nafld组(p0.05)。2.免疫组化结果显示nafld组大鼠肝组织grp78蛋白表达水平高于control组(p0.05);pcr和western-blot结果显示nafld组lo2细胞内upr相关信号分子atf6,grp78和ire1的mrna和蛋白表达水平均高于control组(p0.05);nafld组lo2细胞内固醇调节级联反应相关信号分子scap,srebp-1c和srebp-2的mrna和蛋白表达水平均高于control组(p0.05)。3.雷公藤红素干预实验:油红o染色显示nafld组lo2细胞内含有大量红染颗粒,而cel0.5μg/ml组、cel1μg/ml组及simva组红染颗粒较nafld组有不同程度减少。nafld组lo2细胞tc及tg含量均高于control组(p0.05),cel0.5μg/ml组、cel1μg/ml组及simva组lo2细胞tc及tg含量均较nafld组有不同程度减少(p0.05)。pcr和western-blot结果显示:(1)nafld组lo2细胞内upr相关信号分子atf6,grp78和ire1的mrna和蛋白表达水平均高于control组(p0.05);cel1μg/ml组和simva组lo2细胞内atf6,grp78和ire1的mrna和蛋白表达水平均低于nafld组(p0.05)。(2)nafld组lo2细胞内固醇调节级联反应相关信号分子scap,srebp-1c和srebp-2的mrna和蛋白表达水平均高于control组(p0.05);cel0.5μg/ml、cel1μg/ml组和simva组lo2细胞内scap,srebp-1c和SREBP-2的mRNA和蛋白表达水平均低于NAFLD组(P0.05);Cel 1μg/ml组和SIMVA组LO2细胞内SREBP-1c和SREBP-2的mRNA和蛋白表达水平均低于Cel 0.5μg/ml组(P0.05)。结论:1.ERS密切参与了NAFLD的脂变过程,在其发生发展起着重要作用。2.ERS中的UPR和固醇调节级联反应,及相关的IRE1/XBP-1通路和ATF6通路参与了NAFLD的发生发展。3.雷公藤红素能减轻肝细胞脂代谢紊乱,改善NAFLD,其机制可能与缓解肝细胞的ERS有关。
[Abstract]:Purpose: 1. Objective To study the effect and mechanism of ER on NAFLD by establishing an external model of non-alcoholic fatty liver (NAFLD). In this paper, the effects of tripterine on NAFLD and its mechanism were discussed. Methods: 1. The establishment and identification of external models in NAFLD were randomly divided into control group and non-alcoholic fatty liver group (NAFLD) after 1 week feeding on basic feed. LO2 cells were divided into control group and NAFLD group, and cultured with basal medium and 0. 2mmol/ L palmitic acid medium respectively. The morphological changes of liver tissue were observed by HE staining; the fat accumulation of liver tissue and LO2 cells were observed by oil-red O staining; TC and TG content in liver tissues and LO2 cells were detected by cholesterol (TC) and triglyceride (TG) kits respectively. The mRNA and protein expression levels of ERS-related signal molecules ATF6, GRP78, IRE1, SCAP, SREBP-1c and SREBP-2 in NAFLD cell model were detected by immunohistochemistry and Western-blot. The results showed that L02 cells were divided into control group, NAFLD group, triptolide low dose group (Cel 0.5), tripterine high dose group (Cel 1) and simvastatin group (SIMVA). The contents of TC and TG in LO2 cells were detected by TC and TG kit respectively, and the ERS-related signal molecules ATF6, GRP78, IRE1 and SCAP in LO2 cells were detected by PCR and Western-blot respectively. mRNA and protein expression levels of SREBP-1c and SREBP-2. Results: 1. The establishment and identification of the outer model in Nafld rats showed that the hepatic lobules were normal in control group. The liver tissue and lo2 cell oil red-o staining showed that the control group had fewer red-stained particles, the structure of hepatocytes was normal, and the arrangement was orderly; and the nafld group hepatocytes contained a large number of red-stained particles and fatty degeneration occurred. The content of tc and DDT in liver tissue and lo2 cells in control group was lower than that of the nafld group (P0.05). The expression level of p78 protein in liver tissue of nafld group was higher than that in control group (P0.05). The mrna and protein expression levels of scap, srebp-1c, and srebp-2 were all higher than that of control group (p0.05). The results showed that there was a large number of red-stained particles in the lo2 cells of the nafld group, while the red-dyed particles of the group were decreased in the group of 0. 5 ug/ ml, 1.mu g/ ml and simva group than in the nafld group. The results showed that: (1) the upr-related signaling molecules in the nafld group of lo2 cells were higher than that of the control group (P0.05), the content of the lo2 cells in the nafld group was higher than that of the control group (P0.05), the content of the lo2 cells in the group of 1.mu. g/ ml and the siva group were reduced to a different extent (P0.05). The expression levels of mrna and protein in p78 and re1 were higher than those in control group (p0.05), and the expression levels of mrna and protein were lower in 1ug/ ml group and simva group, respectively, and the expression level of mrna and protein was lower than that of the nafld group (p0.05). (2) The mRNA and protein expression levels of scap, srebp-1c and srebp-2 in the nafld group lo2 cells were higher than those of control group (P0.05); The mRNA and protein expression levels of SREBP-1c and SREBP-2 in the Cel 1 ug/ ml group and the SIMVA group were lower than that of Cel 0.5ug/ ml group (P0.05). Conclusion: 1. ERS is closely involved in the fat-change process of NAFLD and plays an important role in the development of NAFLD. Triptolide can alleviate hepatocyte lipid metabolism disorder and improve NAFLD, and its mechanism may be related to the response of liver cells to ERS.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
本文编号：2254884
[Abstract]:Purpose: 1. Objective To study the effect and mechanism of ER on NAFLD by establishing an external model of non-alcoholic fatty liver (NAFLD). In this paper, the effects of tripterine on NAFLD and its mechanism were discussed. Methods: 1. The establishment and identification of external models in NAFLD were randomly divided into control group and non-alcoholic fatty liver group (NAFLD) after 1 week feeding on basic feed. LO2 cells were divided into control group and NAFLD group, and cultured with basal medium and 0. 2mmol/ L palmitic acid medium respectively. The morphological changes of liver tissue were observed by HE staining; the fat accumulation of liver tissue and LO2 cells were observed by oil-red O staining; TC and TG content in liver tissues and LO2 cells were detected by cholesterol (TC) and triglyceride (TG) kits respectively. The mRNA and protein expression levels of ERS-related signal molecules ATF6, GRP78, IRE1, SCAP, SREBP-1c and SREBP-2 in NAFLD cell model were detected by immunohistochemistry and Western-blot. The results showed that L02 cells were divided into control group, NAFLD group, triptolide low dose group (Cel 0.5), tripterine high dose group (Cel 1) and simvastatin group (SIMVA). The contents of TC and TG in LO2 cells were detected by TC and TG kit respectively, and the ERS-related signal molecules ATF6, GRP78, IRE1 and SCAP in LO2 cells were detected by PCR and Western-blot respectively. mRNA and protein expression levels of SREBP-1c and SREBP-2. Results: 1. The establishment and identification of the outer model in Nafld rats showed that the hepatic lobules were normal in control group. The liver tissue and lo2 cell oil red-o staining showed that the control group had fewer red-stained particles, the structure of hepatocytes was normal, and the arrangement was orderly; and the nafld group hepatocytes contained a large number of red-stained particles and fatty degeneration occurred. The content of tc and DDT in liver tissue and lo2 cells in control group was lower than that of the nafld group (P0.05). The expression level of p78 protein in liver tissue of nafld group was higher than that in control group (P0.05). The mrna and protein expression levels of scap, srebp-1c, and srebp-2 were all higher than that of control group (p0.05). The results showed that there was a large number of red-stained particles in the lo2 cells of the nafld group, while the red-dyed particles of the group were decreased in the group of 0. 5 ug/ ml, 1.mu g/ ml and simva group than in the nafld group. The results showed that: (1) the upr-related signaling molecules in the nafld group of lo2 cells were higher than that of the control group (P0.05), the content of the lo2 cells in the nafld group was higher than that of the control group (P0.05), the content of the lo2 cells in the group of 1.mu. g/ ml and the siva group were reduced to a different extent (P0.05). The expression levels of mrna and protein in p78 and re1 were higher than those in control group (p0.05), and the expression levels of mrna and protein were lower in 1ug/ ml group and simva group, respectively, and the expression level of mrna and protein was lower than that of the nafld group (p0.05). (2) The mRNA and protein expression levels of scap, srebp-1c and srebp-2 in the nafld group lo2 cells were higher than those of control group (P0.05); The mRNA and protein expression levels of SREBP-1c and SREBP-2 in the Cel 1 ug/ ml group and the SIMVA group were lower than that of Cel 0.5ug/ ml group (P0.05). Conclusion: 1. ERS is closely involved in the fat-change process of NAFLD and plays an important role in the development of NAFLD. Triptolide can alleviate hepatocyte lipid metabolism disorder and improve NAFLD, and its mechanism may be related to the response of liver cells to ERS.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
【参考文献】
相关期刊论文 前10条
1 孟冉;阮国良;杨代勤;;内质网应激与脂类代谢[J];生命科学;2014年10期
2 田琳;刘宏;谢友红;;炎症通过SCAP-SREBP2-LDLr途径促进四氯化碳诱导的小鼠脂肪变肝细胞胆固醇内流[J];第三军医大学学报;2014年11期
3 张亚辉;周伏喜;卢放根;;大黄素对大鼠非酒精性脂肪肝及其糖脂代谢紊乱的防治作用[J];海南医学;2013年05期
4 刘江;李伟平;施杰民;戴利成;刘春燕;丁健;唐涛;;内质网应激参与细胞脂肪变性的研究[J];医学研究杂志;2012年12期
5 孙文静;熊吉;陈东风;;内质网-线粒体对话在非酒精性脂肪性肝病发病机制中的作用[J];中华肝脏病杂志;2011年09期
6 郭晓鹤;李贞娟;张彩凤;韩宇;;肿瘤坏死因子-α与非酒精性脂肪肝的关系[J];胃肠病学和肝病学杂志;2011年07期
7 李秀丽;王蒙;;非酒精性脂肪肝病发病机制的研究进展[J];时珍国医国药;2010年10期
8 陈鹏;杨成明;曾春雨;王旭开;熊秀勤;何多芬;;心肌梗死后心力衰竭小鼠心肌组织内质网应激相关凋亡途径的研究[J];中国病理生理杂志;2010年06期
9 沈红;柳涛;张莉;郑培永;季光;邢练军;;非酒精性脂肪肝细胞损伤敏感性的机制[J];世界华人消化杂志;2010年07期
10 严茂祥;陈芝芸;何蓓晖;;山楂叶总黄酮对非酒精性脂肪性肝炎大鼠肝组织NF-κB及其抑制物表达的影响[J];中华中医药杂志;2009年02期
相关硕士学位论文 前2条
1 万颖;内质网应激通过SREBP-1c-FAS途径影响肝细胞脂质沉积[D];重庆医科大学;2013年
2 王万东;内质网应激合并固醇调控在非酒精性脂肪性肝病中的意义[D];第三军医大学;2011年
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