肝脏胆汁酸代谢在Roux-en-Y胃旁路术治疗2型糖尿病中的作用及机制研究

发布时间：2018-01-27 01:25

本文关键词： 胆汁酸 Roux-en-Y胃旁路术法尼醇X受体肥胖糖尿病 HepG2细胞　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着近年经济发展,人们生活水平逐渐提高,糖尿病患者增长极为迅速,在过去20年糖尿病患者人数增长了一倍,已成为严重的公共卫生问题,其中,2型糖尿病(type 2diabetesmellitus,T2DM)患者数量增长惊人,尤其是肥胖合并T2DM患者,然而目前以内科治疗为主的综合治疗,虽然能够短期内控制血糖,但由于需终生服药以及部分患者依从性差,长期血糖控制的效果并不理想。因此如何更好地治疗T2DM是现今医学界最重要的议题之一。从90年代开始,减重手术逐渐成为了治疗T2DM的有效手段,其中,Roux-en-Y胃旁路术(Roux-en-Y gastric bypass,RYGB)表现出了良好的治疗效果和较少的并发症。近年来研究发现,RYGB可明显改善糖代谢和脂代谢,这可能与胆汁酸通路相关,但其具体机制仍不明确。本研究通过建立肥胖合并T2DM大鼠模型并给予大鼠RYGB的方法,探讨RYGB对大鼠肝糖代谢以及肝脏胆汁酸通路的影响,并进一步验证FXR通路改变对HepG2细胞肝糖异生关键酶的影响,从而为RYGB治疗肥胖合并T2DM提供理论依据。材料与方法1.主要材料:健康雄性SD大鼠,共40只;HepG2细胞。2.方法:(1)将SD大鼠随机分为正常对照组、高脂饮食组、假手术组、手术组(每组10只)。定期测量大鼠体重、进食量和空腹血糖;检测术前、术后血清胆固醇及甘油三酯水平;术前、术后行腹腔糖耐量试验(intraperitioneal glucose tolerance test,IPGTT)和胰岛素耐量试验(insulin tolerance test,ITT)评价大鼠糖耐量及胰岛素敏感性,并采用Real-time PCR检测法尼醇X受体(farnesoidX receptor,FXR)、小异二聚体伴侣(small heterodimeric partner,SHP)、胆盐输出泵(bile salt export pump,BSEP)、磷酸烯醇式丙酮酸羧基酶(phosphoenolpyruvate carboxykinase,PEPCK)、葡萄糖-6-磷酸酶(glucose-6-phosphatase,G6Pase)、葡萄糖转运蛋2(Glucose transporter2,GLUT2)、成纤维细胞生长因子 21(Fibroblast growth factor21,FGF21)mRNA 表达水平,采用 Western blot 检测 FXR、PEPCK、G6Pase和FGF21的蛋白表达水平;大鼠肝脏切片予油红-0染色观察组织学变化。(2)HepG2 细胞分别予以 FXR 激动剂(GW4064,5μmol/L)、FXR 抑制剂(Guggulsterone,5μmol/L)处理不同时间(Omin、30min、2h、4h、6h、12h、24h)。并采用 Rea1-time PCR 和 Western blot 检测 FXR、SHP、BSEP、PEPCK、G6Pase mRNA 和 FXR、SHP、PEPCK、G6Pase蛋白表达水平。实验结果1.术前高脂饮食组、假手术组和手术组体重、进食量和空腹血糖无明显差异,术后假手术组体重和进食量下降,至第4周逐渐恢复正常,与高脂饮食组相近。术后手术组体重、进食量和空腹血糖较术前下降,显著低于其他各组。2.正常对照组的IPGTT和ITT曲线下面积明显优于其他各组,术前高脂饮食组、假手术组和手术组的IPGTT和ITT曲线下面积没有明显差别,术后手术组IPGTT和ITT曲线下面积优于高脂饮食组和假手术组,而高脂饮食组和假手术组无明显差别。3.手术组血清中的胆固醇和甘油三酯明显低于高脂饮食组和假手术组。油红-0染色可见高脂饮食组和假手术组红色脂滴较多,正常对照组和手术组较少。4.手术组大鼠肝脏FXR、SHP、BSEP、GLUT2和FGF21的mRNA表达水平和FXR、FGF21得蛋白水平均高于高脂饮食组和假手术组,而PEPCK、G6PasemRNA表达水平和蛋白水平则低于高脂饮食组和假手术组。5.采用FXR激动剂GW4064处理HepG2细胞,FXR、SHP、BSEPmRNA表达水平和FXR、SHP蛋白水平升高,PEPCK、G6Pase mRNA表达水平和蛋白水平升高降低,提示糖异生明显减弱;而采用Guggulsterone抑制FXR通路后,HepG2细胞糖异生明显增强。结论1.RYGB显著降低肥胖合并T2DM大鼠的体重及摄食量,改善糖脂代谢紊乱及肝脏脂肪变性。2.RYGB可通过激活胆汁酸-FXR-SHP通路,改善肝糖异生,维持葡萄糖稳态,从而发挥其治疗肥胖合并糖尿病的作用。3.FXR通路参与HepG2细胞糖异生代谢的调控。
[Abstract]:In recent years, with the development of economy, people's living standards gradually improved, the growth of diabetic patients is very fast in the past 20 years, the number of patients with diabetes has doubled, has become a public health problem, serious among type 2 diabetes patients (type 2diabetesmellitus, T2DM) the number of amazing growth, especially in obese patients with T2DM, however the medical treatment of comprehensive treatment, although the short-term glycemic control, but because of the need for lifelong medication, some patients with poor compliance, long-term glycemic control effect is not ideal. Therefore, how to better treatment of T2DM is one of the most important issues of the present medical science. From the beginning of 90s, bariatric surgery has become an effective means of treatment. The T2DM, Roux-en-Y gastric bypass (Roux-en-Y gastric bypass, RYGB) showed a good therapeutic effect and less complications. In recent years, the study found that RYGB Can significantly improve the glucose metabolism and lipid metabolism, which may be associated with bile acid pathway, but the mechanism is still unclear. This study through the establishment of obesity T2DM rats and model rats were given RYGB, to discuss the effects of RYGB on glucose metabolism in rats and effects of hepatic bile acid pathway, and further validation of the FXR pathway. Effect on HepG2 cell Glyconeogenesis key enzyme, which provides a theoretical basis for the treatment of obesity with RYGB T2DM. Materials and methods of 1. main materials: healthy male SD rats, a total of 40; HepG2 cell.2. method: (1) SD rats were randomly divided into normal control group, high fat diet group, sham operation group, operation group (n = 10). Regular measurement of body weight of rats, food intake and fasting blood glucose; detection of preoperative, postoperative serum cholesterol and triglyceride levels; preoperative, postoperative intraperitoneal glucose tolerance test (intraperitioneal glucose tolerance test, and IPGTT) Insulin tolerance test (insulin tolerance, test, ITT) and the insulin sensitivity evaluation of glucose tolerance in rats, using Real-time and PCR detection of farnesoid X receptor (farnesoidX, receptor, FXR), two small heterodimer partner (small heterodimeric, partner, SHP), bile salt export pump (bile salt export pump, BSEP), phosphoric acid phosphoenolpyruvate carboxylase (phosphoenolpyruvate carboxykinase, PEPCK), glucose -6- phosphatase (glucose-6-phosphatase, G6Pase), glucose transporter 2 (Glucose transporter2, GLUT2 protein), fibroblast growth factor 21 (Fibroblast growth, factor21, FGF21) mRNA expression by Western blot detection of FXR, PEPCK, G6Pase and FGF21 protein expression rat; liver biopsy for oil red -0 staining to observe the histological changes. (2) HepG2 cells which were treated with FXR agonist (GW4064,5 mol/L), FXR inhibitor (Guggulsterone, 5 mol/L). Different time (Omin, 30min, 2h, 4h, 6h, 12h, 24h). And the Rea1-time PCR and Western blot SHP, BSEP detection of FXR, PEPCK, G6Pase, mRNA and FXR, SHP, PEPCK, G6Pase protein expression levels. The experimental results of 1. preoperative high fat diet group, sham operation group and operation group weight, food intake and fasting blood glucose had no significant difference between the sham operation group, the body weight and food intake decreased to fourth weeks after operation, gradually returned to normal, similar to the high fat diet group. Postoperative surgery group weight, food intake and fasting blood glucose decreased, significantly lower than those in other groups.2. normal control group IPGTT and the area under the ITT curve is better than that of other groups, preoperative high fat diet group, there was no significant difference between the area of sham operation group and operation group of IPGTT and ITT under the curve, the operation group IPGTT and the area under the ITT curve is better than the high fat diet group and sham operation group and high-fat diet group and sham operation group significant difference The.3. group the serum cholesterol and triglycerides were significantly lower than the high-fat diet group and sham operation group. Oil red -0 staining showed a high fat diet group and sham operation group of red lipid droplets were the normal control group and operation group were less.4. group rat liver FXR, SHP, BSEP, GLUT2 and FGF21 mRNA expression and FXR, FGF21 protein levels were higher than the high fat diet group and sham operation group, and PEPCK, G6PasemRNA expression and protein level were lower than the high fat diet group and sham operation group.5. with FXR agonist GW4064 treatment of HepG2 cells, FXR, SHP, the expression level of BSEPmRNA and FXR, increased the protein level of SHP PEPCK. Increased G6Pase mRNA expression and protein level decreased, suggesting that gluconeogenesis is weakened; while using Guggulsterone to inhibit the FXR pathway, HepG2 cells significantly enhanced gluconeogenesis. Conclusion 1.RYGB significantly reduced obesity T2DM rats weight and food intake, improve Glucose and lipid metabolism and fatty degeneration of the liver bile acid.2.RYGB can activate -FXR-SHP pathway, improve hepatic gluconeogenesis, glucose homeostasis, and thus play its regulatory role in treatment of obesity and diabetes.3.FXR pathway involved in gluconeogenesis in HepG2 cells.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R656.6

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