【作者】 杨立群；

【导师】 王富军；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:糖尿病心肌病(diabetic cardiomyopathy,DC)是糖尿病心脏病的特异性表现,与糖尿病(diabetes mellitus,DM)病人的心力衰竭发生和死亡密切相关,是糖尿病的特异性慢性并发症,其主要病理特点是心肌细胞肥大增殖,局灶性坏死,间质重构及心肌纤维化等。近年来研究表明间质重构在DC中扮演着不可忽视的角色。细胞外基质(extracellular matrix,ECM)是心肌间质的主体,它处于不断合成,分泌和降解的平衡状态中。存在于心肌组织中的基质金属蛋白酶(matrix metalloproteinases,MMPs)是ECM的主要降解酶,MMPs及其组织抑制因子(tissuc inhibitor of metalloproteinases,TIMPs)的改变是间质重构过程中导致ECM代谢失衡的主要因素。其蛋白表达和活性变化以及对心肌胶原(collagen,CL)含量的影响可能在DM心肌病变的发生、发展过程中起重要作用。MMP-1是基质降解的关键酶之一,目前MMP-1与其特异性抑制剂TIMP-1在糖尿病心肌病变中的研究较少。吡格列酮属于噻唑烷二酮类药物( thiazolidinediones,TZD) ,是近年来应用的一种通过激动PPARγ受体(Peroxisome proliferator activated receptors)的新型降糖药,因其不促进胰岛素过度分泌的特点而倍受关注,近年来的研究表明,它不仅可以降低血糖,改善胰岛素抵抗和脂代谢紊乱,并对多种组织有直接的效应,具有潜在的器官保护作用。但其对糖尿病心肌组织中MMP-1和TIMP-1表达的影响尚未见报道。本研究使用高糖高脂喂养及小剂量链脲佐菌素(streptozotocin ,STZ)复制2型糖尿病大鼠模型,通过对DM大鼠心肌MMP-1及TIMP-1蛋白水平进行检测,了解它们在心肌中的表达,观察心肌组织形态学变化及心肌胶原含量的变化,了解它们与血糖、血脂代谢的关系,探讨糖尿病心肌病变的可能机制。并给予DM大鼠吡格列酮干预治疗,研究其可能通过改变心肌中MMP-1、TIMP-1的表达及胶原含量对DM大鼠心肌的保护作用。方法:1 2型糖尿病大鼠模型的建立健康SD雄性大鼠45只,8周龄(体重220克左右),所有大鼠分笼饲养,每笼5只,适应性喂养1周,随机取15只作为正常对照组(A组),给予普通饲料喂养,其余30只作为糖尿病模型组。给予高糖高脂饲料(普通饲料加15%熟猪油、2.5%胆固醇、20%蔗糖)喂养4周,按李光伟HOMA指数判断出现胰岛素抵抗后,腹腔注射链脲佐菌素(streptozocin,STZ)(0.5%0.1mmol/L柠檬酸缓冲液,pH4.2)30mg/kg,并继续高糖高脂喂养2周,于注射后第2周测空腹血糖大于7.8mmol/L者纳入糖尿病模型组。将糖尿病大鼠随机分为两组:DM组(B组)15只,DM吡格列酮治疗组(C组)15只。两组仍继续高糖高脂饲料喂养,C组给予吡格列酮11mg/kg/d灌胃8周。于8周末处死动物。实验末,除意外死亡和未成模者外,A组剩余15只,B组剩余12只,C组剩余13只。每组大鼠均于实验开始、STZ注射前、注射后2周及实验结束时称重、取血。大鼠自采血前一日20:00起禁食,实验日8:00分别自内眦静脉采血,分离血清,-20℃保存,用于各指标的检测。实验结束时处死动物,留取部分心肌组织4%中性多聚甲醛固定,待组织学检查。2血糖、胰岛素的测定:血糖用葡萄糖氧化酶法测定。空腹胰岛素(FINS)采用放射免疫法测定。应用稳态模型(HOMA)及胰岛素敏感指数(ISI)评价胰岛素抵抗的程度。3血脂的测定:甘油三脂(TG)、总胆固醇(TC)使用比色法测定。游离脂肪酸(FFA)含量使用Cu2+比色法测定。4心肌细胞形态学检查:常规方法制备大鼠心肌光镜组织切片,行HE、Masson染色及MMP-1、TIMP-1免疫组化染色。5统计学处理:所有数据用SPSS11.0软件处理,计量资料以均数±标准差表示,组间差异用两样本均数的t检验进行比较,两组以上比较采用单因素方差分析作统计学处理。检验的显著性用P值表示。P<0.05为差异有显著性,P<0.01为差异有极显著性。结果:1体重:实验初,各组体重无统计学差别,具有可比性。STZ注射前模型组体重明显高于正常对照组,有显著性差异(P<0.01)。STZ注射后两周,模型组体重明显高于正常对照组(P<0.05)。实验末,B组体重明显高于A组(P<0.01)。C组体重高于A组(P<0.05),但低于B组(P<0.05)。2血糖及胰岛素:造模前各组大鼠空腹血糖无差别。造模后,模型组血糖明显高于正常组,有显著性差异(P<0.01)。实验末,B组血糖明显高于A组,有显著性差异(P<0.01)。C组血糖与A组相比较有显著性差异,明显低于B组(P<0.01)。B组胰岛素明显高于A组(P<0.01),C组胰岛素高于A组(P<0.05),明显低于B组(P<0.01)。3血脂:STZ造模后模型组大鼠的TC、TG及FFA均明显升高,与正常组相比有显著性差异(P<0.01)。吡格列酮干预组的TC、TG及FFA比模型组明显降低(P<0.01),但仍高于对照组(P<0.01)。4免疫组化心肌MMP-1、TIMP-1表达水平的变化:实验末,①MMP-1在B组心肌表达水平显著高于A组(P<0.01),C组显著低于B组(P<0.05);但仍显著高于A组(P<0.01);②TIMP-1在模型组心肌表达水平显著高于正常对照组(P<0.01),C组显著低于B组(P<0.05);但仍显著高于A组(P<0.01);③TIMP-1/MMP-1比例改变:B组高于于A组(P<0.01),C组经药物干预后低于B组(P<0.05),但仍高于A组(P<0.05)。5心肌细胞形态:HE染色显示:正常组大鼠心肌纤维排列整齐、横纹清晰,细胞核形态正常,细胞间隙无增宽及狭窄;模型组大鼠心肌纤维排列紊乱、局灶性坏死,心脏成纤维细胞(cardiac fibroblast,CFbs)增多,间质胶原增生,局部脂肪细胞浸润;干预组大鼠心肌病变较模型组有所减轻。MASSON染色光镜下可见正常心肌组织小动脉周围及心肌细胞间隙有少许胶原,模型组肌组织小动脉周围有较多胶原沉积明显,并有向心肌细胞间隙伸展的趋势。同时心肌组织间隙的胶原沉积也显著增加。吡格列酮干预组心肌亦有胶原沉积的表现,但较模型组为轻。结论:1通过高糖高脂饮食及小剂量STZ诱导的T2DM大鼠模型与人类T2DM具有相似特征,可用于T2DM及其并发症的研究。2 MMP-1、TIMP-1的改变和比例失衡导致的胶原改变是2型糖尿病心肌病变发生发展的重要因素。3吡格列酮不但具有改善糖脂代谢、增加胰岛素敏感性、减少胰岛素抵抗的作用,而且可通过降低心肌MMP-1、TIMP-1表达水平,改善TIMP-1/MMP-1平衡,减少心肌间质异常重构,对糖尿病心肌病变起到了保护的作用,减缓了糖尿病心肌病变的发展进程。更多还原

【Abstract】 Objective: Diabetic cardiomyopathy (DC) is one kind of unique cardiovascular complications of diabetes mellitus and it has a close relation with the high rate of heart failure and death in diabetes mellitus (DM) patients. Diabetic cardiomyopathy is a special chronic complication of DM, and its major pathological hallmarks of DC were myocardial hypertrophy, myocardial necrosis, myocardium mesenchyme remodeling, myocardial fibrosis and so on. Recent years, a lot of research revealed that myocardium mesenchyme remodeling play an important role in DC. Extracellular matrix (ECM) is the principal part of myocardium mesenchyme, and keeps the balance in metabolic of continuous synthesis, secretion and degradation. Matrix metalloproteinases (MMPs) exist in myocardial tissue; they are the main enzymes to degrade all the components of ECM. The change of MMPs and their tissue inhabitor -(TIMPs)will make ECM lose its balance. The changes of MMPs and TIMPs protein expression levels and accounts of collagen may play the key role during the occurrence and development of cardiomyopathy in DM.Pioglitazone that belongs to thiazolidinediones (TZDs) is a kind of new drug treating T2DM through activating PPARγ. More and more studies pay attention to thiazolidinediones because their potencial protection to many body organs.In this study type 2 DM (T2DM) rat model was copied by giving Sprague-Dawlay (SD) rats long-term high-fat diet and a low dose intraperitoneal injection of streptozotocin (STZ). Then we detected the protein expression level and activity of MMP-1、TIMP-1 and the content of collagen (CL) in experimental animals myocardial tissue to know the connection with the change of blood glucose and blood lipid in order to discuss the mechanism of DC.Furthermore, Pioglitazone was given to experimental rats to observe the function to MMP-1、TIMP-1 for explaining the mechanism of its protective effects on myocardium .Methods:1. Induction of type 2 diabetic cardiomyopathy in ratsWe chosen 45 healthy male SD rats as experiment objects .After breeded adaptively for 1 week, they were divided randomly into 2 groups: control group (Group A,n=15) fed regular diet and experiment group (n =30) fed high-sugar and high-fat diet (including 2.5% cholesterin, 15% cooked lard, 20% cane sugar and general diet together) for 4 weeks. When the high-fat diet animals appeared the insulin resistance according to Li Guangwei HOMA index formula, STZ was administered via intraperitoneal injection at a dose of 30mg/kg once. Two weeks later, the rats whose fasting blood glucose (FBG) was greater than 7.8mmol/L and insulin sensitivity decreased were considered as T2DM model rats. The model rats were divided randomly into two group: DM model rats (group B) fed the high-sugar and high-fat diet continuously for 8 weeks, and DM model rats treated with Pioglitazone (Group C) which fed a high-sugar and high-fat diet meanwhile pioglitazone was administered by oral gavage (11mg/kg) once daily for 8 weeks. Except for accidental death and abortive sample, there remain 15 rats in A group, 12 rats in B group, 13 rats in C group at the end of the experiment. All the rats were weighed and their blood was taken at the beginning and end of the experiment. The rats were fasted at 20:00 in the day before experiment, blood was taken from the angular vein at 8:00 of experimental day, and the blood serum was separated and stored at -20℃for all the indexes’detection.At the end of experiment, the heart tissues were taken and fixed for histological analysis by neutral paraform.2 Assays of blood glucose and insulinWe measured fasting glucose by glucose oxidase method, and insulin by radioimmunoassay on serum samples. The level of insulin resistance was evaluated according to Homeoestasis Model Assessment (HOMA-IR) and Insulin Sensitivity Index (ISI).3 Assays of blood lipidAt the end of this experiment, Triglyceride (TG), total cholesterol (TC) was measured by chromometry. FFA was measured by Cu2+chromatometry.4 Cell morphology examination of myocardiumThe tissue section was made by routine method for light microscope examination, and proceeds with MASSON specific staining to calculate the content of CL. Detecting the protein expression level and activity of MMP-1 and TIMP-1 in myocardium by immunohistochemistry.5 Data analysis and statistical evaluationAll data were treated with SPSS11.0. Measurement data were expressed as mean values±standard deviation. The difference between means was compared by the Student’s t-test, and analysis of variance for one factors (ANOVA) was used to compare continuous variables among groups. In pykno-ranked data, the difference between two groups was compared by the Wilcoxon’s rank test, and Kruskal-Wallis H’s test was used to compare the difference among groups. The degree of significance was judged by P values when necessary. P<0.05 means the difference is significant, P<0.01 means the difference is highly significant.Results:1 Body weight: There was no statistically significant difference in weight among each group of the rats at the beginning of the experiment. At the end of the experiment, the weight of rats in group B and group C was significantly higher than that in group A (P<0.05), and the weight of group C was lower than that of group B (P>0.05). 2 Blood glucose and insulin: There was no significant difference in fasting glucose among the rats of each group at the beginning of the experiment. After the injection of STZ, the blood glucose level of model rats and the rats treated with pioglitazong significantly raised up from normal baseline (P<0.01). The blood glucose level of rats treated with pioglitazong was lower than that of model rats (P<0.01), but it was still hyperglycemia. The insulin level of model rats was markedly higher than that of rats treated withpioglitazong and control rats (P<0.01), The insulin level of control rats lower than that of rats treated with pioglitazong (P<0.05).3 Blood lipid: The plasma levels of TG, TC and FFA in rats with T2DM were obviously higher than those in control rats (P<0.01). After treated with pioglitazong, their plasma levels decreased to some extent, but they were still higher than those in control rats(P<0.01).4 Immunohistochemistry: The MMP-1 expression of the rat myocardium in group C was significantly higher than that in group A (P<0.01), and lower than group B (P<0.01). The TIMP-1 expression of the rat myocardium in group C was significantly higher than that in group A (P<0.01), and lower than that in group B (P<0.01). The ratio of TIMP-1/ MMP-1 in group C was significantly higher than that in group A (P<0.05), and lower than group B (P<0.05).5 The pathological morphological change of myocardiumHE staining showed that cardiac tube of group A were in order, cross striation was clear, karyon and ECM was normal. While in T2DM model rats, cardic muscle degeneration and ECM accumulation could be observed. Foregoing changes in-group B rats were more severe than thosein group C rats.MASSON-staining method: Under light microscope, most of cardiac muscle fibers were stained with red, only few collagens showed green in control rats. There was more collagen showed green in group B than groupA, less than group C.Conclusions:1 The type 2 diabetic rat model that was induced by high-sugar, high-fat diet and a low dose of STZ had some characteristics similar to human T2DM. It would be available to study T2DM and its complications.2 The protein expression level and activity of MMP-1、TIMP-1 in T2DM rats myocardium was increased. These changes lead collagen hyperplasia and ECM remodeling. It indicated that the balance of TIMP-1/MMP-1 was an important factor to the occurrence and development of DC.3 By activating PPARγ, pioglitazong plays an important role in improving glucolipid metabolism, increasing the sensitivity of insulin and reducing the insulin resistance. Moreover, it can lower the expression of MMP-1 and TIMP-1 in cardiac muscle, improve the balance of TIMP-1/MMP-1, thus it exerts the action against remodeling of ECM and protects cardiac muscle in T2DM, and slows the process of diabetic cardio- myopathy.更多还原