發(fā)布時(shí)間：2018-01-27 01:25

  本文關(guān)鍵詞： 膽汁酸 Roux-en-Y胃旁路術(shù) 法尼醇X受體 肥胖 糖尿病 HepG2細(xì)胞　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：隨著近年經(jīng)濟(jì)發(fā)展,人們生活水平逐漸提高,糖尿病患者增長極為迅速,在過去20年糖尿病患者人數(shù)增長了一倍,已成為嚴(yán)重的公共衛(wèi)生問題,其中,2型糖尿病(type 2diabetesmellitus,T2DM)患者數(shù)量增長驚人,尤其是肥胖合并T2DM患者,然而目前以內(nèi)科治療為主的綜合治療,雖然能夠短期內(nèi)控制血糖,但由于需終生服藥以及部分患者依從性差,長期血糖控制的效果并不理想。因此如何更好地治療T2DM是現(xiàn)今醫(yī)學(xué)界最重要的議題之一。從90年代開始,減重手術(shù)逐漸成為了治療T2DM的有效手段,其中,Roux-en-Y胃旁路術(shù)(Roux-en-Y gastric bypass,RYGB)表現(xiàn)出了良好的治療效果和較少的并發(fā)癥。近年來研究發(fā)現(xiàn),RYGB可明顯改善糖代謝和脂代謝,這可能與膽汁酸通路相關(guān),但其具體機(jī)制仍不明確。本研究通過建立肥胖合并T2DM大鼠模型并給予大鼠RYGB的方法,探討RYGB對大鼠肝糖代謝以及肝臟膽汁酸通路的影響,并進(jìn)一步驗(yàn)證FXR通路改變對HepG2細(xì)胞肝糖異生關(guān)鍵酶的影響,從而為RYGB治療肥胖合并T2DM提供理論依據(jù)。材料與方法1.主要材料:健康雄性SD大鼠,共40只;HepG2細(xì)胞。2.方法:(1)將SD大鼠隨機(jī)分為正常對照組、高脂飲食組、假手術(shù)組、手術(shù)組(每組10只)。定期測量大鼠體重、進(jìn)食量和空腹血糖;檢測術(shù)前、術(shù)后血清膽固醇及甘油三酯水平;術(shù)前、術(shù)后行腹腔糖耐量試驗(yàn)(intraperitioneal glucose tolerance test,IPGTT)和胰島素耐量試驗(yàn)(insulin tolerance test,ITT)評價(jià)大鼠糖耐量及胰島素敏感性,并采用Real-time PCR檢測法尼醇X受體(farnesoidX receptor,FXR)、小異二聚體伴侶(small heterodimeric partner,SHP)、膽鹽輸出泵(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)、葡萄糖轉(zhuǎn)運(yùn)蛋2(Glucose transporter2,GLUT2)、成纖維細(xì)胞生長因子 21(Fibroblast growth factor21,FGF21)mRNA 表達(dá)水平,采用 Western blot 檢測 FXR、PEPCK、G6Pase和FGF21的蛋白表達(dá)水平;大鼠肝臟切片予油紅-0染色觀察組織學(xué)變化。(2)HepG2 細(xì)胞分別予以 FXR 激動劑(GW4064,5μmol/L)、FXR 抑制劑(Guggulsterone,5μmol/L)處理不同時(shí)間(Omin、30min、2h、4h、6h、12h、24h)。并采用 Rea1-time PCR 和 Western blot 檢測 FXR、SHP、BSEP、PEPCK、G6Pase mRNA 和 FXR、SHP、PEPCK、G6Pase蛋白表達(dá)水平。實(shí)驗(yàn)結(jié)果1.術(shù)前高脂飲食組、假手術(shù)組和手術(shù)組體重、進(jìn)食量和空腹血糖無明顯差異,術(shù)后假手術(shù)組體重和進(jìn)食量下降,至第4周逐漸恢復(fù)正常,與高脂飲食組相近。術(shù)后手術(shù)組體重、進(jìn)食量和空腹血糖較術(shù)前下降,顯著低于其他各組。2.正常對照組的IPGTT和ITT曲線下面積明顯優(yōu)于其他各組,術(shù)前高脂飲食組、假手術(shù)組和手術(shù)組的IPGTT和ITT曲線下面積沒有明顯差別,術(shù)后手術(shù)組IPGTT和ITT曲線下面積優(yōu)于高脂飲食組和假手術(shù)組,而高脂飲食組和假手術(shù)組無明顯差別。3.手術(shù)組血清中的膽固醇和甘油三酯明顯低于高脂飲食組和假手術(shù)組。油紅-0染色可見高脂飲食組和假手術(shù)組紅色脂滴較多,正常對照組和手術(shù)組較少。4.手術(shù)組大鼠肝臟FXR、SHP、BSEP、GLUT2和FGF21的mRNA表達(dá)水平和FXR、FGF21得蛋白水平均高于高脂飲食組和假手術(shù)組,而PEPCK、G6PasemRNA表達(dá)水平和蛋白水平則低于高脂飲食組和假手術(shù)組。5.采用FXR激動劑GW4064處理HepG2細(xì)胞,FXR、SHP、BSEPmRNA表達(dá)水平和FXR、SHP蛋白水平升高,PEPCK、G6Pase mRNA表達(dá)水平和蛋白水平升高降低,提示糖異生明顯減弱;而采用Guggulsterone抑制FXR通路后,HepG2細(xì)胞糖異生明顯增強(qiáng)。結(jié)論1.RYGB顯著降低肥胖合并T2DM大鼠的體重及攝食量,改善糖脂代謝紊亂及肝臟脂肪變性。2.RYGB可通過激活膽汁酸-FXR-SHP通路,改善肝糖異生,維持葡萄糖穩(wěn)態(tài),從而發(fā)揮其治療肥胖合并糖尿病的作用。3.FXR通路參與HepG2細(xì)胞糖異生代謝的調(diào)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1;R656.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;中國2型糖尿病合并肥胖綜合管理專家共識[J];中華內(nèi)分泌代謝雜志;2016年08期

2 Jun Yao;Chun-Suo Zhou;Xiong Ma;Bai-Qing Fu;Li-Sheng Tao;Miao Chen;Ya-Ping Xu;;FXR agonist GW4064 alleviates endotoxin-induced hepatic inflammation by repressing macrophage activation[J];World Journal of Gastroenterology;2014年39期

，

本文編號：1467162