mTORC1在慢性肾脏病胰岛素抵抗中的作用

发布时间：2018-04-11 06:12

本文选题：慢性肾脏病 + 胰岛素抵抗　；参考：《中国人民解放军医学院》2017年硕士论文

【摘要】：目的:代谢综合征(MetS)在慢性肾脏病(CKD)患者中普遍存在,与CKD预后密切相关。胰岛素抵抗(IR)作为MetS的基础,是非糖尿病CKD患者疾病进展的独立危险因素。而哺乳动物雷帕霉素靶蛋白复合物1 (mTORC1)信号通路可感受多种环境变化调节机体的生长代谢稳态,参与肿瘤、肥胖、2型糖尿病等多种疾病发生发展过程。过量的氨基酸、葡萄糖等引起mTORC1信号通路的过度激活,通过负反馈作用,抑制胰岛素信号通路,被认为是2型糖尿病IR的重要机制之一。但在CKD疾病状态下,mTORC1表达与IR的关系尚不十分清楚。因此,本实验旨在研究慢性肾脏病动物模型即5/6肾切除大鼠,发生IR状态下,mTORC1信号通路在脂肪、肌肉、肝脏组织中的变化,及其血清刺激肌肉细胞、脂肪细胞所产生的IR及与mTORC1的关系。初步探讨CKD疾病状态下mTORC1与IR的关系。方法:1、建立慢性肾脏疾病大鼠模型,即进行5/6肾切除手术(CKD组),即先进行左肾的2/3切除,一周以后摘除右肾,对照组同期行肾脏被膜剥离手术,以二次手术后时间为起始点,饲养20周;2、禁食水12小时后行腹腔注射糖耐量试验(IPGTT),即按2g/Kg葡萄糖行腹腔葡萄糖注射,分别测0分、30分、60分、90分、120分血糖值,并通过曲线下面积统计手术组与对照组差异;继续饲养2天后,麻醉下下腔静脉取血处死,取腓肠肌组织、附睾脂肪组织、肝脏组织液氮冰冻,-80℃保存;3、检测血清生化及24h尿变化,ELISA法检测空腹胰岛素,稳态模式评估法(HOMA-IR)检测胰岛素敏感性,Western blotting方法检测手术组与对照组脂肪、肌肉、肝脏组织中胰岛素信号通路蛋白p-y-IRS1、p-AKT及mTORC1信号通路关键因子p-mTOR、p-p70s6k表达差异;4、采用C2C12细胞模型及3T3L1细胞模型,分别诱导为成熟的肌肉细胞及脂肪细胞,通过CKD模型鼠血清刺激,观察胰岛素及mTORC1信号通路变化,并采用mTORC1激活剂磷脂酸(PA)刺激,上调mTORC1,观察胰岛素信号通路变化。结果:1、CKD 组血肌酐(93.63±8.05)、尿素氮(15.81 ±2.07)、尿酸(90.33±3.26)、胆固醇(2.73±0.29),尿蛋白(45.32±11.68)明显高于对照组血肌酐(35.10±4.07)、尿素氮(5.55±0.35)、尿酸(77.27±4.34)、胆固醇(1.56±0.07),尿蛋白(11.17±4.06),p0.05;白蛋白(31.69±1.19)低于对照组白蛋白(36.57±0.55),p0.05; 2、IPGTT显示CKD组糖耐量明显低于对照组,p0.05; HOMA-IR显示CKD组HOMA-IR(4.45±0.73)明显高于对照组(1.87±0.54), p0.05,即CKD组胰岛素敏感性明显低于对照组;3、CKD组脂肪组织、肌肉组织胰岛素信号通路蛋白p-y-IRS1、p-AKT及mTORC1信号通路蛋白p-mTOR、p-p70s6k明显低于对照组,p0.05;肝脏p-AKT及p-mTOR未见明显差异;4、CKD组大鼠血清体外刺激肌细胞及脂肪细胞导致胰岛素信号通路蛋白表达下降,伴随mTORC1信号通路蛋白表达下降,通过上调mTORC1信号通路,体外肌细胞细胞胰岛素抵抗有所改善,对脂肪细胞影响不大。结论:1、CKD大鼠IR状态下肌肉、脂肪组织发生胰岛素抵抗伴mTORC1下调。2、体外CKD大鼠血清刺激肌细胞、脂肪细胞可产生IR。3、体外激活mTORC1可改善CKD大鼠血清刺激肌细胞产生的IR,但没有对脂肪细胞产生明显影响。
[Abstract]:Objective: metabolic syndrome (MetS) in patients with chronic kidney disease (CKD) are common in patients is closely related with the prognosis of CKD. Insulin resistance (IR) as the basis of MetS are independent risk factors for disease progression in CKD patients with diabetes. And mTOR complex 1 (mTORC1) growth and metabolic homeostasis, signal pathway can experience a variety of environmental changes to regulate the body involved in tumor development process, obesity, type 2 diabetes and other diseases. Excess amino acids, glucose caused by excessive activation of the mTORC1 signaling pathway, through the negative feedback function, inhibition of insulin signaling, is considered to be one of the important mechanisms of IR of type 2 diabetes. But in CKD disease under the condition of relationship between the expression of mTORC1 and IR is not clear. Therefore, the purpose of this experiment was to study the chronic kidney disease animal model of 5/6 nephrectomy rats, IR state, mTORC1 signaling pathway in fat Fat, muscle, changes in liver tissue, and serum stimulated muscle cells produced by fat cells and the relationship between mTORC1 and IR. To investigate the relationship between mTORC1 and IR CKD disease. Methods: 1 to establish a rat model of chronic kidney disease, namely 5/6 nephrectomy (group CKD), i.e. the first 2/3 of left kidney resection, a week after the removal of the right kidney, the control group underwent renal capsule stripping operation in time after the two operation as a starting point for 20 weeks; 2, fasting 12 hours after intraperitoneal glucose tolerance test (IPGTT), which is based on 2g/Kg intraperitoneal glucose glucose injection, measured 0 points, 30 points, 60 points, 90 points, 120 points of blood glucose, and the area under the curve of statistical operation group compared with control group; after 2 days, blood from the inferior vena cava were sacrificed under anesthesia, gastrocnemius muscle tissue, adipose tissue, liver tissue frozen in liquid nitrogen, -80 C 3, detection of blood preservation; Clear and 24h urine biochemical changes, fasting insulin ELISA, homeostasis model assessment of insulin sensitivity (HOMA-IR) detection method for the detection of blotting, Western group and the control group of fat, muscle, insulin signaling protein p-y-IRS1 in liver tissue, p-AKT and mTORC1 signal pathway key factor p-mTOR, the difference of p-p70s6k expression; 4, by C2C12 cells model and 3T3L1 cell model were induced into mature muscle cells and fat cells in CKD rats by serum stimulation, insulin and mTORC1 signaling pathway changes, and the use of mTORC1 activator phosphatidic acid (PA) stimulation, upregulation of mTORC1, observe the changes of insulin signaling pathway. Results: 1 CKD group, serum creatinine (93.63. 8.05), urea nitrogen (15.81 + 2.07), uric acid (90.33 + 3.26), cholesterol (2.73 + 0.29), urine protein (45.32 + 11.68) was significantly higher than the control group (35.10 + 4.07) serum creatinine, urea nitrogen (5.55 + 0.35), Uric acid (77.27 + 4.34), cholesterol (1.56 + 0.07), urine protein (11.17 + 4.06), P0.05 (31.69 + 1.19); albumin albumin was lower than that of control group (36.57 + 0.55), P0.05; 2, IPGTT showed significantly lower than the control group, CKD group of glucose tolerance P0.05; group CKD (HOMA-IR HOMA-IR 4.45 + 0.73) was significantly higher than the control group (1.87 + 0.54), P0.05 CKD group, the insulin sensitivity was significantly lower than the control group; 3, fat tissue in group CKD, p-y-IRS1 protein in insulin signaling pathway in muscle tissue, p-AKT and mTORC1 signaling pathway protein p-mTOR, p-p70s6k was significantly lower than the control group, P0.05; p-AKT and p-mTOR had no significant difference in the liver; 4, muscle cells and fat cells led to the expression of insulin signaling proteins decreased stimulation in serum of rats in the CKD group, with the protein expression of mTORC1 signaling pathway decreased by upregulation of mTORC1 signaling pathway, insulin resistance body muscle cells improved to fat cells. Not influence. Conclusion: 1. CKD rats under the condition of IR muscle, adipose tissue insulin resistance associated with down-regulation of mTORC1.2 insulin, CKD in vitro rat serum stimulation of muscle cells, fat cells can produce IR.3 activation in vitro, mTORC1 can improve the serum CKD of rats stimulated muscle cells to produce IR, but had no obvious effect on fat cells.

【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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