發(fā)布時間：2018-01-24 14:08

  本文關(guān)鍵詞： 胰高血糖素樣肽-1 糖尿病 心肌 自噬　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:比較胰高血糖素樣肽-1(GLP-1)受體激動劑利拉魯肽(Liraglutide)干預(yù)前后糖尿病大鼠心肌自噬標(biāo)記分子LC3BmRNA表達及其蛋白含量,光鏡下觀察心肌脂肪沉積及纖維結(jié)構(gòu)的改變,探討GLP-1對糖尿病大鼠心肌自噬調(diào)節(jié)的機制。阻斷自噬通路后,繼續(xù)觀察利拉魯肽對糖尿病大鼠心肌脂質(zhì)沉積及纖維結(jié)構(gòu)的影響,進一步驗證GLP-1是通過調(diào)節(jié)自噬而發(fā)揮對糖尿病心肌細胞的保護作用。方法:1、研究設(shè)計:應(yīng)用高糖高脂飲食聯(lián)合小劑量鏈脲佐菌素建立糖尿病大鼠模型,成模后予以利拉魯肽干預(yù)4周,觀察大鼠一般特征,檢測內(nèi)臟脂肪相對含量、血糖、血脂,測定自噬標(biāo)志物L(fēng)C3BmRNA表達及其蛋白含量,光鏡下觀察大鼠心肌脂肪沉積及纖維結(jié)構(gòu)的改變,對自噬水平與脂質(zhì)沉積程度進行相關(guān)性分析,并與未干預(yù)組相比較。應(yīng)用氯喹抑制自噬體與溶酶體結(jié)合阻斷自噬通路后,再比較各組大鼠一般特征、內(nèi)臟脂肪含量、血糖、血脂、LC3BmRNA表達及其蛋白含量、心肌脂質(zhì)沉積和纖維結(jié)構(gòu),并與對照組和未阻斷組相比較。2、實驗方法(1)糖尿病大鼠模型制備:36只4~5周齡的健康雄性Wistar大鼠,體重180~200g購于北京動物飼養(yǎng)中心,每籠6只,適應(yīng)性喂養(yǎng)一周后,采用簡單隨機抽樣法分為非造模組(10只)和造模組(26只)。對照組以基礎(chǔ)飼料喂養(yǎng),每克飼料提供熱量13.4k J,其中脂肪占10.2%,蛋白質(zhì)23.3%,碳水化合物66.5%;造模組以高糖高脂飼料喂養(yǎng),每克飼料提供熱量21.8k J,脂肪占56.0%,蛋白質(zhì)7%,碳水化合物37.0%。早晚各喂養(yǎng)1次,自由飲水。飼養(yǎng)環(huán)境:光照12小時,室溫18~22℃,相對濕度30%~70%。每2周測大鼠體重、空腹血糖(FBG)。12周時,非造模組大鼠FBG為4.4~4.9 mmol/L,造模組大鼠FBG為5.3~5.9mmol/L,隔夜禁食12h后,造模組大鼠按25mg/Kg腹腔注射鏈脲佐菌素溶液,非造模組大鼠腹腔注射等體積檸檬酸-檸檬酸鈉緩沖液。72h后鼠尾采血測定兩組大鼠FBG,以FBG≥7.8mmol/L并持續(xù)一周以上為糖尿病造模成功,其中造模組成模25只,死亡1只。非造模組大鼠FBG均在正常范圍內(nèi)。(2)大鼠分組及檢測指標(biāo):非造模組大鼠設(shè)為正常對照組(NC組n=10),繼續(xù)予基礎(chǔ)飼料喂養(yǎng)。成模的25只糖尿病大鼠采用簡單隨機抽樣法分為:糖尿病對照組(DMNS組,n=9)、利拉魯肽干預(yù)組(LIR組,n=8)及利拉魯肽+自噬抑制劑氯喹組(LIR+CQ組,n=8),三組繼續(xù)予高糖高脂飼料喂養(yǎng)。LIR組以100ug/Kg每天兩次腹部皮下注射利拉魯肽溶液,LIR+CQ組以100ug/Kg每天兩次腹部皮下注射利拉魯肽溶液的同時以50mg/Kg每兩天一次腹腔注射氯喹,DMNS組、NC組腹部注射等體積生理鹽水。每周六測定各組大鼠體重及FBG。上述藥物干預(yù)大鼠4周后,隔夜禁食12h,測定體重,鼠尾采血測定FBG、50%葡萄糖注射液灌胃(4ml/kg)測定餐后2小時血糖(2h BG)。次日空腹腹腔注射20%烏拉坦溶液(4ml/kg)麻醉大鼠,開腹后經(jīng)腹主動脈取血,迅速離心后測定甘油三酯(TG)、總膽固醇(TC)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL-C)。采血后迅速分離心臟,PBS液沖洗后,切取三塊約100mg的心臟組織,錫箔紙包裹放于凍存管,迅速投入液氮罐中保存,待行實時熒光定量PCR測定LC3BmRNA水平;取左心室部分組織浸泡于10%甲醛液,固定48h后石蠟包埋,備做病理切片,用于免疫組化測定LC3B蛋白的表達及光學(xué)顯微鏡下觀察心臟脂肪沉積及心肌纖維情況。分離內(nèi)臟脂肪(睪丸脂肪墊、雙側(cè)腎周脂肪、腹膜處及腸管間脂肪),稱重,并計算內(nèi)臟脂肪與體重的比值即內(nèi)臟脂肪相對含量(VF/W)。(3)統(tǒng)計學(xué)方法:所有計量資料以均數(shù)±標(biāo)準(zhǔn)差表示,運用SPSS17.0統(tǒng)計軟件分析,組間比較采用單因素方差分析(oneway-ANOVA)、LSD-t檢驗,兩變量相關(guān)性采用Pearson相關(guān)分析,以P≤0.05為差異有統(tǒng)計學(xué)意義。結(jié)果:1.各組大鼠一般特征比較:適應(yīng)性喂養(yǎng)期間,大鼠飲食量、體重?zé)o明顯差異,精神狀態(tài)良好,毛色光澤。造模期間,造模組大鼠較非造模組大鼠飲食量、體重均增加。造模成功后,藥物干預(yù)期間,NC組大鼠飲食量較之前無變化,體重呈穩(wěn)定增長,精神狀態(tài)良好,毛色光澤;DMNS組大鼠較同期NC組大鼠體重增加,少動;LIR組大鼠較同期NC組大鼠飲食量及體重?zé)o差異,較同期DMNS組大鼠飲食量、體重均下降,精神狀態(tài)好轉(zhuǎn);LIR+CQ組大鼠精神萎靡,體毛蓬松失去光澤,有脫毛現(xiàn)象,較同期NC組大鼠飲食量、體重增加,與同期DMNS組大鼠比較,飲食量、體重?zé)o差別。與NC組比較,DMNS組、LIR+CQ組大鼠內(nèi)臟脂肪相對含量升高,差異有統(tǒng)計學(xué)意義(P0.05),LIR組內(nèi)臟脂肪相對含量差異無統(tǒng)計學(xué)意義(P0.05)。與DMNS組比較,LIR+CQ組大鼠內(nèi)臟脂肪相對含量差異無統(tǒng)計學(xué)意義(P0.05),LIR組內(nèi)臟脂肪相對含量降低,差異有統(tǒng)計學(xué)意義(P0.05)。2.各組大鼠生化指標(biāo)比較:與NC組比較,DMNS組、LIR+CQ組大鼠FBG、2h BG、TG、TC、LDL-C升高,HDL降低,差異均有統(tǒng)計學(xué)意義(P0.05);LIR組上述指標(biāo)差異均無統(tǒng)計學(xué)意義(P0.05)。與DMNS組比較,LIR組大鼠FBG、2h BG、TG、TC、LDL-C降低,HDL升高,差異均有統(tǒng)計學(xué)意義(P㩳0.05);LIR+CQ組上述指標(biāo)差異無統(tǒng)計學(xué)意義(P0.05)。與LIR組比較,LIR+CQ組FBG、2h BG、TG、TC、LDL-C升高,HDL降低,差異有統(tǒng)計學(xué)意義(P㩳0.05)3.各組大鼠心肌自噬水平及脂肪沉積比較:與NC組比較,DMNS組、LIR+CQ組大鼠LC3B蛋白及mRNA表達降低,泡沫細胞/總心肌細胞數(shù)比值升高,差異有統(tǒng)計學(xué)意義(P㩳0.05),LIR組大鼠LC3B蛋白及mRNA表達差異無統(tǒng)計學(xué)意義(P0.05),泡沫細胞/總心肌細胞數(shù)差異無統(tǒng)計學(xué)意義(P0.05)。與DMNS組比較,LIR組大鼠LC3B蛋白及mRNA表達升高、泡沫細胞/總心肌細胞數(shù)下降,差異有統(tǒng)計學(xué)意義(P㩳0.05),LIR+CQ組大鼠LC3B蛋白及mRNA表達、泡沫細胞/總心肌細胞數(shù)差異無統(tǒng)計學(xué)意義(P0.05)。4.心肌自噬水平與脂質(zhì)沉積相關(guān)性分析:對泡沫細胞/總心肌細胞數(shù)比值與LC3BmRNA及其蛋白含量作Pearson相關(guān)性分析,結(jié)果提示心肌LC3BmRNA表達、LC3B蛋白含量與泡沫細胞/總心肌細胞數(shù)比值呈顯著負相關(guān)(P㩳0.05)。5.各組大鼠心肌病理學(xué)比較:NC組大鼠心肌纖維排列整齊,細胞結(jié)構(gòu)完整,染色均勻,細胞核呈圓形或橢圓形居中,未見泡沫細胞。DMNS組大鼠心肌滿布泡沫細胞,細胞核被擠向細胞一側(cè),心肌纖維排列紊亂、斷裂。LIR組大鼠心肌細胞結(jié)構(gòu)恢復(fù),細胞內(nèi)未見明顯核固縮、核溶解。LIR組與DMNS組比較,心肌泡沫細胞數(shù)量減少,心肌纖維排列較整齊;與NC組比較,心肌泡沫細胞數(shù)量增多,心肌纖維排列無明顯變化。LIR+CQ組與NC組比較,心肌泡沫細胞數(shù)量增多,肌纖維斷裂、排列紊亂;與DMNS組比較,心肌泡沫細胞數(shù)量及心肌纖維結(jié)構(gòu)無明顯差異。結(jié)論:1、糖尿病階段存在心肌自噬減弱。2、GLP-1減輕糖尿病大鼠糖脂代謝紊亂。3、GLP-1通過增強自噬減輕糖尿病心肌脂質(zhì)沉積和纖維結(jié)構(gòu)紊亂。
[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，

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