發(fā)布時間：2018-05-08 22:28

  本文選題：糖尿病性心肌病 + 3硝基酪氨酸　； 參考：《南昌大學(xué)》2015年碩士論文

【摘要】：目的:通過腹腔注射鏈脲佐菌素(STZ)的方法建立糖尿病性心肌病(Diabetic cardiomyopathy,DCM)大鼠模型,應(yīng)用氮氧自由基化合物Tempol對DCM大鼠進行干預(yù),觀察DCM大鼠心肌3硝基酪氨酸(3-nitmtynosine,3-NT)及轉(zhuǎn)化因子β1(Transforming growth factor betal-1,TGF-β1)的表達情況,探究Tempol對DCM大鼠的作用及可能機制。方法:1、隨機取42只健康雄性SD大鼠,體重為200-250g,適應(yīng)性喂養(yǎng)1周后隨機取26只分為模型組,按55mg/kg腹腔注射鏈脲佐菌素的方法先建立大鼠糖尿病(Diabetes mellitus,DC)模型。余下16只大鼠分為健康對照組,對照組腹腔注射等劑量檸檬酸-檸檬酸鈉緩沖液。2、4周后隨機將2只造模大鼠處死,取心肌組織進行HE染色及Masson三色染色觀察大鼠心肌結(jié)構(gòu),如果HE染色發(fā)現(xiàn)心肌細胞排列紊亂,心肌出現(xiàn)不同程度的變性、壞死,且Masson染色提示心肌細胞間成纖維細胞、膠原纖維增生,則表明已成功建立DCM動物模型。3、16只健康大鼠及24只造模成功的大鼠再次進行分組。分組及處理方式如下:(1)健康生理鹽水組(NC+NS):8只健康對照組大鼠,常規(guī)飲食,并按18mg/kg/d劑量給予生理鹽水灌胃8周。(2)健康Tempol干預(yù)組(NC+Tempol):另外8只健康對照組大鼠給以常規(guī)飲食,同時給予18mg/kg/d劑量的Tempol進行灌胃干預(yù)8周。(3)DCM模型組(DCM):12只DCM大鼠常規(guī)飲食,給予18mg/kg/d劑量的生理鹽水灌胃8周。(4)DCM模型Tempol干預(yù)組(DCM+Tempol):給以常規(guī)飲食,同時給予18mg/kg/d劑量的Tempol進行灌胃干預(yù)8周。4、8周后采用HE染色及Masson染色分別對實驗SD大鼠心肌進行形態(tài)學(xué)觀察;用酶聯(lián)免疫吸附法(ELISA)檢測實驗SD大鼠血清3-NT及TGF-β1的表達;用免疫組化分析法檢測SD大鼠心肌3-NT及TGF-β1的表達。結(jié)果:1、利用STZ腹腔注射法成功建立了糖尿病性心肌病模型。2、DCM模型組及DCM模型干預(yù)組隨機血糖均顯著高于健康對照組(NC+NS對照組、NC+Tempol對照組),P0.05;DCM模型組及DCM模型干預(yù)組間隨機血糖無明顯差異,P0.05;DCM模型組及DCM模型干預(yù)組體重較健康對照組明顯降低,P0.05;DCM模型干預(yù)組體重下降程度較DCM模型組小,P0.05;健康對照組間血糖及體重均無明顯差異,P0.05。3、顯微鏡下觀察結(jié)果示:HE染色提示健康對照組間心肌細胞排列整齊,無心肌壞死及肌纖維的溶解;DCM模型組心肌細胞排列紊亂,可見肌纖維溶解、斷裂,部分心肌細胞腫脹、壞死;DCM模型干預(yù)組心肌損傷程度較DCM模型組減輕。Masson染色結(jié)果提示健康對照組大鼠心肌細胞及血管周圍僅有少許正常膠原纖維,無間質(zhì)纖維化;DCM模型組、DCM模型干預(yù)組心肌較健康組存在明顯纖維化;DCM模型干預(yù)組心肌纖維化程度比DCM模型組輕。4、4組大鼠血清ELISA結(jié)果提示:NC+NS對照組、NC+Tempol對照組、DCM模型組、DCM模型干預(yù)組血清3-NT濃度分別為:79.11±25.37nmol/L、99.98±24.99nmol/L、371.56±95.64nmol/L、246.31±81.37nmol/L;血清TGF-β1濃度分別為:107.02±30.53pg/ml、136.69±19.68pg/ml、347.07±57.85pg/ml、218.66±26.62pg/ml。DCM模型組與DCM模型干預(yù)組血清3-NT、TGF-β1的表達均明顯高于健康對照組,差異有統(tǒng)計學(xué)意義,P0.05;DCM模型干預(yù)組血清3-NT、TGF-β1的表達水平低于DCM模型組,P0.05;NC+NS對照組與NC+Tempol對照組間血清3-NT、TGF-β1的表達水平均無明顯差異,P0.05。5、4組大鼠心肌組織免疫組化結(jié)果提示:NC+NS對照組、NC+Tempol對照組、DCM模型組、DCM模型干預(yù)組心肌3-NT表達量分別為:2512.38±1636.43、2935.69±1492.17、11684.89±2590.43、5625.41±1501.94;心肌TGF-β1表達量分別為:894.67±152.29、1001.72±215.04、8805.64±2893.75、4707.79±1121.13。DCM模型組與DCM模型干預(yù)組心肌3-NT、TGF-β1表達均明顯高于健康對照組,P0.05;DCM模型干預(yù)組心肌3-NT、TGF-β1的表達水平低于DCM模型組,P0.05;NC+NS對照組與NC+Tempol對照組間心肌3-NT、TGF-β1的表達量均無明顯差異,P0.05。結(jié)論:1、通過腹腔注射STZ法誘導(dǎo)建立DC大鼠模型后,長期的高血糖狀態(tài)可引起大鼠心肌不同程度的損傷。2、DC高糖狀態(tài)下氧化應(yīng)激產(chǎn)生的3-NT及TGF-β1可引起心肌細胞壞死、纖維化,3-NT及TGF-β1可能參與了DCM的發(fā)生發(fā)展。3、2,2,6,6-四甲基-4-哌啶醇(Tempol)對正常大鼠心肌無明顯影響,且Tempol可能通過抑制氧化應(yīng)激反應(yīng),下調(diào)大鼠心肌及血清3-NT、TGF-β1的表達來減輕DCM大鼠心肌的損傷,發(fā)揮對DCM大鼠心肌的保護作用。
[Abstract]:Objective: to establish a rat model of diabetic cardiomyopathy (Diabetic cardiomyopathy (DCM) by intraperitoneal injection of streptozotocin (STZ), and the application of nitrogen oxygen free radical compound Tempol to DCM rats, and to observe the myocardial 3 nitrotyrosine (3-nitmtynosine, 3-NT) and transforming factor beta 1 (Transforming growth factor) in DCM rats. - the expression of - beta 1, explore the effect and possible mechanism of Tempol on DCM rats. Methods: 1, 42 healthy male SD rats were randomly selected and the body weight was 200-250g. After 1 weeks of adaptive feeding, 26 rats were randomly divided into model groups. The rat model of diabetes (Diabetes mellitus, DC) was first established by intraperitoneal injection of streptozotocin in 55mg/kg. The remaining 16 were large. Rats were divided into a healthy control group, and 2 rats were killed at random after.2,4 weeks by intraperitoneal injection of citric acid sodium citrate buffer. Myocardial tissue was stained with HE and Masson trichromatic staining was used to observe the structure of the rat myocardium. If HE staining was used to detect the disorder of cardiac myocytes, the myocardium appeared to varying degrees of degeneration, necrosis and Masson. The staining suggested that the fibroblasts and collagen fibers proliferated in the cardiac myocytes, which showed that the DCM animal model was successfully established in.3,16 rats and 24 successful rats were grouped again. (1) the healthy saline group (NC+NS): 8 healthy control rats, regular diet, and given a dose of 18mg/kg/d. (2) healthy Tempol intervention group (2) the healthy Tempol intervention group (NC+Tempol): the other 8 healthy control rats were given a routine diet, while the 18mg/kg/d dose of Tempol was given for 8 weeks. (3) the DCM model group (DCM): the normal diet of the DCM rats was given for 8 weeks. (4) the DCM model Tempol intervention group (DCM+Tempol) The normal diet was given, and the 18mg/kg/d dose of Tempol was given at the same time for 8 weeks after.4,8 weeks. HE staining and Masson staining were used to observe the myocardial morphology of the experimental SD rats, and the serum 3-NT and TGF- beta 1 of SD rats were detected by enzyme linked immunosorbent assay (ELISA), and the myocardial 3-NT in SD rats was detected by immunohistochemical method. And the expression of TGF- beta 1. Results: 1, diabetic cardiomyopathy model.2 was successfully established by intraperitoneal injection of STZ. The random blood sugar of DCM model group and DCM model intervention group was significantly higher than that of the healthy control group (NC+NS control group, NC+Tempol control group), P0.05, DCM model group and DCM model intervention group, there was no significant difference in blood glucose between the DCM model group and DCM model group, P0.05; DCM model group. The body weight of the DCM model group was significantly lower than that of the healthy control group, P0.05. The weight loss of the DCM model intervention group was smaller than that of the DCM model group, P0.05. There was no significant difference in blood sugar and weight between the healthy control groups, P0.05.3. The observation under microscope showed that the HE staining showed that the myocardial cells in the healthy control group were arranged neatly, no myocardial necrosis and muscle fibers were found. The myocardial cells in the DCM model group were disorganized and the myocytes were disorganized and the muscle cells were dissolved, broken, and some of the cardiomyocytes were swollen and necrotic. The degree of myocardial injury in the DCM model intervention group was less than that of the DCM model group and the.Masson staining results showed that there were only a few normal collagen fibers around the blood vessels in the healthy control group and the peripheral blood vessels, and the DCM model was no interstitial fibrosis; the DCM model was no interstitial fibrosis. The myocardial fibrosis in the DCM model intervention group was more obvious than that in the healthy group, and the serum ELISA results of the myocardial fibrosis in the DCM model group were compared with the DCM model group, and the serum 3-NT concentration in the NC+NS control group, the NC+Tempol control group, the DCM model group and the DCM model intervention group were 79.11 + 25.37nmol/L, 99.98 + 24.99nmol/L, and 371.56 +. Mol/L, 246.31 + 81.37nmol/L, serum TGF- beta 1 concentration was 107.02 + 30.53pg/ml, 136.69 + 19.68pg/ml, 347.07 + 57.85pg/ml, 218.66 + 26.62pg/ml.DCM model group and DCM model intervention group serum 3-NT, the expression of TGF- beta 1 was significantly higher than the healthy control group, the difference was statistically significant, P0.05; DCM model intervention group, the expression of beta 1 The level of serum 3-NT and TGF- beta 1 in the NC+NS control group was lower than that of the DCM model group, and the expression level of TGF- beta 1 was not significantly different. The results of myocardial tissue immunization in the P0.05.5,4 group showed that the myocardial 3-NT expression in the NC+NS control group, the NC+Tempol control group, the DCM model group and the DCM model intervention group were 2512.38 + + 149. 2.1711684.89 + 2590.435625.41 + 1501.94; the expression of TGF- beta 1 in myocardium were respectively: 894.67 + 152.291001.72 + 215.048805.64 + 2893.754707.79 + 1121.13.DCM model group and DCM model intervention group. The expression of TGF- beta 1 was significantly higher than that of the healthy control group, P0.05, and the expression level of beta 1 was lower than that of the model. In group, P0.05, NC+NS control group and NC+Tempol control group, there was no significant difference in the expression of 3-NT and TGF- beta 1. P0.05. conclusion: 1. After intraperitoneal injection of STZ to induce the model of DC rat, long-term hyperglycemia can cause myocardial damage to different degrees in rats, and 3-NT and TGF- beta 1 can cause cardiac arrest under DC high glucose state. Myonecrosis, fibrosis, fibrosis, 3-NT and TGF- beta 1 may be involved in the development of DCM,.3,2,2,6,6- four methyl -4- piperidine (Tempol) has no obvious effect on normal rat myocardium, and Tempol may reduce myocardium and serum 3-NT, TGF- beta 1 to reduce myocardium damage in DCM rats by inhibiting oxidative stress response, and play the heart of DCM rat heart. The protective effect of muscle.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.2

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