【作者】 李倩；

【导师】 汪翼；

【作者基本信息】 山东大学 ， 儿科学， 2008， 博士

【摘要】 研究背景及目的近年来,儿童糖尿病的发病率正在逐渐上升,其中发病年龄较早、病情较重的多为1型糖尿病,即胰岛素依赖性糖尿病（insulin-dependentdiabetic mellitus,IDDM）。糖尿病心肌病是一种独立的糖尿病并发症,系由血糖代谢紊乱引起心肌功能障碍及心肌细胞学改变,从而出现亚临床的心功能异常,以后渐进为心脏小血管病变、微循环障碍及自主神经病变,最后导致心功能不全。其临床特征主要以左心室肥厚和舒张功能缺损为主,随着病程的进展也会出现收缩功能不全。青少年糖尿病患者在病程早期即出现明显的心脏舒张功能下降,随着病程的延长,心脏左室结构会发生改变,最终可导致充血性心力衰竭,甚至死亡。因此,早期发现糖尿病患者,并给予适当的干预,对于延缓心功能减退和预防糖尿病心肌病具有重要意义。糖尿病心肌病变在组织形态学上的特点主要为心肌细胞增生、肥大,心肌间质胶原沉积、纤维化。以往认为糖尿病心肌病变是以心肌细胞的肥大增殖为主,但近年来已认识到在心脏细胞数量上占2/3的非心肌细胞（主要是成纤维细胞）和细胞外间质（主要成分是心肌间质胶原）在心肌重塑中起着不可忽视的作用。心脏间质、血管周围纤维化、局部微瘢痕形成可导致心脏肥厚、心功能下降,并最终导致心力衰竭,是糖尿病的主要致死原因之一。因此糖尿病心脏间质重塑成为近年来糖尿病并发症研究领域的一个热点。心肌间质胶原的蓄积一方面有胶原的过量生成,另一方面也有胶原的降解受抑制。胶原合成多于降解是心肌间质纤维化的主要形成原因之一。心肌间质胶原蛋白主要由成纤维细胞合成分泌。在胶原的合成过程中,Ⅰ型前胶原羧基端肽（typeⅠprocollagen carboxyterminal propeptide,PⅠCP）和Ⅲ型前胶原氨基端肽（typeⅢprocollagen aminoterminal propeptide,PⅢNP）的释放与胶原纤维的生成呈1:1的关系,因此血清PⅠCP和PⅢNP被视为体内胶原过度合成的间接标志,可作为体内组织器官纤维化非侵入性的检测方法,已被证实在高血压、心力衰竭、急性心肌梗塞等心血管疾病中具有重要意义。研究发现,糖尿病心肌病变时,成纤维细胞增殖,胶原合成增多,心肌间质胶原沉积。然而,糖尿病患者血清胶原末端肽的变化及其与心肌间质纤维化之间的关系尚不明确,有待进一步探究。胶原降解主要在基质金属蛋白酶（Matrix metalloproteinases,MMPs）及其组织抑制因子（Tissue inhibitors of metalloproteinases,TIMPs）的作用下进行。MMPs是目前所知最重要的一类降解ECM（extracellular matrix,ECM）的蛋白酶类,在正常心肌组织中主要以非活性状态存在,通常与内源性TIMPs保持动态平衡,共同调节ECM合成、降解以及组织重塑等。MMPs家族成员众多,根据作用底物不同分为四种亚型,即胶原酶、明胶酶、基质溶解素和膜型MMPs。其中MMP-2（明胶酶-A）是基质胶原降解成小分子多肽的关键酶,在生理状态下与其特异性抑制剂TIMP-2处于相对的平衡状态,使ECM的降解与合成达到动态的平衡。研究表明,MMPs/TIMPs参与了多种心血管疾病的发病过程,然而关于其在糖尿病心肌病的变化及其作用的报道少见,需要进一步研究。本实验通过链脲佐菌素（streptozocin,STZ）建立1型糖尿病动物模型,检测血清PⅢNP、心肌Ⅲ型胶原（collagen typeⅢ,Ⅲ-C）蛋白表达以及心肌胶原容积分数（collagen volume fraction,CVF）的变化,从多个角度观察糖尿病大鼠不同时期心肌间质胶原代谢的情况,同时从基因表达、蛋白及生物学活性三个水平检测大鼠血液和心肌MMP-2、TIMP-2,分析其相关关系,探讨MMPs/TIMPs在糖尿病心肌间质重塑过程中的作用,进一步研究糖尿病心肌病变的可能发病机制。近来研究证实肾素-血管紧张素系统（renin-angiotensin system,RAS）在糖尿病心血管病变发展中起着重要作用,通过血管紧张素转化酶抑制剂（angiotensin-conVerting enzyme inhibitor,ACEI）抑制RAS可有效延缓糖尿病心血管病变发展。苯那普利即是目前常用的一种ACEI类药物,它竞争性抑制血管紧张素转换酶（angiotensin-conVerting enzyme,ACE）的活性而减少血管紧张素Ⅱ（angiotensin,AngⅡ）,从而阻断由RAS激活所介导的一系列连锁反应。然而有关RAS在糖尿病心肌病ECM重塑中的作用以及与MMPs/TIMPs之间的关系却罕见研究报道。因此,本实验在STZ诱导建立1型糖尿病动物模型的基础上,从病理形态学和分子生物学水平观察苯那普利对心肌胶原代谢、MMP-2/TIMP-2系统的影响,探讨苯那普利在糖尿病心肌病间质重塑中的作用。研究方法:1.建立IDDM大鼠模型及分组:选用6～8周龄雄性Wistar大鼠46只,体重200～250g,随机分为正常对照组（16只）和糖尿病组（30只）。糖尿病模型以一次性腹腔注射60mg/kg STZ诱发,注射72h后取尾静脉血检测血糖≥16.7mmol/L、尿糖+++以上者表示造模成功,成功率为100%。将正常对照组大鼠按体重随机再分为4周组（NC4W）和12周组（NC12W）,每组8只;糖尿病大鼠随机分为三组:糖尿病4周组（DM4W）,糖尿病12周组（DM12W）和糖尿病苯那普利干预组（DB12W）,每组10只。DB12W组予苯那普利10mg·kg^-1·d^-1灌胃,持续12周,NC组和DM组灌予等量自来水。DM4W、DM12W和DB12W组各取8只进行取材和检测。2.血清PⅢNP水平:放射免疫法检测。3.血清MMP-2、TIMP-2含量:酶联免疫吸附实验（enzyme-linkedimmunosorbent assay,ELISA）定量检测。4.心肌组织的形态学检测:（1）Van Gieson（VG）染色:观察心肌间质胶原含量的变化,并测量CVF进行半定量分析;（2）透射电镜铀—铅双重染色:观察心肌超微结构的改变。5.心肌组织MMP-2活性:明胶酶谱分析法检测。6.心肌组织MMP-2、TIMP-2、Ⅲ-C的蛋白表达:免疫组化方法检测。7.心肌组织中MMP-2、TIMP-2mRNA表达:逆转录—聚合酶链反应（reversetranscription-polymerase chain reaction,RT-PCR）方法测定。研究结果:1.血糖、心重、体重及心脏重量指数的变化:与同期正常对照组相比较,糖尿病大鼠在病程第4周和第12周时血糖水平明显升高,心重、体重显著下降,心脏重量指数升高。随着病程的延长,糖尿病大鼠心重、体重逐渐下降,但心重/体重逐渐升高。ACEI干预治疗对上述指标影响不明显。2.心肌超微结构的改变:正常对照组大鼠心肌肌原纤维丰富,肌丝排列紧密整齐;线粒体嵴多、排列有序。糖尿病大鼠4周时肌原纤维呈灶性变性、断裂、溶解,排列紊乱,线粒体数目增多;12周时肌节对位不齐,线粒体增多肿胀,有断嵴、空泡样变性现象,间质纤维增生,糖原颗粒增多。苯那普利治疗组大鼠心肌病理改变明显减轻。3.胶原代谢的变化:（1）心肌胶原分布的改变:正常大鼠心肌细胞之间和血管周围仅见少许红染的胶原组织,糖尿病组心肌细胞间及血管周围胶原分布明显增多,CVF显著升高;随病程延长,糖尿病大鼠心肌间质纤维化愈加明显,CVF随之相应增加。苯那普利治疗后心肌纤维化程度明显改善,CVF同步下降。（2）心肌Ⅲ-C蛋白表达的变化:与同期正常对照组相比,糖尿病大鼠心肌Ⅲ-C分布明显增多,且随病程延长而加重;ACEI干预后显著减少,但仍高于正常。（3）血清PⅢNP水平的变化:病程4周时糖尿病大鼠血清PⅢNP明显高于同期正常对照组,12周时这一变化进一步加剧,经苯那普利治疗后显著降低。相关分析显示血清PⅢNP与心肌CVF、Ⅲ-C均呈显著正相关关系。4.血清MMP-2、TIMP-2水平的变化:与同期正常对照组相比,糖尿病大鼠病程4周时血清MMP-2有增高趋势,但差异尚无统计学意义,12周时则明显升高;糖尿病组血清TIMP-2在病程4周和12周时均显著高于同期正常对照组。5.心肌MMP-2活性的改变:糖尿病大鼠心肌组织MMP-2活性表达在病程4周和12周时均明显低于同期正常对照组,且随着病程的延长持续减弱;苯那普利干预后MMP-2活性轻度升高,但差异尚无统计学意义。6.心肌MMP-2、TIMP-2蛋白及mRNA表达的变化:与同期正常对照组相比,糖尿病大鼠在病程4周和12周时心肌组织MMP-2基因和蛋白表达均明显减弱;TIMP-2 mRNA表达均显著升高;TIMP-2蛋白表达在病程4周时有升高趋势,但与正常组的差异尚不具统计学意义,至12周时则显著增强。ACEI干预后,糖尿病大鼠心肌MMP-2 mRNA和蛋白表达明显上升,TIMP-2基因和蛋白表达均显著下降。7.相关分析显示,血清MMP-2与心肌MMP-2活性、基因表达负相关,与MMP-2蛋白表达无相关关系,与血清PⅢNP、心肌CVF、Ⅲ-C蛋白表达呈正相关关系;血清TIMP-2与心肌CVF、Ⅲ-C蛋白表达呈正相关,但与血清PⅢNP、心肌TIMP-2基因和蛋白表达无相关关系。8.相关分析显示,心肌CVF、Ⅲ-C蛋白表达均与MMP-2活性、mRNA和蛋白表达呈高度负相关,与TIMP-2基因、蛋白表达呈明显正相关关系。研究结论:1.糖尿病大鼠在病程早期已出现心肌重塑,表现为心脏重量指数升高、心肌细胞增生肥大和变性坏死、心肌间质胶原纤维化明显,并随病程延长加重。2.糖尿病大鼠在病程早期即出现血清PⅢNP水平显著升高,心肌Ⅲ-C蛋白表达增多,心肌胶原容积分数升高等心肌胶原代谢紊乱表现,进而心肌胶原网络结构受到破坏,胶原代谢增强并过度沉积,形成心肌纤维化。3.糖尿病大鼠血清PⅢNP水平的升高与心肌胶原含量和Ⅲ-C蛋白表达的增加相一致,提示血清PⅢNP可以用于预测糖尿病大鼠心肌间质纤维化的程度,作为糖尿病心肌病变的无创检测指标之一。4.糖尿病大鼠在病程早期即开始有心肌MMP-2mRNA和蛋白表达、活性的明显降低,TIMP-2mRNA和蛋白表达的增强,并且相关分析显示心肌CVF、Ⅲ-C与MMP-2和TIMP-2的表达密切相关,提示心肌MMP-2/TIMP-2比例下降导致胶原降解减少,出现胶原沉积,进而造成心肌间质重塑。5.血清MMP-2和TIMP-2水平与心肌CVF、Ⅲ-C蛋白表达呈正相关,提示血清MMP-2和TIMP-2均可作为心肌胶原合成或纤维化程度的预测指标,有助于糖尿病心肌病的早期发现。6.ACEI类药物苯那普利干预不仅可使糖尿病大鼠心肌细胞病变减轻,还可使血清PⅢNP水平、心肌间质胶原含量以及Ⅲ-C蛋白表达下降,减少间质胶原的聚积,减轻心肌纤维化程度。提示ACEI类药物可同时干预心肌实质和间质重塑。7.应用ACEI类药物干预可使糖尿病大鼠心肌MMP-2表达上调和TIMP-2表达下调,改善MMP-2/TIMP-2系统的失衡,促进心肌胶原的降解,减轻心肌间质胶原沉积,延缓心肌间质重塑的进展。创新和意义:1.本研究应用STZ诱导建立1型糖尿病组大鼠模型,从多角度、多水平观察其心肌胶原代谢变化及其与MMP-2/TIMP-2系统的关系,进一步揭示了MMPs/TIMPs在糖尿病心肌间质重塑过程中的作用,为以MMPs为治疗靶点的新药在糖尿病心肌病中的应用提供了有力的理论依据。2.首次在同一动物个体内发现血清PⅢNP、MMP-2、TIMP-2与心肌胶原含量密切相关,提出上述指标可用于预测糖尿病患者心肌间质纤维化的程度,作为糖尿病心肌病变的无创检测指标,有助于糖尿病心肌病的早期发现。3.本研究应用苯那普利干预治疗,观察其对于糖尿病大鼠心肌胶原代谢以及与MMPs/TIMPs系统表达的影响,证实ACEI类药物可以通过改善MMP-2/TIMP-2系统的失衡,促进间质胶原降解,达到延缓甚至逆转糖尿病心肌间质重塑的目的,为临床应用ACEI类药物治疗糖尿病心肌病提供新的论点和实验依据。更多还原

【Abstract】 Backgroud and objectivesType 1 diabetic mellitus (DM1), that is insulin-dependent diabetic mellitus (IDDM) in children has an increasing morbidity in recent years. Diabetic cardiomyopathy (DC) is one of the independent complications of diabetes mellitus. It is caused by metabolism disturbance of blood glucose, subsequenced by cardiac dysfunction and cellular changes, and then accompanied with small vascular lesions, microcirculation disturbances and autonomic neuropathy, finally leading to cardiac insufficiency. The clinic manifestation of DC mainly includes left ventricular hypertrophy and impairment of diastolic function, and systolic malfunction appears in the late course of DC. Early signs of diastolic malfunction are first observed in children and adolescents with DM1, continued with left ventricular remodeling and finally congestive cardiac failure in the progression of disease. Therefore, early diagnosis and suitable intervention are very important in patients with DC.Diabetic cardiomyopathy is expressed histomorphologically mainly through proliferation and hypertrophy of cardiocytes and interstitial fibrosis. Changes of cardiocyte have been regarded as the focus of ventricular remodeling of DC in the past. However, more and more experts are putting an emphasis on non-cardocytes (mainly fibroblasts) and excellular matrix (mainly collagen) recently. Cardiac interstitial fibrosis might cause ventricular hypertrophy, impairment of cardiac function, and lead to heart failure in the end. So the pathological mechanisms and preventions and treatments of interstitial remodeling in DC become the key target in the fields of diabetic complications.Accumulation of cardiac interstitial collagen is caused by either excess production or weakened degradation of collagens. If collagen synthesis exceeds its degradation, interstitial fibrosis occurs. Collagen proteins are synthesized and secreted mainly in cardiac fibroblasts. Type I procollagen carboxyterminal propeptide (PICP) and type III procollagen aminoterminal propeptide (PIIINP) are released into blood during the synthesis of collagen. Serum PICP and PIIINP have been regarded as indirect indexes of oversynthesis of collagen in vivo. And they have been found to play an important role in cardiovascular diseases such as hypertension, heart failure and acute myocardial infarction. Some studies reported that there were fibroblast proliferation, oversynthesis of collagen and interstitial fibrosis in DC. However, changes of serum PICP and PIIICP and their relationship with cardiac fibrosis in diabetic patients remain uncertain.Collagens are degraded mainly under the control of the family of zincdependent, redox sensitive, endopeptidases: MMPs. Generally metabolism of extracellular matrix and tissue remodeling are maintained under the balance of MMPs and tissue inhibitors of metalloproteinases (TIMPs). There are many members in the MMP family. They are classified into subgroups according to substrate specificity and structure, including the collagenase, the gelatinase, the stromelysin and the membrane-type MMPs. Gelatinases MMP-2 is involved in degrading gelatins (collagen fragments) and cleaving basement membrane matrix proteins. MMPs and TIMPs have been found to take part in the genesis and development of many cardiovascular diseases. However, the molecular changes and the roles of MMPs/TIMPs in diabetic cardiomyopathy remain unclear yet.The present study was designed to investigate the changes of cardiac collagen metabolism by monitoring serum PIIINP levels, cardiac collagen type III (III-C) protein expression and collagen vollume fraction (CVF) , and determine their relationships with MMPs/TIMPs system in a rat model of DM1 induced by streptozocin (STZ), in order to probe what roles MMPs/TIMPs system might play in the pathophysiolgic course of DC.Studies have demonstrated that renin-angiotensin system (RAS) plays an important role in diabetic angiocardiopathy, and angiotensin-converting enzyme inhibitors (ACEIs), which reduce the production of Ang-II by inhibiting ACE, could prevent the progression of cardiovascular complications in DM. However, what role RAS plays in ECM remodeling and how it regulates the expressions of MMPs/TIMPs in DC remains unknown. Benazapril is a potent ACEI with prolonged action and hence used in our study to observe its therapeutic effects in cardiac remodeling in diabetes. The present study was designed to explore the effects of ACEIs on myocardial collagen metabolism and MMPs/TIMPs to gain insight into the potential molecular mechanism of ACEIs therapy in delaying cardiac interstitial fibrosis in streptozotocin-induced typel diabetic rats.Methods1. The rat diabetic model: 46 healthy male Wistar rats (220-250g) were divided randomly into control group (NC, n=16) and diabetes group (n=30). Diabetic models were induced by a single intraperitoneal injection of STZ 60 mg/kg and in the following 72 hours blood glucose level was measured. Rats with blood glucose≥16.7 mmol/L and urine glucose≥3+ were indicated as diabetes. And then diabetic rats were subdivided into 4-week diabetes group (DM4W) , 12-week diabetes group (DM12W) and 12-week diabetes group with benazepril treatment (DB12W) . DB12W rats were treated with benazepril (10mgkg-1·d-1, qd by intragastric administration). The whole study last 12 weeks. Eight NC and DM rats were killed on week 4 and 12 respectively; and DB12W rats on week 12.2. The level of serum PIIINP was measured by radioimmunity methods.3. The levels of serum MMP-2 and TIMP-2 were detected quantitatively by ELISA method.4. Histopathologic evaluation: (l)Myocardial ultrastructure were observed by transmission electron microscopy. (2)Areas integra of collagen fiber were calculated by Van Gieson staining.5. Cardiac MMP-2 activity was determined by zymography.6. The protein abundence for myocardial MMP-2, TIMP-2 and collagen III (III-C) were observed by immunohistochemical stain.7. Determination of the mRNA expression of MMP-2 and TIMP-2 by RT-PCR method.Results1. Blood glucose, heart weight, body weight and the ratio of heart weight/body weight: At 4 and 12 weeks, diabetic rats exhibited decreased heart weight (HW) and body weight (BW) but increased HW/BW ratio compared with those of NC. However, the indexes mentioned above had no statistical difference between DM12W and DB12W groups.2. Myocardial ultrastructure: Obviously focal degeneration and rupture of myofibrils and increased mitochondria appeared in the myocardium at 4 weeks. Degeneration and necrosis of mitochondria, accumulation of cardiac fibers were significant at 12 weeks. These changes were decreased significantly in ACEI-treated group.3. Collagen metabolism:(1)Myocardial collagen contents: Considerable fibrosis was seen mainly between cardiomyocytes and around vessels in diabetic hearts at 4 weeks compared with the values in NC group. Much more fibrosis appeared in diabetic hearts at 12 weeks. These structural changes were reduced significantly in DB12W group.(2)Protein expressions of III-C in left ventricular: In DM group, the cardiac III-C protein expression was higher than that in NC group at 4 weeks, and kept increasing with the longer course. The indicator values in DB groups were markedly lower than those in DM group.(3)Serum PIIINP levels: PIIINP level in plasma was markedly higher from DM rats than in NC group at 4 weeks, ,and kept increasing with the longer course. Treatment with benazepril significantly reduced the levels of PIIINP in diabetic group.4.The level of serum MMP-2 and TIMP-2: compared with that of control group, serum MMP-2 level was slightly elevated but not statistically significantly in diabetic group at 4 weeks, and markedly increased at 12 weeks . Increased concentrations of TIMP-2 in blood were evident in diabetic rats at 4 and 12 weeks.5. MMP-2 activity of left ventricular tissue: MMP-2 activity assessed by gelatin zymography was weaker obviously in the hearts of diabetic rats compared to normal values at 4 and 12 weeks. MMP-2 activity was mildly but not significantly higher in DB group than that in DM group.6. MMP-2 and TIMP-2 mRNA and protein expression of left ventricular tissue: MMP-2 mRNA and protein expression was down-regulated and TIMP-2 mRNA expression were up-regulated in diabetic hearts compared with NC group at 4 and 12 weeks. TIMP-2 protein expression was slightly elevated but not statistically significantly at 4 weeks, but markedly increased at 12 weeks in diabetic group. The above changes were prevented with benazepril treatment in DB rats .7. Correlative relationship analysis: serum MMP-2 were significantly negatively correlated with cardiac activity and gene expression of MMP-2 and positively correlated with CVF, III-C and serum PIIINP. Serum TIMP-2 were significantly positively correlated with CVF, III-C, but not correlated with TIMP-2 mRNA and protein expression and serum PIIINP.8. Correlative relationship analysis: CVF and III-C were significantly negatively correlated with cardiac activity, gene and protein expression of MMP-2 and positively correlated with gene and protein expression of TIMP-2.Conclusions 1. Cardiac hypertrophy and the imbalance of collagen metabolism occurred in the early stages of diabetes at least in a rat animal model, with degeneration and necrosis of cardiocytes and accumulation of interstitial collagens.2. Serum PIIINP, cardiac III-C protein expression and CVF increased gradually with the DM course prolonging, indicating that collagen dysmetabolism may be a possible pathway to accumulate ECM in DC.3. Serum PIIINP increased gradually with the DM course prolonging and correlated positively with cardiac III-C protein expression and CVF respectively. It meaned that serum PIIINP may be a possible index of collagen fibrosis in DC.4. MMP-2 activity, mRNA and protein expression was downregulated and TIMP-2 mRNA and protein expression were upregulated in diabetic hearts. CVF and III-C were significantly negatively correlated with cardiac activity, gene and protein expression of MMP-2 and positively correlated with gene and protein expression of TIMP-2. These indicated that a decrease in MMP-2/TIMP-2 complex might contribute to an impairment in ECM degradation, eventually resulting in cardiac fibrosis in diabetic myocardiopathy.5. Serum MMP-2 and TIMP-2 levels were significantly positively correlated with CVF, III-C, which indicated that both serum MMP-2 and TIMP-2 might be used to predict the extent of collagen fibrosis in DC.6. Benazepril treatment helps to the recovery of cardiac substantial and interstitial remodeling by ameliorating hypertrophy and necrosis of cardiocytes and accumulation and fibrosis of collagen fibers.7. Recovery of diabetic changes of cardiac MMP-2 and TIMP-2 with benazepril treatment indicated that the drug may promote collagen degradation and inhibit its accumulation by improving MMP-2/TIMP-2 imbalance.Innovations and meanings1. This study investigated the relationships between cardiac collagen metabolism and MMP-2/TIMP-2 imbalance in the same animal model of DM1, disclosed the roles of MMP-2/TIMP-2 in interstitial remodeling of DC. Thus potent theoretical evidences were offered for MMPs as a new therapeutic target in DC.2. This study firstly viewed serum MMP-2, TIMP-2 and PIIINP were significantly correlated with cardiac collagen accumulation in the same diabetic models, therefore these indexes could be used to predict the extent of collagen fibrosis and contribute to early discovery and early intervention of DC.3. This study explored the effects of benazepril on in vivo collagen metabolism and MMP-2/TIMP-2 in diabetic animals. ACEIs were confirmed to inhibit diabetic collagen accumulation and cardiac remodeling by regulating the expressions of MMPs and TIMPs system. It offered new theoretic evidence for ACEIs in the treatment of DC in clinic.更多还原