内质网应激、胰岛素抵抗与鹅肥肝形成的关系研究

发布时间：2018-02-26 08:02

本文关键词： 鹅肥肝胰岛素抵抗内质网应激衣霉素葡萄糖耐受试验胰岛素耐受试验　出处：《扬州大学》2016年硕士论文　论文类型：学位论文

【摘要】：非酒精性脂肪肝(Nonalcoholic fatty liver disease, NAFLD)的日渐流行给人类的健康造成了巨大威胁。随着相关研究的增多,对人类及哺乳类模式动物NAFLD形成机制的认识已取得较大的进展。研究表明哺乳动物肝细胞内质网应激(endoplasmic reticulum stress, ERS)对胰岛素抵抗(Insulin resistance, IR)的形成十分重要,而IR可诱发高胰岛素血症,将促进肝脏中的脂肪合成。相对于哺乳动物,作为重要禽产品的鹅肥肝,有关其形成机制的研究和认识还相当匮乏。鹅与哺乳类动物在进化树上存在较大的遗传距离,在营养代谢特别是脂肪代谢方面有些不同的特点。因此,作为哺乳动物NAFLD重要形成机制的ERS和IR是否同样适用于鹅肥肝还不清楚。本试验围绕这一关键问题在细胞与活体两个层面上展开研究：鹅原代肝细胞在高能营养因子(高葡萄糖和高饱和脂肪酸)刺激后,检测ERS标记基因(如Grp78和Xbp1)在这些细胞中的mRNA水平；采用药物和强制饲喂两种方式处理鹅一定时间后,对试验鹅进行血糖、葡萄糖耐受试验、胰岛素耐受试验、剖检以及基因表达分析。主要研究结果如下：1、高能营养因子的刺激可诱导鹅原代肝细胞产生ERS。本研究利用高能营养因子(高葡萄糖和高饱和脂肪酸)处理鹅原代肝细胞,并在此基础上添加不饱和脂肪酸(如油酸、亚油酸),荧光定量PCR检测ERS标记基因-Grp78和Xbp1的mRNA水平。结果显示高葡萄糖和高棕榈酸处理鹅原代肝细胞后,Grp78和Xbp1的mRNA水平都有不同程度的上调,表明高能营养因子能诱导鹅原代肝细胞产生ERS；油酸和亚油酸处理细胞后,棕榈酸所诱导的Grp78和Xbp1的mRNA表达增加受到明显抑制(P0.05)。以上结果说明,高能营养因子可引起鹅原代肝细胞产生ERS,而不饱和脂肪酸可抑制这种诱导。2、药物诱导产生的ERS可引起鹅全身性IR和脂肪肝的形成。本试验选用两种内质网应激诱导剂即衣霉素和毒胡萝卜素(主要作用于肝脏),处理扬州鹅20 d,并对试验鹅进行血糖、葡萄糖耐受试验、剖检分析以及检测Grp78和Xbp1在肝脏中的]mRNA水平。结果显示相对于对照组,Grp78和Xbp1在衣霉素肝脏中的mRNA表达水平极显著上调(P0.01)；20天衣霉素饲喂也引起鹅对葡萄糖耐受能力明显下降,且鹅肝也从深红色转变为脂肪肝的土黄色,这表明ERS可引起鹅产生IR,并促进脂肪肝的形成；毒胡萝卜素注射试验的结果进一步证实衣霉素试验的结论。3、填肥可引起鹅IR和脂肪肝形成,但并不通过ERS的途径。对70日龄朗德鹅进行正常或强制饲喂,在77和84日龄对禁食的试验鹅进行GTT试验,并在85日龄时测定了未禁食试验鹅的血糖水平,在88日龄禁食的试验鹅进行ITT试验以及在强制饲喂一段时间后,屠宰部分试验鹅取肝样用于检测ERS标记基因的mRNA水平。结果表明相对于正常饲养的对照组,填肥没有诱导ERS,可能与脂肪酸去饱和酶的上调有关。不过,填肥鹅在强制饲喂7天后就开始出现葡萄糖耐受能力下降的现象。在14天时,这种现象更加明显。在填肥15天时,非禁食状态下填肥鹅的基础血糖值也极显著高于对照组(P0.01)。在18天时,ITT试验结果进一步说明填肥鹅在脂肪肝形成过程中出现了胰岛素抵抗。这种IR现象和填肥鹅肝脏呈现严重的土黄色脂肪肝密切关联。综上所述,内质网应激可引起鹅IR和脂肪肝,但强制填饲所引起的鹅IR和脂肪肝并不依赖于ERS途径。鹅胰岛素抵抗与脂肪肝的形成密不可分,胰岛素抵抗是脂肪肝形成的重要机制。
[Abstract]:Non alcoholic fatty liver (Nonalcoholic fatty liver disease, NAFLD) is endemic to the human health threat. With the increase of relevant research, understanding the formation mechanism of human and mammalian animal model of NAFLD has made great progress. The research showed that mammalian animal liver cell endoplasmic reticulum stress (endoplasmic reticulum, stress, ERS insulin resistance (Insulin) of resistance, IR) formation is very important, but IR can induce hyperinsulinemia, promote fat synthesis in the liver. Compared with mammals, as an important bird of goose liver products, research and understanding about its mechanisms is still quite scarce. Goose and mammalian animal genetic distance the larger the evolutionary tree in nutrient metabolism especially fat metabolism are different. Therefore, as an important mechanism for the formation of the mammalian NAFLD ERS and IR The same applies to the goose liver is not clear. This experiment focuses on the key issues in two aspects: living cells and goose primary hepatocytes in high nutritional factors (high glucose and high saturated fatty acid) after stimulation, detection of ERS marker genes (Grp78 and Xbp1) in these cells. The mRNA level of processing time; goose using drugs and forced feeding in two ways, the blood sugar test on goose, glucose tolerance test and insulin tolerance test, autopsy and gene expression analysis. The main results are as follows: 1, high-energy nutrition factor stimulation can produce high-energy nutrient factors in this study using ERS. goose primary hepatocytes (induced by high glucose and high saturated fatty acid) in primary processing of goose liver cells, and on the basis of adding unsaturated fatty acids (such as oleic acid, linoleic acid), fluorescence quantitative PCR detection of ERS marker gene -Grp78 and Xbp1 mRNA Ping. The results showed that high glucose and palmitate treatment of goose primary hepatocytes after Xbp1, Grp78 and mRNA levels have different degrees of increase, indicating that high-energy nutritional factors can induce goose primary hepatocytes ERS; oleic acid and linoleic acid treated cells after induced by palmitic acid Grp78 and Xbp1 mRNA expression the increase was significantly inhibited (P0.05). These results indicated that high-energy nutritional factors can cause goose primary hepatocytes to produce ERS, and unsaturated fatty acid can inhibit the.2 induced drug induced ERS can cause the formation of goose fatty liver and systemic IR. This experiment selects two kinds of endoplasmic reticulum stress induced agent tunicamycin and thapsigargin (mainly in liver), Yangzhou goose was 20 D, and blood glucose test on goose, glucose tolerance test, autopsy analysis and detection of Grp78 and Xbp1 in the liver]mRNA level. The results showed that compared with the control group Xbp1, Grp78 and tunicamycin in liver mRNA expression level was significantly increased (P0.01); 20 Tianyi mycin also caused goose feeding glucose tolerance decreased significantly, and also from the deep into the red goose fatty liver khaki, which indicates that ERS can cause the goose to produce IR, and promote the formation of fat liver; thapsigargin injection test results confirmed that tunicamycin test conclusion.3, overfeeding can cause goose IR and fatty liver, but not by ERS way. The normal or forced feeding on 70 day old Landes, at 77 and 84 days of fasting test goose GTT test and determination of blood glucose levels without fasting test geese at the age of 85 days, the ITT test at the age of 88 days of fasting and geese in forced feeding after a period of time, the slaughter of the goose liver like part of the test for the detection of ERS marker gene mRNA level. The results show that the phase for it The control group is raised, overfeeding did not induce ERS may be related to the upregulation of fatty acid desaturase. However, overfeeding geese in forced feeding 7 days began to decline glucose tolerance phenomenon. In 14 days, this phenomenon is more obvious. Fill in fertilizer 15 days, non fasting blood sugar under the condition of filling fat goose value is significantly higher than the control group (P0.01). On the 18 day, the ITT test result shows that fill fat goose appeared in the process of the development of insulin resistance in fatty liver. The IR phenomenon and overfeeding goose liver showed serious yellow fatty liver was closely related. In summary, the endoplasmic reticulum stress can cause the goose IR and fatty liver, but forced feeding caused by goose IR and fatty liver is not dependent on ERS pathway. The formation of goose insulin resistance and fatty liver is closely related to insulin resistance is an important mechanism for the formation of fatty liver.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.33

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