胰高血糖素样肽-1对糖尿病大鼠心脏自噬调节机制的研究
发布时间：2018-01-24 14:08
本文关键词： 胰高血糖素样肽-1 糖尿病 心肌 自噬 出处：《山西医科大学》2017年硕士论文 论文类型：学位论文
【摘要】：目的:比较胰高血糖素样肽-1(GLP-1)受体激动剂利拉鲁肽(Liraglutide)干预前后糖尿病大鼠心肌自噬标记分子LC3BmRNA表达及其蛋白含量,光镜下观察心肌脂肪沉积及纤维结构的改变,探讨GLP-1对糖尿病大鼠心肌自噬调节的机制。阻断自噬通路后,继续观察利拉鲁肽对糖尿病大鼠心肌脂质沉积及纤维结构的影响,进一步验证GLP-1是通过调节自噬而发挥对糖尿病心肌细胞的保护作用。方法:1、研究设计:应用高糖高脂饮食联合小剂量链脲佐菌素建立糖尿病大鼠模型,成模后予以利拉鲁肽干预4周,观察大鼠一般特征,检测内脏脂肪相对含量、血糖、血脂,测定自噬标志物LC3BmRNA表达及其蛋白含量,光镜下观察大鼠心肌脂肪沉积及纤维结构的改变,对自噬水平与脂质沉积程度进行相关性分析,并与未干预组相比较。应用氯喹抑制自噬体与溶酶体结合阻断自噬通路后,再比较各组大鼠一般特征、内脏脂肪含量、血糖、血脂、LC3BmRNA表达及其蛋白含量、心肌脂质沉积和纤维结构,并与对照组和未阻断组相比较。2、实验方法(1)糖尿病大鼠模型制备:36只4~5周龄的健康雄性Wistar大鼠,体重180~200g购于北京动物饲养中心,每笼6只,适应性喂养一周后,采用简单随机抽样法分为非造模组(10只)和造模组(26只)。对照组以基础饲料喂养,每克饲料提供热量13.4k J,其中脂肪占10.2%,蛋白质23.3%,碳水化合物66.5%;造模组以高糖高脂饲料喂养,每克饲料提供热量21.8k J,脂肪占56.0%,蛋白质7%,碳水化合物37.0%。早晚各喂养1次,自由饮水。饲养环境:光照12小时,室温18~22℃,相对湿度30%~70%。每2周测大鼠体重、空腹血糖(FBG)。12周时,非造模组大鼠FBG为4.4~4.9 mmol/L,造模组大鼠FBG为5.3~5.9mmol/L,隔夜禁食12h后,造模组大鼠按25mg/Kg腹腔注射链脲佐菌素溶液,非造模组大鼠腹腔注射等体积柠檬酸-柠檬酸钠缓冲液。72h后鼠尾采血测定两组大鼠FBG,以FBG≥7.8mmol/L并持续一周以上为糖尿病造模成功,其中造模组成模25只,死亡1只。非造模组大鼠FBG均在正常范围内。(2)大鼠分组及检测指标:非造模组大鼠设为正常对照组(NC组n=10),继续予基础饲料喂养。成模的25只糖尿病大鼠采用简单随机抽样法分为:糖尿病对照组(DMNS组,n=9)、利拉鲁肽干预组(LIR组,n=8)及利拉鲁肽+自噬抑制剂氯喹组(LIR+CQ组,n=8),三组继续予高糖高脂饲料喂养。LIR组以100ug/Kg每天两次腹部皮下注射利拉鲁肽溶液,LIR+CQ组以100ug/Kg每天两次腹部皮下注射利拉鲁肽溶液的同时以50mg/Kg每两天一次腹腔注射氯喹,DMNS组、NC组腹部注射等体积生理盐水。每周六测定各组大鼠体重及FBG。上述药物干预大鼠4周后,隔夜禁食12h,测定体重,鼠尾采血测定FBG、50%葡萄糖注射液灌胃(4ml/kg)测定餐后2小时血糖(2h BG)。次日空腹腹腔注射20%乌拉坦溶液(4ml/kg)麻醉大鼠,开腹后经腹主动脉取血,迅速离心后测定甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL-C)。采血后迅速分离心脏,PBS液冲洗后,切取三块约100mg的心脏组织,锡箔纸包裹放于冻存管,迅速投入液氮罐中保存,待行实时荧光定量PCR测定LC3BmRNA水平;取左心室部分组织浸泡于10%甲醛液,固定48h后石蜡包埋,备做病理切片,用于免疫组化测定LC3B蛋白的表达及光学显微镜下观察心脏脂肪沉积及心肌纤维情况。分离内脏脂肪(睾丸脂肪垫、双侧肾周脂肪、腹膜处及肠管间脂肪),称重,并计算内脏脂肪与体重的比值即内脏脂肪相对含量(VF/W)。(3)统计学方法:所有计量资料以均数±标准差表示,运用SPSS17.0统计软件分析,组间比较采用单因素方差分析(oneway-ANOVA)、LSD-t检验,两变量相关性采用Pearson相关分析,以P≤0.05为差异有统计学意义。结果:1.各组大鼠一般特征比较:适应性喂养期间,大鼠饮食量、体重无明显差异,精神状态良好,毛色光泽。造模期间,造模组大鼠较非造模组大鼠饮食量、体重均增加。造模成功后,药物干预期间,NC组大鼠饮食量较之前无变化,体重呈稳定增长,精神状态良好,毛色光泽;DMNS组大鼠较同期NC组大鼠体重增加,少动;LIR组大鼠较同期NC组大鼠饮食量及体重无差异,较同期DMNS组大鼠饮食量、体重均下降,精神状态好转;LIR+CQ组大鼠精神萎靡,体毛蓬松失去光泽,有脱毛现象,较同期NC组大鼠饮食量、体重增加,与同期DMNS组大鼠比较,饮食量、体重无差别。与NC组比较,DMNS组、LIR+CQ组大鼠内脏脂肪相对含量升高,差异有统计学意义(P0.05),LIR组内脏脂肪相对含量差异无统计学意义(P0.05)。与DMNS组比较,LIR+CQ组大鼠内脏脂肪相对含量差异无统计学意义(P0.05),LIR组内脏脂肪相对含量降低,差异有统计学意义(P0.05)。2.各组大鼠生化指标比较:与NC组比较,DMNS组、LIR+CQ组大鼠FBG、2h BG、TG、TC、LDL-C升高,HDL降低,差异均有统计学意义(P0.05);LIR组上述指标差异均无统计学意义(P0.05)。与DMNS组比较,LIR组大鼠FBG、2h BG、TG、TC、LDL-C降低,HDL升高,差异均有统计学意义(P㩳0.05);LIR+CQ组上述指标差异无统计学意义(P0.05)。与LIR组比较,LIR+CQ组FBG、2h BG、TG、TC、LDL-C升高,HDL降低,差异有统计学意义(P㩳0.05)3.各组大鼠心肌自噬水平及脂肪沉积比较:与NC组比较,DMNS组、LIR+CQ组大鼠LC3B蛋白及mRNA表达降低,泡沫细胞/总心肌细胞数比值升高,差异有统计学意义(P㩳0.05),LIR组大鼠LC3B蛋白及mRNA表达差异无统计学意义(P0.05),泡沫细胞/总心肌细胞数差异无统计学意义(P0.05)。与DMNS组比较,LIR组大鼠LC3B蛋白及mRNA表达升高、泡沫细胞/总心肌细胞数下降,差异有统计学意义(P㩳0.05),LIR+CQ组大鼠LC3B蛋白及mRNA表达、泡沫细胞/总心肌细胞数差异无统计学意义(P0.05)。4.心肌自噬水平与脂质沉积相关性分析:对泡沫细胞/总心肌细胞数比值与LC3BmRNA及其蛋白含量作Pearson相关性分析,结果提示心肌LC3BmRNA表达、LC3B蛋白含量与泡沫细胞/总心肌细胞数比值呈显著负相关(P㩳0.05)。5.各组大鼠心肌病理学比较:NC组大鼠心肌纤维排列整齐,细胞结构完整,染色均匀,细胞核呈圆形或椭圆形居中,未见泡沫细胞。DMNS组大鼠心肌满布泡沫细胞,细胞核被挤向细胞一侧,心肌纤维排列紊乱、断裂。LIR组大鼠心肌细胞结构恢复,细胞内未见明显核固缩、核溶解。LIR组与DMNS组比较,心肌泡沫细胞数量减少,心肌纤维排列较整齐;与NC组比较,心肌泡沫细胞数量增多,心肌纤维排列无明显变化。LIR+CQ组与NC组比较,心肌泡沫细胞数量增多,肌纤维断裂、排列紊乱;与DMNS组比较,心肌泡沫细胞数量及心肌纤维结构无明显差异。结论:1、糖尿病阶段存在心肌自噬减弱。2、GLP-1减轻糖尿病大鼠糖脂代谢紊乱。3、GLP-1通过增强自噬减轻糖尿病心肌脂质沉积和纤维结构紊乱。
[Abstract]:Objective: To compare the effects of glucagon like peptide -1 (GLP-1) receptor agonist liraglutide (Liraglutide) before and after the intervention of myocardial expression of autophagy marker LC3BmRNA in diabetic rats and its protein content, myocardial fat deposition and fibrous structure changes under the light microscope, to explore the mechanism of regulation of GLP-1 on myocardial autophagy in diabetic rats. Blocking the autophagy pathway after, continue to observe the effects of liraglutide on diabetic rat myocardial lipid deposition and fiber structure, we proved that GLP-1 exerts a protective effect on diabetic myocardial cells by regulating autophagy. Methods: 1. Study design: application of high glucose and high fat diet combined with small dose of streptozotocin diabetic rat model, model after be liraglutide 4 weeks, observe the general characteristics of rats, to detect the content of visceral fat, blood glucose, blood lipids, determination of autophagy marker LC3BmRNA expression and protein content, light To observe the changes of myocardial fat deposition and fiber structure of rats under microscope, analyzed the correlation of autophagy level and the degree of lipid deposition, and compared with the untreated group. Application of chloroquine inhibition of autophagosomes and lysosomes with blocking the autophagy pathway, and then compare the general characteristics of the rats, visceral fat content, blood glucose, blood lipid, LC3BmRNA expression and the content of protein, myocardial lipid deposition and fiber structure, and compared with the control group and non blocking group compared to.2, experimental methods (1) the model of diabetic rats by 36 4~5 week old healthy male Wistar rats, weight 180~200g was purchased from Beijing animal breeding center, 6 in each cage, a week after adaptive feeding, divided into the model group by simple random sampling method (10 rats) and model group (26 rats). The control group was given basic feed, feed per gram of 13.4k J to provide heat, which accounted for 10.2% of fat, 23.3% protein, 66.5% carbohydrate; The model by high-fat diet, per gram of feed to provide heat 21.8k J, 56% fat, 7% protein and carbohydrate 37.0%. each morning and evening feeding 1 times, free drinking water. The breeding environment: 12 hours of light, the room temperature is 18~22 DEG C, relative humidity of 30%~70%. every 2 weeks, the body weight, fasting blood glucose (FBG).12 at 12 weeks, the rats in non FBG 4.4~4.9 mmol/L, the rats in FBG 5.3~5.9mmol/L, 12h after overnight fasting, the rats in 25mg/Kg by intraperitoneal injection of streptozotocin solution, non made intraperitoneal injection in the rat model and the volume of citric acid sodium citrate buffer.72h after rat tail blood determination two rats in group FBG, FBG = 7.8mmol/L and lasted for more than a week for a successful diabetes model, which consists of 25 modular modeling, 1 rats died. The rats in non FBG were within the normal range. (2) the grouping and detection index of rats: model group rats as normal control group (group NC, n= 10), continue to basal diet. All 25 rats using simple random sampling method is divided into: diabetic control group (group DMNS, n=9), liraglutide group (LIR group, n=8) and liraglutide + chloroquine group (group LIR+CQ, n=8), the three Group continued given a high-fat diet for group.LIR to 100ug/Kg two times daily subcutaneous injection of liraglutide solution, group LIR+CQ with 100ug/Kg two times daily subcutaneous injection of liraglutide solution at the same time to 50mg/Kg every two days by intraperitoneal injection of chloroquine, DMNS group, NC group, abdominal injection volume of saline were determined every Saturday. The body weight and FBG. of the drug intervention in rats after 4 weeks, overnight fasting 12h, body weight, tail blood was 50% FBG, Glucose Injection (4ml/kg) by gavage 2 hour postprandial blood glucose (2H BG). Fasting intraperitoneal injection of 20% urethane solution (4ml/kg) anesthesia rats, open After abdominal aortic blood, rapid determination of triglycerides after centrifugation (TG), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL-C). After the rapid separation of heart blood, PBS after washing liquid, cut three pieces of approximately 100mg of cardiac tissue, the foil wrapped put in the freezing tube, quickly put in lines to be preserved in liquid nitrogen tank, real-time fluorescence quantitative PCR determination of LC3BmRNA level; the left ventricular tissue soaked in 10% formalin fixed, paraffin embedded 48h, prepared for pathological observation for cardiac fat deposition and myocardial fiber were determined by immunohistochemistry. The expression of LC3B protein and optical microscope. The separation of visceral fat (testis fat pad, bilateral perirenal fat, peritoneal and intestinal fat), weighing, and calculated the ratio of visceral fat and body weight or the content of visceral fat (VF/W). (3) statistical methods: all measurement data were expressed as the mean + SD. The use of SPSS17.0 statistical software, analysis, comparison between groups using single factor analysis of variance (oneway-ANOVA), LSD-t test, two variable correlation analysis using Pearson correlation with P lower than 0.05 for the difference was statistically significant. Results: compared with the general characteristics of the 1. rats in each group: adaptive feeding period, the rats diet, there were no significant differences in weight a good mental state, hair color, luster. During the modeling, the rats in model group rats compared with non food intake, body weight increased. After the modeling, drug intervention period, the rats in group NC diet than before no changes in body weight showed a steady growth, a good mental state, hair color gloss; DMNS rats compared with NC rats weight gain less; rats in the LIR group compared with the same period there was no difference between group NC rats food intake and body weight, compared with group DMNS rats food intake, body weight decreased, improving the mental state; LIR+CQ rats, listlessness, fluffy hair loss of light 娉，
本文编号：1460230
本文链接：https://www.wllwen.com/yixuelunwen/mazuiyixuelunwen/1460230.html
最近更新
教材专著
