同型半胱氨酸通过内质网应激致肝脂质蓄积作用

发布时间：2018-03-27 07:42

本文选题：蛋氨酸　切入点：同型半胱氨酸　出处：《重庆医科大学》2015年硕士论文

【摘要】：背景：同型半胱氨酸(Homocysteine, Hcy)是蛋氨酸代谢途径的中间代谢产物,高同型半胱氨酸血症(Hyperhomocysteinemia, HHcy)与心血管疾病、大脑认知障碍、骨质疏松和非酒精性脂肪肝密切相关。HHcy是动脉粥样硬化的一个独立危险因素,近年来有关此方面的报道已有很多,但Hcy对非酒精性脂肪肝的影响及其分子机制尚不明确。本研究旨在采用蛋氨酸负荷的方法建立高同型半胱氨酸血症动物模型,结合原代肝细胞体外培养实验以探讨Hcy诱发肝脏脂质代谢紊乱的作用及分子机制。方法：选取4周龄健康雄性C57BL/6J小、鼠20只,随机分为对照组(Control)和蛋氨酸组(Methionine),每组各10只；在自由进食和饮水条件下,适应性喂养14天至6周龄时,对照组小鼠喂养标准饲料,蛋氨酸组小鼠喂养在标准饲料基础上添加2%蛋氨酸饲料,持续喂养16周,每周记录体重；对照组和蛋氨酸组动物在22周龄时处死,称取肝脏、附睾周围脂肪重量；收集血液进行生化检测,采用ELISA试剂盒测定血清Hcy水平；采用HE染色观察肝细胞形态、脂质沉积程度；Real-time PCR检测肝脏组织目标基因表达水平；Western Blotting检测肝脏GRP78/BIP、PERK、p-PERK、eIF2a、p-eIF2a和nSREBP1c蛋白表达水平；原代肝细胞用高浓度Hcy、Met、PBA和TM处理后进行油红O染色观察细胞内红染脂质数量；Real-time PCR检测肝细胞目标基因表达水平；Western blotting检测原代肝细胞GRP78/BIP、PERK、 p-PERK、eIF2α、p-eIF2α蛋白表达水平。结果：高蛋氨酸饮食对动物体重增长无显著影响,但肝脏／体重比值和附睾周围脂肪／体重比值均显著高于对照组；肝脏组织切片H.E染色结果显示蛋氨酸组小鼠肝组织出现显著的肝细胞脂肪变性,血清AST、TG水平显著增高,HDL水平显著降低；Real-time PCR结果显示蛋氨酸组小鼠肝脏组织CBS的mRNA表达水平较对照组明显下调；Western blotting结果提示高蛋氨酸饮食组小鼠肝脏GRP78/BIP、活化的p-PERK、活化的p-eIF2a和成熟形式的n-SREBP-1c表达水平增高；油红O染色结果表明原代肝细胞在同型半胱氨酸和衣霉素作用下发生明显脂质蓄积,PBA预处理后再加入同型半胱氨酸干预肝细胞脂质蓄积明显减轻；Real-time PCR结果显示原代肝细胞在同型半胱氨酸和衣霉素作用下GRP78、CHOP、FAS、ACClα和HMG-CoAr的mRNA表达水平较对照组明显上调,PBA预处理后再加入同型半胱氨酸干预肝细胞GRP78、FAS和ACC1α表达水平较同型半胱氨酸组明显下调,但CHOP和HMG-CoA r表达水平与同型半胱氨酸组无明显变化；Western blotting结果提示原代肝细胞在同型半胱氨酸和衣霉素作用下GRP78/BIP、p-PERK和p-eIF2a蛋白表达显著增强,PBA预处理后再加入同型半胱氨酸干预肝细胞上述内质网应激标志物蛋白表达明显减弱。结论：高蛋氨酸饮食可通过升高血液中同型半胱氨酸水平导致肝脏发生脂肪变性。同型半胱氨酸可通过诱导内质网应激激活脂质代谢相关基因的表达进而导致肝脏脂肪变性。同型半胱氨酸水平升高可通过调节肝细胞脂质代谢关键转录因子的活化,从而对肝脏脂质代谢造成影响。
[Abstract]:Background: homocysteine (Homocysteine, Hcy) is the intermediate metabolite methionine metabolism, hyperhomocysteinemia (Hyperhomocysteinemia, HHcy) and cardiovascular disease, cognitive impairment, osteoporosis and nonalcoholic fatty liver is closely related to.HHcy is an independent risk factor for atherosclerosis, in recent years the related reports there are many, but the effect of Hcy on nonalcoholic fatty liver and its molecular mechanism is still unclear. Methods: This study aims to establish the methionine load homocysteine in animal models, combined with in vitro culture of primary hepatocytes to investigate the effects and molecular mechanisms of lipid metabolism disorders induced by Hcy liver. Methods: select healthy 4 week old male C57BL/6J were small, 20 rats were randomly divided into control group (Control) and methionine group (Methionine), 10 rats in each group; and in freedom. Under the condition of drinking water, fed for 14 days to 6 weeks of age, the control group of mice fed the standard diet, mice were fed with 2% methionine methionine feed with standard diet, lasting for 16 weeks, body weight was recorded weekly; the control group and the methionine group animal were sacrificed at 22 weeks old, weighing around liver, epididymal fat weight collect blood; biochemical test, serum Hcy levels were measured by ELISA kit; HE staining was used to observe the morphology of hepatocytes, lipid deposition; detecting liver tissue Real-time expression level of PCR gene; Western Blotting detection PERK, liver GRP78/BIP, p-PERK, eIF2a, p-eIF2a and nSREBP1c protein expression; primary hepatocytes with high concentration Hcy, Met, PBA and TM after treatment were observed in cells stained with oil red O stained red lipid quantity; Real-time PCR detection of liver cell target gene expression level; Western blotting The primary detection of liver cell GRP78/BIP, PERK, p-PERK, eIF2 alpha, the expression level of p-eIF2 protein. Results: high methionine diet had no significant effect on animal growth, but the liver / body weight ratio and epididymis around fat / body weight ratio were significantly higher than control group; liver tissue slices H.E staining showed that methionine group of mice liver tissue there is a significant fatty degeneration of liver cells, serum AST, TG levels were significantly increased, HDL levels decreased significantly; Real-time PCR showed that the expression level of methionine group of mice liver tissue CBS mRNA significantly decreased compared with the control group; Western blotting showed a high methionine diet group mouse liver GRP78/BIP, activation of p-PERK, increased the expression level of p-eIF2a and the activation of the mature form of n-SREBP-1c; oil red O staining results showed that hepatocytes in homocysteine and tunicamycin markedly lipid accumulation PBA, pre treated with homocysteine significantly reduce lipid accumulation in liver cells; Real-time PCR showed GRP78, primary hepatocytes in homocysteine and tunicamycin under the action of CHOP, FAS, the expression level of ACCl alpha and HMG-CoAr mRNA increased significantly compared with the control group, PBA pretreatment and then adding the same type cysteine intervention of liver GRP78 cells, the expression level of FAS and ACC1 alpha is homocysteine group significantly decreased, but CHOP and HMG-CoA expression of R and homocysteine group had no obvious change; Western blotting showed GRP78/BIP hepatocytes in homocysteine and tunicamycin, the expression of p-PERK and p-eIF2a protein significantly enhanced after pretreatment with PBA added homocysteine above liver cell markers of ER stress protein expression significantly decreased. Conclusion: high methionine diet by elevated blood homocysteine Cystine levels lead to liver fatty degeneration. Homocysteine can then lead to hepatic steatosis by expression of genes related to lipid metabolism activation induced by endoplasmic reticulum stress. Elevated homocysteine levels can live through the regulation of lipid metabolism in liver cells of the key transcription factor, resulting in hepatic lipid metabolism.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589

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