糖尿病大鼠心肌损伤及发病机理的实验研究
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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。
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 parts
1 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.
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。
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 parts
1 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.
引文
1 Schannwell CM, Schneppenheim M, Perings S, et al. Cardiol-logy, 2002;98(1-2):33-39
2 Lon Wu, Wendell, Nicholson, Susan, ET al. Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines. [J] Biol, Chem, 2004,279(13): 12126-12134
3 Strosova M, Skuciovo M, Horakova L, et al. Oxidative damage to Ca++-ATPase sarcoplasmic reticulum by HOC1 and protective effect of some antioxidants. [J] Bio, 2005, 24(1-4):111-6
4 朱禧星,周晓明,何德华,等.链脲佐菌素糖尿病大鼠的心肌病变。中华内分泌代谢杂志,1992,8(4):222
5 Rubller S, Dulgush J, Yuceogl YZ, et al. New type of cardiomyopathy associated with diabetic Glomerulosclerosis.Am J Cardiol.1972, 30: 595
6 Hamby RI, Zoneraich S, Sherman L. Diabetic cardiomy -opathy[J]. JAMA, 1974, 229(13):1749-1754
7 Fein FS, Komstein LB, Strobeck JK, et al. Altered myocardial mechanics in diabetic rats. Circ Res, 1980, 47:922-933
8 Morgan JP, Emy RE, Allen PD, et al. Abnormal intracellular calcium handing, a major cause of systolic and diastolic dysfunction in ventricular myocardium from patients with heart failure [J]. Circulation, 1990, 81(supple):III21-III32
9 Zhong Y, Ahmed S, Grupp IL, et al. Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts [J]. Am J Physiol, 2001, 281(3): H1137-H1147
10 陈群清.心肌肌浆网 Ca++ATPase 与心肌功能的调控[J].第一军医大学学报,1999,19(6):583
11 McCall E, Ginsberg KS, Bassani RA, et al. Ca-flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes. Am J Physiol, 1998, 274: H1348-H1360.
12 He H, Giordano TJ, Hilal-Dandan R et al. Over-expression of the sarcoplasmic reticulum Ca++-ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation. J Clin Tnvest,1997,100:380-389
13 Ji Y, Lalli MJ, Babu GJ, et al. Diaruption of a single copy of the SERCA2 gene results in altered Ca++ homeostasis andcardiomyocyte function. [J] Biol Chem, 2000, 275:38073 -38080
14 Toyofuko T, Kurzydlowski K, Lyton J, et al. The nucleotidebin dingthingle domain plays a crucial roal in determining isoform specific Ca++ dependence for ganellar Ca++-ATPase[J]. Bio, Chem,1999,267:14490-14496
15 Penpargkul S, Fein FS, et al. Depressed cardiac sarcoplamic reticular function from diabetic rats. [J] Mel and Cell Cardiology,1981,13:303-309
16 Ganguly PK, Pierce GN, et al. Defective sarcoplasmic reticular calcium transport in diabetic cardiomyopathy. Am J Physiol, 1983,244:E528-535
17 Teshima Y, Takahashi N, Saikawa T, et al. Piminished expression of sarcoplamic reticulum Ca++-ATPase and ryanodine sensitive Ca++ channel mRNA in treptozocin -induced diabetic rat heart.[J].Mol Cell Cardiol,2000, 32(4):655-664
18 Kim HW, Ch YS, Lee HR, et al. Diabetic alterations in cardiac sarcoplamic reticulum Ca++-ATPase and phosphol -amban protein expression. Life Sci,2001, 70(4): 367-379
19 Netticadan T, Temansah RM, Kent A, et al. Depressed levels of Ca++cycling proteins may underlie sarcoplasmic reticulum dysfunction in the diabetic hear. Diabetes, 2001,50(9): 2133-2138
20 Houser SR, Piacentino V, Weisser J. Abnormalities of calcium cycling in the hypertrophied and failing heart[J]. JMol Cell Cardiol,2000,32(9):1595
21 耿召华,李隆贵,吴强.卡维地洛治疗心力衰竭对大鼠心肌细胞调亡和 Bcl-2、Bax 蛋白表达水平的影响[J]。重庆医学
22 Stocker R and Keaney JF Jr. Role of oxidative modifications in atherosclerosis [J]. Physiol Rev, 2004,84(4): 1381-478
23 Hanman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol, 1956,11(3):298-300
24 McCord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein(hemocuprein). J Biol Chem,1969,244(22):6049-6055
25 Santanam-N, Ramachandran-s, Parthasarathy-S. Oxygen radicals, antioxidants, and lipid peroxidation. Semin-Reprod -Endocrinol. 1998, 16(4): 275-80
26 曹锡清.脂质过氧化对细胞与机体的作用.生物化学与生物物理进展杂志.1986,2:17-23
27 Chen Y, Saari JT, Kang YJ. Weak antioxidant defenses make the heart a target for damage in copper-deficient rats[J]. Free Radic Biol Med,1994,17:529-536
28 Wautier MP, Chappey O, Corda S, et al. Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE[J]. Am J Physiol Endocrinol Metab,2001,280(5):685-94
29 鲁超,邹宇宏,王建青,等.非酒精性脂肪性肝炎病因及发病机制的研究进展[J].安徽医药,2006,10(2):43-55
30 Moorthi VR, Bobby Z, Selvaraj N, et al. Vitamin E protectsthe insulin sensitivity and redox balance in rat L6 muscle cell exposed to oxidative stress [J]. Clin Chim Acta,2006, 367(1-2):132-6
31 Fariss MW, Chan CB, Atel M, et al. Role of mitochondria in toxic oxidative stress[J]. Mol Interv,2005,5(2):94-111
32 Rolo AP and Palmeira CM. Diabetes and mitochondrial function: role of hypercemia and oxidative stress[J]. Toxicol Appl Phaemacol,2006,212(2):167-78
33 Anderwald C, and Roden M. Adipotoxicity and the insulin resistance syndrome[J]. Pediatr Endocinol Rev,2004, 1(3) :310-9
34 Anabela PR, Carlos MP. Diabetes and mitochodrial function: Role of hyperglycemia and oxidative stress [J]. Toxicol Appl Pharm,2006, 212(2):167-78
35 Bruner SD, Norman DP, Verdine GL. Structural basia for recognition and reparie of the endogenous mutagen 8-oxogua-nine in DNA [J]. Nature, 2000,403(6772): 859-866
36 Makropoulos W, Kocher K, Heintz B, etal. Urinary thymidine glycol as a biomarker for oxidative stress after kidney transplantation[J]. Renal Failure,2000,22(4):499-510
37 方允中,生物体内自由基的产生和清除.见:方允中,王文杰.自由基与酶:基础理论及其生物学和医学中的应用[M].北京科学出版社,1989:147
38 Scherer NM, Deamer DW. Oxidative stress impairs the function of sarcoplasmic reticulum by oxidation ofsulfhydryl groups in the Ca++-ATP. Arch Biochem Biophys JT.1986,246(2):289-601
39 Horakova L, Strosova M, Skuciova M. Antioxidants prevented oxidative injury of SR induced by Fe++/H2O2 /ascorbate system but failed to prevent Ca++-ATPase activity decrease. Biofactors JT.2005, 24(1-4):105-9
40 Kaplan P, Babusikova E, Lehotsky J, et al. Free r5adical-induced protein modification and inhibition of Ca++-ATPase of cardiac sarcoplasmic reticulum. Mol Cell Biochem JT,2003,248(1-2):41-7
1 Rullber S, Dulgush J, Yuceogl YZ. et al. New type ofcardio -myopathy associated with diabetic Glomerulosclerosis. AmJ Cardiol 1972; 30: 595
2 Galderisi M.Diastolic dysfunction and diabetic cardiom -yopathy : evaluation by Doppler echocardiography[J].J Am Coll Cardiol,2006;481548-1551
3 Ren J, Gintant GA, Miller RE, et al.High extracellular glucose impairs cardiac E2c coupling in a glycosylation dependent manner[J].Am J Physiol,1997,273:H2876-2883
4 Way KJ,Katai N,King GL. Protein Kinase C and the development of diabetic vascular complications[J].Diabet Med,2001;18:945-959
5 Joffe RT, Travers KE, Micale CL, et al.Abnormal cardiac function in the streptozotocin-induced non-insulin -dependent diabetic rat:noninvasive assessment with Doppler echocardiography and contribution of the nitric oxide pathway[J].J Am Coll Cardiol,1999;34:2111-2119
6 De Flines J, Scheen AJ. Diabetes mellitus and congestive heart failure: physiopathology and treatment. Rev Med Suisse,2006;2:1893-1896,1898-1900
7 Poirier P,Bogaty P,Garneau C,et al. Diabetic dysfuction in normotensive men with well-controlled type 2 diabetes : importance of maneuvers in echocardiographic screening for preclinical diabetic cardiomopathy. Diabetes Care,2001;24:5-10
8 梁自文,张忠辉,张平,等.糖尿病性心肌病大鼠的心肌结构、代谢和功能的变化.第三军医大学学报 1996;18 (8): 306-308
9 朱禧星,周晓明,何德华,等.链脲佐菌素糖尿病大鼠的心肌病变.中华内分泌代谢杂志 1992; 8(4): 222
10 王瑞平,钱贻简,陈曼丽.老年人糖尿病心肌病的临床与病理改变.中华内科杂志 1998; 37: 677-679
11 任俊,糖尿病心肌病的基础与临床研究现状。心血管病学进展 2001;22(5):299-302
12 Nielen LB,Bartels ED,Bollano E. Over expression of apolipoprotein B in the heart impedes cardiac triglyceride accumulation and development of cardiac dysfuction in diabetic mice. [J]. Bid Chem,2002;277(30):27014-27020
13 Fiordaliso F, Bianchi R, Staszewsky L, et al.Antioxidant treatment attenuates hyperglycemia-inducedcardiomyocyte death in rats. J Mol Cell cardiol,2004;37:959-968
14 Cai L,Liw,Wang G,et al. Diabetes,2002;51(6):1938-1948
15 Cai L,Kang YJ. Cardiovasc Toxicol,2001;1(3):181-193
16 Golfman LS, Wilson CR, Sharma S, et al .Activation of Ppargamma enhances myocardial glucose oxidation and improves contractile function in isolated working hearts of ZDF rats. Am J. Physiol Endocrinol Metab,2005;289: E328 -E336
17 陈力平.糖尿病心肌病.中国厂矿医学 2001; 14(1): 69-71
18 Listenberger LL,Schaffer JE.Trends. CardiovascMed,2002;12(3):134-138
19 Vecchini A,Del Rosso F,Binoglia L,et al. J Mol cell Caroliol,2002;32(6):1061-1074
20 Young LH. Diastolic function and type 1 diabetes. Diabetes Care,2004,27:2081-2083
21 Trost SV,Belke DD,Bluhm WF,et al. Overexpression of the sarcoplasmic reticulum Ca2+-ATPase improves myocardial contractility in diabetic cardiomyopathy [J] . Diabetes,2002; 51(4):1166-1171
22 Brown L, Wall D, Marchant C, et al. Tissuespecificchanges in angiotensin Ⅱreceptors in streptozotoc indiabetic rats. JEndocrinol 1997; 154: 355
23 边延涛,王国宏,袁申元,等.氯沙坦对实验性糖尿病大鼠心肌的保护作用.中华心血管病杂志,2000; 28(4): 293-296
24 Ledet T,Vuust J. Arterial procollagen typeⅠ ,and type Ⅲ,and fibronectin:effects of diabetic serum,glucose insulin ,ketone, and growth hormone studied on rabbit aortic myomedial cell cultures [J]. Diabetes,1997;26(8):798-803
25 Chen Y, Saari JT, Kang YJ. Weak antioxidant defenses make the heart a target for damage in copper-deficient rats[J]. Free Radic Biol Med,1994,17:529-536
26 Vecchini A,Del Rosso F,Binaglia L,et al. Molecular defects in sardemmal glycerophospholipid subclasses in diabetic cardiomoopathy. J Moll Cell Cardio,2002;28:293-296
27 从丽,俞茂华,李益明,等.心肌胶原代谢变化在糖尿病心肌病变发生中作用的实验研究 [J] 中国临床康复,2004;8(9):1655
28 Nishikawa N, Yamamoto K, Sakata Y, et al. Differential activation of matrix metalloproteinase in heart failure without ventricular dilatation [J] . Cardiovasc Res,2003, 57(3):766-774
29 Hileeto D, Cukiernik M, Mukherjee S, et al. Diabetes Metab Res Rev,2002;18(5):386-394
30 Sun Y, Zhang J, Zhang JQ, et al. Local angiotensinII and transforming growth factor β1 in renal fibrosis of rats. Hypertension,2004,35:1078-1084
31 Li JP, Petrov V, Rumilla K, et al . Transforming growth factor beta-1 promotes contraction of collagen gel by cardia fibroblasts through their differentiation into myofibroblasts. Methods Find Exp Clin Pharma-col,2003,25:79-86
32 Colligan PB, Relling DP, Ren J. Cell Mol Biol(Noisy-legrand),2002;48 Online Pub:OL251-OL257
33 Krishnawamy G, Hall K, Youngberg G, et al. J Interferon Cytokine Res,2002;22(3):379-388
2 Lon Wu, Wendell, Nicholson, Susan, ET al. Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines. [J] Biol, Chem, 2004,279(13): 12126-12134
3 Strosova M, Skuciovo M, Horakova L, et al. Oxidative damage to Ca++-ATPase sarcoplasmic reticulum by HOC1 and protective effect of some antioxidants. [J] Bio, 2005, 24(1-4):111-6
4 朱禧星,周晓明,何德华,等.链脲佐菌素糖尿病大鼠的心肌病变。中华内分泌代谢杂志,1992,8(4):222
5 Rubller S, Dulgush J, Yuceogl YZ, et al. New type of cardiomyopathy associated with diabetic Glomerulosclerosis.Am J Cardiol.1972, 30: 595
6 Hamby RI, Zoneraich S, Sherman L. Diabetic cardiomy -opathy[J]. JAMA, 1974, 229(13):1749-1754
7 Fein FS, Komstein LB, Strobeck JK, et al. Altered myocardial mechanics in diabetic rats. Circ Res, 1980, 47:922-933
8 Morgan JP, Emy RE, Allen PD, et al. Abnormal intracellular calcium handing, a major cause of systolic and diastolic dysfunction in ventricular myocardium from patients with heart failure [J]. Circulation, 1990, 81(supple):III21-III32
9 Zhong Y, Ahmed S, Grupp IL, et al. Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts [J]. Am J Physiol, 2001, 281(3): H1137-H1147
10 陈群清.心肌肌浆网 Ca++ATPase 与心肌功能的调控[J].第一军医大学学报,1999,19(6):583
11 McCall E, Ginsberg KS, Bassani RA, et al. Ca-flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes. Am J Physiol, 1998, 274: H1348-H1360.
12 He H, Giordano TJ, Hilal-Dandan R et al. Over-expression of the sarcoplasmic reticulum Ca++-ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation. J Clin Tnvest,1997,100:380-389
13 Ji Y, Lalli MJ, Babu GJ, et al. Diaruption of a single copy of the SERCA2 gene results in altered Ca++ homeostasis andcardiomyocyte function. [J] Biol Chem, 2000, 275:38073 -38080
14 Toyofuko T, Kurzydlowski K, Lyton J, et al. The nucleotidebin dingthingle domain plays a crucial roal in determining isoform specific Ca++ dependence for ganellar Ca++-ATPase[J]. Bio, Chem,1999,267:14490-14496
15 Penpargkul S, Fein FS, et al. Depressed cardiac sarcoplamic reticular function from diabetic rats. [J] Mel and Cell Cardiology,1981,13:303-309
16 Ganguly PK, Pierce GN, et al. Defective sarcoplasmic reticular calcium transport in diabetic cardiomyopathy. Am J Physiol, 1983,244:E528-535
17 Teshima Y, Takahashi N, Saikawa T, et al. Piminished expression of sarcoplamic reticulum Ca++-ATPase and ryanodine sensitive Ca++ channel mRNA in treptozocin -induced diabetic rat heart.[J].Mol Cell Cardiol,2000, 32(4):655-664
18 Kim HW, Ch YS, Lee HR, et al. Diabetic alterations in cardiac sarcoplamic reticulum Ca++-ATPase and phosphol -amban protein expression. Life Sci,2001, 70(4): 367-379
19 Netticadan T, Temansah RM, Kent A, et al. Depressed levels of Ca++cycling proteins may underlie sarcoplasmic reticulum dysfunction in the diabetic hear. Diabetes, 2001,50(9): 2133-2138
20 Houser SR, Piacentino V, Weisser J. Abnormalities of calcium cycling in the hypertrophied and failing heart[J]. JMol Cell Cardiol,2000,32(9):1595
21 耿召华,李隆贵,吴强.卡维地洛治疗心力衰竭对大鼠心肌细胞调亡和 Bcl-2、Bax 蛋白表达水平的影响[J]。重庆医学
22 Stocker R and Keaney JF Jr. Role of oxidative modifications in atherosclerosis [J]. Physiol Rev, 2004,84(4): 1381-478
23 Hanman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol, 1956,11(3):298-300
24 McCord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein(hemocuprein). J Biol Chem,1969,244(22):6049-6055
25 Santanam-N, Ramachandran-s, Parthasarathy-S. Oxygen radicals, antioxidants, and lipid peroxidation. Semin-Reprod -Endocrinol. 1998, 16(4): 275-80
26 曹锡清.脂质过氧化对细胞与机体的作用.生物化学与生物物理进展杂志.1986,2:17-23
27 Chen Y, Saari JT, Kang YJ. Weak antioxidant defenses make the heart a target for damage in copper-deficient rats[J]. Free Radic Biol Med,1994,17:529-536
28 Wautier MP, Chappey O, Corda S, et al. Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE[J]. Am J Physiol Endocrinol Metab,2001,280(5):685-94
29 鲁超,邹宇宏,王建青,等.非酒精性脂肪性肝炎病因及发病机制的研究进展[J].安徽医药,2006,10(2):43-55
30 Moorthi VR, Bobby Z, Selvaraj N, et al. Vitamin E protectsthe insulin sensitivity and redox balance in rat L6 muscle cell exposed to oxidative stress [J]. Clin Chim Acta,2006, 367(1-2):132-6
31 Fariss MW, Chan CB, Atel M, et al. Role of mitochondria in toxic oxidative stress[J]. Mol Interv,2005,5(2):94-111
32 Rolo AP and Palmeira CM. Diabetes and mitochondrial function: role of hypercemia and oxidative stress[J]. Toxicol Appl Phaemacol,2006,212(2):167-78
33 Anderwald C, and Roden M. Adipotoxicity and the insulin resistance syndrome[J]. Pediatr Endocinol Rev,2004, 1(3) :310-9
34 Anabela PR, Carlos MP. Diabetes and mitochodrial function: Role of hyperglycemia and oxidative stress [J]. Toxicol Appl Pharm,2006, 212(2):167-78
35 Bruner SD, Norman DP, Verdine GL. Structural basia for recognition and reparie of the endogenous mutagen 8-oxogua-nine in DNA [J]. Nature, 2000,403(6772): 859-866
36 Makropoulos W, Kocher K, Heintz B, etal. Urinary thymidine glycol as a biomarker for oxidative stress after kidney transplantation[J]. Renal Failure,2000,22(4):499-510
37 方允中,生物体内自由基的产生和清除.见:方允中,王文杰.自由基与酶:基础理论及其生物学和医学中的应用[M].北京科学出版社,1989:147
38 Scherer NM, Deamer DW. Oxidative stress impairs the function of sarcoplasmic reticulum by oxidation ofsulfhydryl groups in the Ca++-ATP. Arch Biochem Biophys JT.1986,246(2):289-601
39 Horakova L, Strosova M, Skuciova M. Antioxidants prevented oxidative injury of SR induced by Fe++/H2O2 /ascorbate system but failed to prevent Ca++-ATPase activity decrease. Biofactors JT.2005, 24(1-4):105-9
40 Kaplan P, Babusikova E, Lehotsky J, et al. Free r5adical-induced protein modification and inhibition of Ca++-ATPase of cardiac sarcoplasmic reticulum. Mol Cell Biochem JT,2003,248(1-2):41-7
1 Rullber S, Dulgush J, Yuceogl YZ. et al. New type ofcardio -myopathy associated with diabetic Glomerulosclerosis. AmJ Cardiol 1972; 30: 595
2 Galderisi M.Diastolic dysfunction and diabetic cardiom -yopathy : evaluation by Doppler echocardiography[J].J Am Coll Cardiol,2006;481548-1551
3 Ren J, Gintant GA, Miller RE, et al.High extracellular glucose impairs cardiac E2c coupling in a glycosylation dependent manner[J].Am J Physiol,1997,273:H2876-2883
4 Way KJ,Katai N,King GL. Protein Kinase C and the development of diabetic vascular complications[J].Diabet Med,2001;18:945-959
5 Joffe RT, Travers KE, Micale CL, et al.Abnormal cardiac function in the streptozotocin-induced non-insulin -dependent diabetic rat:noninvasive assessment with Doppler echocardiography and contribution of the nitric oxide pathway[J].J Am Coll Cardiol,1999;34:2111-2119
6 De Flines J, Scheen AJ. Diabetes mellitus and congestive heart failure: physiopathology and treatment. Rev Med Suisse,2006;2:1893-1896,1898-1900
7 Poirier P,Bogaty P,Garneau C,et al. Diabetic dysfuction in normotensive men with well-controlled type 2 diabetes : importance of maneuvers in echocardiographic screening for preclinical diabetic cardiomopathy. Diabetes Care,2001;24:5-10
8 梁自文,张忠辉,张平,等.糖尿病性心肌病大鼠的心肌结构、代谢和功能的变化.第三军医大学学报 1996;18 (8): 306-308
9 朱禧星,周晓明,何德华,等.链脲佐菌素糖尿病大鼠的心肌病变.中华内分泌代谢杂志 1992; 8(4): 222
10 王瑞平,钱贻简,陈曼丽.老年人糖尿病心肌病的临床与病理改变.中华内科杂志 1998; 37: 677-679
11 任俊,糖尿病心肌病的基础与临床研究现状。心血管病学进展 2001;22(5):299-302
12 Nielen LB,Bartels ED,Bollano E. Over expression of apolipoprotein B in the heart impedes cardiac triglyceride accumulation and development of cardiac dysfuction in diabetic mice. [J]. Bid Chem,2002;277(30):27014-27020
13 Fiordaliso F, Bianchi R, Staszewsky L, et al.Antioxidant treatment attenuates hyperglycemia-inducedcardiomyocyte death in rats. J Mol Cell cardiol,2004;37:959-968
14 Cai L,Liw,Wang G,et al. Diabetes,2002;51(6):1938-1948
15 Cai L,Kang YJ. Cardiovasc Toxicol,2001;1(3):181-193
16 Golfman LS, Wilson CR, Sharma S, et al .Activation of Ppargamma enhances myocardial glucose oxidation and improves contractile function in isolated working hearts of ZDF rats. Am J. Physiol Endocrinol Metab,2005;289: E328 -E336
17 陈力平.糖尿病心肌病.中国厂矿医学 2001; 14(1): 69-71
18 Listenberger LL,Schaffer JE.Trends. CardiovascMed,2002;12(3):134-138
19 Vecchini A,Del Rosso F,Binoglia L,et al. J Mol cell Caroliol,2002;32(6):1061-1074
20 Young LH. Diastolic function and type 1 diabetes. Diabetes Care,2004,27:2081-2083
21 Trost SV,Belke DD,Bluhm WF,et al. Overexpression of the sarcoplasmic reticulum Ca2+-ATPase improves myocardial contractility in diabetic cardiomyopathy [J] . Diabetes,2002; 51(4):1166-1171
22 Brown L, Wall D, Marchant C, et al. Tissuespecificchanges in angiotensin Ⅱreceptors in streptozotoc indiabetic rats. JEndocrinol 1997; 154: 355
23 边延涛,王国宏,袁申元,等.氯沙坦对实验性糖尿病大鼠心肌的保护作用.中华心血管病杂志,2000; 28(4): 293-296
24 Ledet T,Vuust J. Arterial procollagen typeⅠ ,and type Ⅲ,and fibronectin:effects of diabetic serum,glucose insulin ,ketone, and growth hormone studied on rabbit aortic myomedial cell cultures [J]. Diabetes,1997;26(8):798-803
25 Chen Y, Saari JT, Kang YJ. Weak antioxidant defenses make the heart a target for damage in copper-deficient rats[J]. Free Radic Biol Med,1994,17:529-536
26 Vecchini A,Del Rosso F,Binaglia L,et al. Molecular defects in sardemmal glycerophospholipid subclasses in diabetic cardiomoopathy. J Moll Cell Cardio,2002;28:293-296
27 从丽,俞茂华,李益明,等.心肌胶原代谢变化在糖尿病心肌病变发生中作用的实验研究 [J] 中国临床康复,2004;8(9):1655
28 Nishikawa N, Yamamoto K, Sakata Y, et al. Differential activation of matrix metalloproteinase in heart failure without ventricular dilatation [J] . Cardiovasc Res,2003, 57(3):766-774
29 Hileeto D, Cukiernik M, Mukherjee S, et al. Diabetes Metab Res Rev,2002;18(5):386-394
30 Sun Y, Zhang J, Zhang JQ, et al. Local angiotensinII and transforming growth factor β1 in renal fibrosis of rats. Hypertension,2004,35:1078-1084
31 Li JP, Petrov V, Rumilla K, et al . Transforming growth factor beta-1 promotes contraction of collagen gel by cardia fibroblasts through their differentiation into myofibroblasts. Methods Find Exp Clin Pharma-col,2003,25:79-86
32 Colligan PB, Relling DP, Ren J. Cell Mol Biol(Noisy-legrand),2002;48 Online Pub:OL251-OL257
33 Krishnawamy G, Hall K, Youngberg G, et al. J Interferon Cytokine Res,2002;22(3):379-388