糖尿病性心肌病大鼠3-NT和TGF-β1的表达及氮氧自由基化合物Tempol对其干预研究

发布时间：2018-05-08 22:28

本文选题：糖尿病性心肌病 + 3硝基酪氨酸　；参考：《南昌大学》2015年硕士论文

【摘要】：目的:通过腹腔注射链脲佐菌素(STZ)的方法建立糖尿病性心肌病(Diabetic cardiomyopathy,DCM)大鼠模型,应用氮氧自由基化合物Tempol对DCM大鼠进行干预,观察DCM大鼠心肌3硝基酪氨酸(3-nitmtynosine,3-NT)及转化因子β1(Transforming growth factor betal-1,TGF-β1)的表达情况,探究Tempol对DCM大鼠的作用及可能机制。方法:1、随机取42只健康雄性SD大鼠,体重为200-250g,适应性喂养1周后随机取26只分为模型组,按55mg/kg腹腔注射链脲佐菌素的方法先建立大鼠糖尿病(Diabetes mellitus,DC)模型。余下16只大鼠分为健康对照组,对照组腹腔注射等剂量柠檬酸-柠檬酸钠缓冲液。2、4周后随机将2只造模大鼠处死,取心肌组织进行HE染色及Masson三色染色观察大鼠心肌结构,如果HE染色发现心肌细胞排列紊乱,心肌出现不同程度的变性、坏死,且Masson染色提示心肌细胞间成纤维细胞、胶原纤维增生,则表明已成功建立DCM动物模型。3、16只健康大鼠及24只造模成功的大鼠再次进行分组。分组及处理方式如下:(1)健康生理盐水组(NC+NS):8只健康对照组大鼠,常规饮食,并按18mg/kg/d剂量给予生理盐水灌胃8周。(2)健康Tempol干预组(NC+Tempol):另外8只健康对照组大鼠给以常规饮食,同时给予18mg/kg/d剂量的Tempol进行灌胃干预8周。(3)DCM模型组(DCM):12只DCM大鼠常规饮食,给予18mg/kg/d剂量的生理盐水灌胃8周。(4)DCM模型Tempol干预组(DCM+Tempol):给以常规饮食,同时给予18mg/kg/d剂量的Tempol进行灌胃干预8周。4、8周后采用HE染色及Masson染色分别对实验SD大鼠心肌进行形态学观察;用酶联免疫吸附法(ELISA)检测实验SD大鼠血清3-NT及TGF-β1的表达;用免疫组化分析法检测SD大鼠心肌3-NT及TGF-β1的表达。结果: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模型组及DCM模型干预组随机血糖均显著高于健康对照组(NC+NS对照组、NC+Tempol对照组),P0.05;DCM模型组及DCM模型干预组间随机血糖无明显差异,P0.05;DCM模型组及DCM模型干预组体重较健康对照组明显降低,P0.05;DCM模型干预组体重下降程度较DCM模型组小,P0.05;健康对照组间血糖及体重均无明显差异,P0.05。3、显微镜下观察结果示:HE染色提示健康对照组间心肌细胞排列整齐,无心肌坏死及肌纤维的溶解;DCM模型组心肌细胞排列紊乱,可见肌纤维溶解、断裂,部分心肌细胞肿胀、坏死;DCM模型干预组心肌损伤程度较DCM模型组减轻。Masson染色结果提示健康对照组大鼠心肌细胞及血管周围仅有少许正常胶原纤维,无间质纤维化;DCM模型组、DCM模型干预组心肌较健康组存在明显纤维化;DCM模型干预组心肌纤维化程度比DCM模型组轻。4、4组大鼠血清ELISA结果提示:NC+NS对照组、NC+Tempol对照组、DCM模型组、DCM模型干预组血清3-NT浓度分别为:79.11±25.37nmol/L、99.98±24.99nmol/L、371.56±95.64nmol/L、246.31±81.37nmol/L;血清TGF-β1浓度分别为:107.02±30.53pg/ml、136.69±19.68pg/ml、347.07±57.85pg/ml、218.66±26.62pg/ml。DCM模型组与DCM模型干预组血清3-NT、TGF-β1的表达均明显高于健康对照组,差异有统计学意义,P0.05;DCM模型干预组血清3-NT、TGF-β1的表达水平低于DCM模型组,P0.05;NC+NS对照组与NC+Tempol对照组间血清3-NT、TGF-β1的表达水平均无明显差异,P0.05。5、4组大鼠心肌组织免疫组化结果提示:NC+NS对照组、NC+Tempol对照组、DCM模型组、DCM模型干预组心肌3-NT表达量分别为:2512.38±1636.43、2935.69±1492.17、11684.89±2590.43、5625.41±1501.94;心肌TGF-β1表达量分别为:894.67±152.29、1001.72±215.04、8805.64±2893.75、4707.79±1121.13。DCM模型组与DCM模型干预组心肌3-NT、TGF-β1表达均明显高于健康对照组,P0.05;DCM模型干预组心肌3-NT、TGF-β1的表达水平低于DCM模型组,P0.05;NC+NS对照组与NC+Tempol对照组间心肌3-NT、TGF-β1的表达量均无明显差异,P0.05。结论:1、通过腹腔注射STZ法诱导建立DC大鼠模型后,长期的高血糖状态可引起大鼠心肌不同程度的损伤。2、DC高糖状态下氧化应激产生的3-NT及TGF-β1可引起心肌细胞坏死、纤维化,3-NT及TGF-β1可能参与了DCM的发生发展。3、2,2,6,6-四甲基-4-哌啶醇(Tempol)对正常大鼠心肌无明显影响,且Tempol可能通过抑制氧化应激反应,下调大鼠心肌及血清3-NT、TGF-β1的表达来减轻DCM大鼠心肌的损伤,发挥对DCM大鼠心肌的保护作用。
[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.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

【参考文献】