【作者】 卢泽芬；

【导师】 房辉；

【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（DM）是目前影响人类健康的常见疾病之一,糖尿病病人发生充血性心力衰竭的危险性显著增高,糖尿病心肌病（DCM）是危险性增高的主要原因。糖尿病心肌病通常表现为心肌舒张和（或）收缩功能障碍,而心肌细胞收缩和舒张与细胞内游离Ca⁺⁺的浓度密切相关,其中起主要调控作用的是肌浆网Ca⁺⁺-ATPase（SERCA）,目前研究的重点也集中于高血糖状况下心肌SERCA活性的变化对心肌收缩和舒张功能的影响,及影响SERCA活性的相关因素。目前研究显示:在糖尿病状态下,体内氧化应激增强,进而影响心肌SERCA功能,使心肌细胞内Ca⁺⁺浓度失调,引起糖尿病心肌病。本研究主要就此展开,共分为四部分:1复制糖尿病大鼠模型:在标准条件下,应用一次性腹腔注射大剂量链脲佐菌素（STZ）的方法复制糖尿病大鼠模型,通过大鼠临床症状和监测静脉血糖确定模型复制成功。2光镜下观察糖尿病大鼠心肌病理学改变:将福尔马林固定的心肌组织经过石蜡包埋、切片,经苏木素、伊红液染色,在光镜下观察、摄片。3测定糖尿病大鼠心肌肌浆网Ca⁺⁺-ATPase（SERCA）活性、代表氧化应激损伤的中间产物丙二醛（MDA）水平及超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）两种抗氧化酶类的活性。4测定糖尿病大鼠心肌肌浆网Ca⁺⁺-ATPase（SERCA2α）mRNA的基因表达水平。方法:本研究在成功复制糖尿病动物模型的基础上,利用病理学实验技术、生物化学及分子生物学方法,系统观察了糖尿病大鼠心肌光镜下的病理学改变、心肌SERCA活性变化及其亚型SERCA2αmRNA基因表达水平、以及氧化应激相关产物和抗氧化酶类活性的变化。探讨糖尿病心肌病发病机制,为临床预防及治疗提供依据。本研究共分为四部分:1糖尿病动物模型的复制:50只SD大鼠适应性喂养1周后,按体重随机分为正常对照组（N组）及糖尿病模型复制组（D组）,N组20只,D组30只,将N组随机分为4周组（N4）、8周组（N8）,D组分为4周组（D4）、8周组（D8）。禁食不禁水12小时,D组大鼠按60mg/kg STZ溶液一次性腹腔注射（临用前用0.1mol/L枸橼酸缓冲液溶解,PH4.5）,N组腹腔注射等量上述的枸橼酸缓冲液。72h后尾静脉采血测定空腹血糖及定性尿糖,空腹血糖≥16.7mmol/L,尿糖定性≥+++,动物多饮、多食和尿量增加者确定为D组模型。饲养期间N组和D组均给予相同的饮食和水。每周测一次体重和血糖,测血糖前12小时禁食不禁水,所有动物均单笼饲养,自由进食及饮水,共观察8周,其间未用过胰岛素治疗,D组有2只大鼠血糖未达到以上标准,4只大鼠在饲养过程中因感染、酮症等原因死亡,剔除出本试验。大鼠饲养至相应周数处死取材。2心肌切片HE染色观察心肌光镜下的病理学改变:将福尔马林固定的心肌组织经过石蜡包埋、切片,经苏木素使胞核着色、伊红液使胞质着色,在光镜下观察、摄片。3心肌SERCA活性及氧化应激中间产物、相关抗氧化酶水平的测定:分别取4周、8周糖尿病组大鼠及相应正常对照组大鼠的心肌组织,制备成10%的心肌组织匀浆,利用生物化学的方法测定组织匀浆中的丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）水平,将10%组织匀浆高速离心制备出心肌肌浆网,利用生物化学的方法测定SERCA活性。4心肌SERCA2αmRNA基因表达水平的测定:取-80℃保存的新鲜左心室心肌组织100mg,利用Trizol一步法提取总RNA,再逆转成cDNA,应用RT-PCR的方法扩增目的基因SERCA2α,利用管家基因β-action作为内参照,1.2%琼脂糖凝胶电泳后,应用MIAS-5.0进行灰度测量,结果取目的基因与内参照的比值进行半定量分析。结果:1糖尿病组大鼠表现为多饮、多尿、体重较相应对照组降低,血糖≥16.7mmol/L,判断糖尿病大鼠模型复制成功。2与正常对照组大鼠相比,4周糖尿病组大鼠心脏光镜下无明显病理学改变,8周组出现心肌细胞肿胀、核仁固缩、小动脉壁细胞核层数增多、核深染、部分小动脉玻璃样变。3与正常对照组大鼠相比,D组SERCA活性明显下降（P<0.01）,D8组比D4组下降更显著（P<0.05）。氧化应激中间产物MDA在D组明显升高（P<0.01）,D8组比D4组上升更显著（P<0.05）;抗氧化酶SOD、GSH-Px明显下降,D8组比D4组下降更显著（P<0.05）。4与正常对照组大鼠相比,D组SERCA2αmRNA表达水平明显下降（P<0.01）,D8组比D4组下降更显著（P<0.05）。结论:1利用一次性腹腔注射大剂量STZ能够成功复制糖尿病大鼠模型。2糖尿病大鼠光镜下出现心肌细胞肿胀、核仁固缩、小动脉壁细胞核层数增多、核深染、部分小动脉玻璃样变等心肌损伤的病理学改变。3糖尿病大鼠体内氧化应激增强,抗氧化能力下降,心肌存在过氧化损伤,随着病程的延长过氧化损伤加重。4糖尿病大鼠SERCA生物学活性下降,其亚型SERCA2αmRNA表达减少,随着病程的延长减少更明显,引起心肌细胞Ca⁺⁺调节异常,心肌舒张及收缩功能受损,导致糖尿病心肌病变。更多还原

【Abstract】 Objectives: Diabetes Mellitus is a common disease which influences human health, and the risk of congestive heat-failure in diabetic patients is more than healthy people. It is clear that the diabetic cardiomyopathy is a important reason. The clinic expression of the diabetic cardiomyopathy is the disorder of relaxation and （or） contraction in myocardial muscle, which is associated with the concentration of Calcium in cell, and the sarcoplasmic reticulum plays important role. The focal points of researches at present are the influences of the relaxation and contraction in myocardial when the activity of SERCA changes, and another focal points are the influential factors to SERCA. The results display: In diabetic states, the oxidative stress is stranger, then the function of SERCA is influences, and the concentration of Calcium in cell is disturbance, and leads to cardiomyopathy. The research is divided into 4parts:1 To establish the model of diabetic rats: under the standard environment, STZ were injected into the rats, the model was consisted successful through the clinical symptom and vein glucose.2 To investigate the pathologic changes of myocardium in diabetic rats by LM: To embed the tissues which fixed by formalin in paraffin, section them into slice, stain them by hematoxylin and eosin, then take photos through light micrographic.3 In order to observe the activity of cardiac sarcoplasmic reticulum Ca⁺⁺-ATPase、super-oxide dismutase （SOD） and glutathion peroxidase（GSH-Px）、the medium production of oxidative stress injury—malondialdehyde（MDA） in diabetic rats.4 To investigate the gene expression of SERCA2а.Methods: At the base of animal models of diabetic rats, four parameters are observed by the use of biochemistry、molecule-biology and experimental pathology technology. These parameters were as follows: the pathologic changes of myocardium in diabetic rats under LM、the alterations of the activity of SERCA、the changes of mRNA expression level and the changes of the productions associated with oxidative stress and the activity of anti-oxidase. This study consisted of four parts1 50 SD rats were fed adaptively for 7days, then divided them randomly into diabetic group （D group） and normal control group （N group）, there were 20 rats in N group and 30 rats in D group, divided N group and D group into N4、N8、D4、D8 group randomly. There were 12 hours before the establishment of the model. In this period, there was no food but water for the rats and through night. Rats in D group were injected STZ（60mg/kg） which had been mixed in citric acid buffer （0.05M,PH=4.5）; The rats in N group were injected citric acid buffer with the same volume. 72 hours after the injection, the vein blood and urine of the rats were took to detect the blood and urine glucose. The criterion of diabetic rat was: the blood glucose must be more than 16.7mmol/l, and the urine glucose≥+++, and they ate more, drunk more and the urine of them was more than N group . Detected the body-weight and blood glucose of the rats once a week, and 12 hours before the establishment, they were fed no food but water. All the rats were fed in individual cage, and they were permitted to eat and drink by themselves. They were observed for 8 weeks, and were not treated by insulin. There were 2 rats in D group were knocked-out experiment for the glucose of them<16.7 mmol/L, and 4 rats were dead for infection and ketotic acidot.2 The Pathologic alterations of myocardium were observed under LM by using HE: To embed the tissues which fixed by formalin in paraffin, section them into slice, stain them by hematoxylin and eosin, then take photos through light micrographic.3 The evaluation of the activity of SERCA、MDA、SOD and GSH-Px: The myocardium of 4-week-group and 8-week-goup rats（included diabetic rats and normal rats）were took, and were pulverized into homogenate which density was 10%. To determinate the activity of SERCA, the homogenate were centrifugated in high speed to reperated the sarcoplasmic reticulum , the biochemistry used to assay the activity of SOD、 GSH-Px and MDA.4 The expression of SERCA2αgene in DM rats: 100 kg fresh muscle which preserved in -80℃were took, the total RNA were extracted by using Trizol one-step-method, the RNA was reversed into cDNA, the cDNA was amplificated and different prime were mixed in PCR to amplificated the target gene SERCA2αand house geneβ-actin, the electrophoretic bands were observed, and the ratios of the intension target gene to house gene were calculated.Results: 1 It was believed that the model of diabetic rats was established successfully, for it was observed that: rats in diabetic group ate more, drunk more, but the weight of them decreased. The blood glucose of diabetic rats was more than 16.7 mmol/L and the urine glucose≥+++. Then the conclusion was that the modal of diabetic rats was successful.2 Compared with normal rats, there was no pathological changes of myocardium in 4-week-DM rats, but in 8-week-DM rats, the changes were obviously: the swollen cardiac muscle cells、the pyknotic nucleoli、the hyperchromatic nucleus and the hyalined small arteries.3 Compared with normal control rats, the activity of SERCA、SOD and GSH-Px were descending in diabetic rats, and it was more obvious in 8-week-DM rats. But the quantity of MDA was ascending in diabetic rats, and it was more obvious in 8-week-DM rats.4 Compared with normal control rats, the gene level results indicated: the level of SERCA2αwas decreased in 4-week-DM rats and in 8-week-DM-rats, and it was more obvious in 8-week-DM rats.Conclusions: 1 The model of diabetic rat can be successfully established by injecting STZ.2 There were pathological changes in DM rats: the swollen cardiac muscle cells、the pyknotic nucleoli、the hyperchromatic nucleus and the hyalined small arteries.3 The level of oxidative stress in diabetic rats was increased, and the ability of anti-oxidase was descending, there was peroxidative injury in the cardiac muscle of diabetic rats.4 The activity of SERCA and the mRNA were descending in diabetic rats, and as the extending of the course of the disease, the decreasing was more obviously. The regulation of Ca⁺⁺ was abnormal, the relaxation and contraction of cardiac muscle was injury, cardiomyopathy arose.更多还原