【作者】 杨红英；

【导师】 李树清； 俞志成；

【作者基本信息】 昆明医学院 ， 外科学， 2009， 博士

【摘要】 第一部分实验性2型糖尿病大鼠模型及观测指标的建立目的建立具有胰岛素抵抗特征及病理过程近似人类2型糖尿病的大鼠模型,并监测血糖、胰岛素和血脂等与血管神经病变密切相关的指标。方法8周龄SD雄性大鼠120只,随机分为30只对照组和90只造模组。对照组大鼠用普通饲料喂养。造模组大鼠用高脂高糖膳食喂养4周,第一次腹腔注射链脲佐菌素（STZ,25mg/kg）,检测随机血糖水平。四周后第二次腹腔注射（STZ,40mg/kg）,检测随机血糖水平,酶比色法检测糖化血红蛋白。全自动生化分析仪检测血脂水平,放射免疫分析法测定空腹胰岛素并计算胰岛素敏感指数（insulin sensitivityindex,ISI）。以随机血糖≥13.8mmol/L者为成模标准。结果糖尿病大鼠模型血脂升高:LDL为0.75±0.32mmol/L,TC为1.43±1.07mmol/L,TG为1.72±0.6mmol/L,与对照组相比有统计学意义（P＜0.05）;胰岛素敏感性降低,敏感性指数为:-2.61±0.34,与对照组相比有统计学意义（P＜0.05）;糖化血红蛋白升高:GHb为6.71±1.37%,与对照组相比有统计学意义（P＜0.05）。结论1.高脂膳食喂养结合不同STZ剂量两次注射能成功复制既近似于人类2型糖尿病致病机制又相对简便易得的实验性2型糖尿病大鼠模型。2.模型具有高血糖、高血脂、胰岛素抵抗等2型糖尿病特征,是研究2型糖尿病发病机制和药物研究的理想动物模型。3.模型存在血脂代谢障碍,高血糖和胰岛素抵抗等致血管神经病变并发症发生的重要因素,为观察分析糖尿病实验动物血管和神经病变奠定了良好基础。模型长期稳定性良好,适用于糖尿病慢性并发症的发病机制研究。第二部分实验性2型糖尿病大鼠脑组织病变与氧化应激关系的研究目的:糖尿病脑神经病变是其重要并发症,但机理仍未完全清楚。利用糖尿病鼠模型,观测氧自由基、抗氧化酶和醛糖还原酶（aldose reductase,AR）与脑组织损伤的关系以及血糖浓度对各观测指标的影响,揭示高血糖、氧化应激与微血管损伤及脑组织病理改变之间的内在联系。阐明与糖尿病血管神经病变密切相关的因素。为糖尿病脑神经病变并发症的预防和治疗提供可靠的实验室依据。方法:正常对照组大鼠60只,糖尿病鼠143只为糖尿病组,糖尿病组以空腹血糖分组:13.8mmol/L＜血糖＜16.7mmol/L为L组;血糖＞16.7mmol/L为H组。分别在8周、12周、16周采集血和脑组织标本,制作病理切片,待检。①光学显微镜和电镜观察脑组织病理损伤;②免疫组化和免疫印迹技术检测超氧化物歧化酶（superoxide dismutase,SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase,GP_x）、过氧化氢酶（catslase,CAT）、AR在脑组织的表达;③RT-PCR检测血SOD、GP_x以及血、脑AR mRNA表达;④紫外分光光度法及化学比色法测定血清O₂^-、H₂O₂、SOD、CAT水平,NADPH减少法测定脑组织和血清中的AR活性;⑤荧光免疫和酶法检测抗中性粒细胞胞浆抗体。结果:1.糖尿病鼠脑组织病理改变:光学显微镜显示16周脑组织血管内皮细胞肿胀,胞核不清,胞浆稀疏,周细胞退行性变。部分微血管管壁玻璃样变性,管壁塌陷呈波状。胶质细胞局灶性增生,颗粒层变薄。电子显微镜显示脑组织血管和神经组织损伤随大等病变。病程初期大部分神经细胞正常,12周少数神经细胞皱缩,16周神经细胞皱缩多见,核浓缩,胞膜及核膜皱缩。线粒体显示由增生、数目增多、轻度肿大到肿胀明显、固缩的病理损伤过程。不同血糖浓度引起的病理改变在同样病程不同组表现不同,H组的病理损伤比L组严重。2.糖尿病组血O₂^-活性明显高于对照组（p＜0.05）,H组与L组相比无统计学意义（p＞0.05）,糖尿病组血H₂O₂含量明显高于对照组,有非常显著的差异（p＜0.01）,H组与L组相比差异有统计学意义（p＜0.05）。3.糖尿病组血GP_x、Cu-Zn-SOD、Mn-SOD mRNA表达均明显高于对照组（p＜0.05）,L组与H组相比无显著差异（p＞0.05）,糖尿病组血和脑组织匀浆AR mRNA表达明显高于对照组（p＜0.05）,L组与H组相比,血与脑组织相比均无统计学意义（p＞0.05）。4.糖尿病鼠血GP_x、SOD活性明显低于对照组（p＜0.05）、CAT活性与对照组相比无统计学意义（p＞0.05）,AR活性明显高于对照组（p＜0.05）,SOD活性H组明显低于L组（p＜0.05）,GP_x、CAT及AR活性H组与L组比较无统计学意义（p＞0.05）,糖尿病鼠脑组织AR活性明显高于对照组（p＜0.05）,L组与H组相比无统计学意义（p＞0.05）,糖尿病鼠血与脑组织AR活性增长率比较无统计学意义（p＞0.05）。5.SOD、GP_x、CAT在脑组织的表达随病程进展逐渐减少。AR的表达在病程8周和12周表达量明显比正常增加,16周较前减少。GP_x、CAT、SOD及AR在糖尿病鼠和对照组大鼠的脑皮质、海马和血管均有表达,SOD主要在胞浆及胞膜表达,CAT和GP_x主要在胞浆表达,AR主要在胞核及核膜表达。6.人和鼠正常对照组ANCA均未检出阳性,糖尿病患者中检出4例阳性,糖尿病鼠中检出1例阳性。结论:1.糖尿病鼠脑血管及神经组织均出现程度不等的病理改变,且随病程进展日趋明显。血糖浓度较高的H组比L组病理损伤发生早、进展快。表明存在明显的脑组织损伤且与病程和血糖水平密切相关。2.糖尿病鼠血氧自由基水平升高,抗氧化酶基因表达增加,抗氧化酶活性降低。表明糖尿病鼠全身存在氧化应激增强的证据。SOD、GP_x、CAT在脑组织的表达呈现病程初期增高随病程进展逐渐下降的趋势,表明糖尿病氧化应激在脑组织有明显表现。3.糖尿病鼠血H₂O₂含量和SOD活性在不同血糖浓度组差异有统计学意义,表明其活性改变与血糖浓度密切相关。O₂^-及GPx、CAT活性;SOD、GPx基因表达均显示不同血糖浓度组差异无统计学意义,提示血糖浓度改变对这些因素无明显影响。可能是单纯控制血糖不能有效治疗和预防糖尿病及其并发症的原因之一。4.糖尿病鼠血及脑组织AR mRNA表达、血AR活力均明显高于对照组,脑组织AR表达随病程进展增加,与同时发生的脑组织病变密切相关,表明AR活性升高是脑组织损伤的重要原因。脑组织AR表达量16周时H组低于L组,提示AR与血糖浓度的关系不是随之增长的线性关系。5.人和鼠正常对照组ANCA均未检出阳性,糖尿病患者检出4例阳性和糖尿病鼠各检出1例阳性。提示ANCA在糖尿病微血管炎症中不起主要作用,但可能是病因之一。糖尿病大鼠脑组织存在明显的氧化应激和AR含量改变且与脑组织血管和神经病变密切相关,氧化应激与AR交互作用共同促进了糖尿病脑损伤的发生。氧自由基、抗氧化酶、AR的变化与血糖浓度的关系呈现不同的规律性,可能是抗氧化和ARI治疗糖尿病神经病变效果出现差异的重要原因。更多还原

【Abstract】 Objective To establish an animal model similar to the metabolic abnormalities of human type 2 diabetes mellitus with insulin resistance,hyperglycemia,hyperlipemia and hyperinsulinism which could induce complication of angioneuropathy.Methods Eight-week-old SD male rats（n=120） were radomly divided into control group and model group.The model group male SD rats（n=90） were fed with high-fat diet that contained unsaturated fatty acid for 4 weeks and then streptozotion（STZ, one-step,25mg/kg） was injected intraperitoneally,then streptozotion（STZ,40 mg/kg） was injected intraperitoneally again 4 weeks later to result in insulin-secretion defect and hyperglycemia.The control group（n=30） were fed with normal diet.Blood lipid was detected by automatic biochemical equipments.Results Biochemical parameters proved that the two-step-treatment methods could develop the type 2 diabetic rat model,the successfully executive rate was 76%.The model was similar to the metabolic abnormalities of human type 2 diabetes mellitus with insulin resistance、hyperglycemia、hyperlipemia and hyperinsulinism.In the type 2 diabetic group,LDL was 0.75±0.32mmol/L,TC was 1.43±1.07 mmol/L,TG was 1.72±0.6 mmol/L,insulin was 42.36±9.57 mIU/L,GHb was 6.71±1.37%.There were statistical significance of these indexes between type 2 diabetic group and control group（P value＜0.05）.Conclusion1.Relatively convenient and easily got experimental type 2 diabetic rat model which reproduced human type 2 diabetic pathogenic mechanism was successfully induced by high-fat diet and twice different dose of STZ injection.2.The model exhibited type 2 diabetic characters of hyperglycemia,hyperlipemia and insulin resistance.It was the ideal animal model for investigating mechanism of type 2 diabetic pathogenic mechanism and medicine research.3.The model presented lipoidosis metabolic obstruction,hyperglycemia and insulin resistance which were the key factors of vasoneuropathy complication.This model was of long term stability and suitable for pathogenesy research of diabetic chronic complications which established a good foundation for observing vasoneuropathy of diabetic experimental animals. Objective:Diabetic neuropathy is an important complication of type two diabetes,but its mechanism is still unclear.We have establish a type 2 diabetic rat model in order to investigate the relationship of aldose reductase（AR）,O₂^—,catalase（CAT） and glutathion peroxidase（GPx） with brain tissue damage,and observe the impact of blood glucose concentration on each observed index to illuminate internal association between hyperglycosemia,oxidative stress,vasculitides and brain pathology,and to reveal close factors of diabetic vasoneuropathy in order to provide reliable laboratory proof for prevention and treatment of diabetic brain neuropathy.Methods:203 healthy adult male SD rats were randomly divided into control group（n=60） and diabetic group（n=143）.The diabetic group was further divided into L group（13.8mmol/L＜blood glucose＜16.7mmol/L） and H group（blood glucose＞16.7 mmol/L）.Blood and brain tissues were taken at 8th week,12th week and 16th week and have been made into pathological sections.Pathological injurey of brain tissue was observed through optical microscope and electron microscope.Expressions of hyperoxide,glutathion peroxidase,catalase and aldose reductase in brain were detected by immunohistochemistry and immunoblot essay.Messenger ribonucleic acid expressions of hyperoxide,glutathion peroxidase and aldose reductase were tested by reverse transcription polymerase chain reaction.The serum O₂^—、H₂O₂、SOD、CAT levels were measured by ultraviolet spectrophotometry and activity of aldose reductase was tested by NADPH decreasing method.Antineutrophil cytoplasmic antibody was detected by immunofluorescence and enzymic method.Results:1.There were brain tissue vascular endothelial cell swelling,dim nucleus, sparseness of endochylema,perithelial cell’s degeneration,glassy degeneration of capillary vessel wall,crinkle collapse of vessel wall,focal hyperplasia of gliocyte and attenuation of GRL under optical microscope at the 16th week.Injury of brain blood vessel and neuron was gradually aggravated following course of disease.Pathology induced by different blood glucose concentration exhibited diversely in different groups. Pathology of H group was more severe than that of L group.2.The O₂^- activity in diabetic groups were significantly lower than that in control group（P＜0.05）.There was no statistical difference of O₂^- activity between diabetic group L and diabetic group H（P＞0.05）.The H₂O₂ activity in diabetic groups were significantly higher than that in control group（P＜0.01）.There was statistical difference of H₂O₂ activity between diabetic group L and diabetic group H（P＜0.05）.3.The serum messenger ribonucleic acid expressions of GP_x、Cu-Zn-SOD、Mn-SOD in diabetic group were all higher than that in control group（p＜0.05）.There was no statistical difference of mRNA expressions between diabetic group L and diabetic group H（P＞0.05）.Aldose reductase mRNA expression of serum and brain tissue bomogenate in diabetic group was obviously higher than that in control group（p＜0.05）,but there was no statistical difference of aldose reductase mRNA expression between diabetic group L and diabetic group H（P＞0.05） both in serum and in brain tissue bomogenate.4.Both AR mRNA expression and AR activity of serum and brain tissue in diabetic group were significantly higher than that in control group.The increased AR expression of brain tissue had close correlation with pathological changes in brain,and it indicated high AR expression was a main reason of brain tissue damage.AR expression of brain tissue in diabetic H group was lower than in L group,which hinted it was not a simple linear relationship between AR and blood glucose.5.The expressions of SOD,GPX and CAT in brain tissue grew downwards gradually with the course of disease.AR expressions rised at the 8th and 12th week,and declined at the 16th week.There were both expressions of GP_x,CAT,SOD and AR in diabetic rats and control group rats’pallium,cornu ammonis and blood vessels,SOD expression mainly located in kytoplasm and cellular membrane,CAT and GP_x expressed in kytoplasm,and expression of AR concentrated in nucleus and nuclear membrane. 6.Antineutrophil cytoplasmic antibody（ANCA） was detected neither in human nor in rat control group,but there was one probable positive case diabetic rat group.and four positive case in diabetic human group.Conclusion1.There were pathological changes in brain,blood vessels,and nerve tissue of diabetic rats to different extents,and which turned to be more obvious with the developmental course of disease.Pathological changes of H group with higher blood glucose took place more early and quickly than in L group,which indicated apparent brain tissue damages correlated intimately with course of disease and blood glucose level.2.In diabetic rats,oxygen free radical level rised,gene expression of antioxidase increased but activity of antioxidase decreased,and the difference had statistical significance compared with control group,which offered a proof of strengthened oxidative stress in the whole body of diabetic rats.SOD、GP_x and CAT expressions in brain tissue were raised up at initial stage and decreased gradually with development of course of disease,which indicated obviously strengthened oxidative stress in brain tissue.3.There was statistical difference of H₂O₂ contents and SOD activity in rats serum between diabetic groups with different blood glucose concentration,and it indicated a close correlation between activity of H₂O₂ and SOD with blood glucose concentration. There were no statistical difference of O₂^—,GPx,CAT activity and gene expressions of SOD and GPx between diabetic groups with different blood glucose concentration. Blood glucose concentration didn’t have obvious effects on these factors,and it might be one of the reasons for bad preventive and curing effects on diabetic complications just merely by blood glucose control.4.Both AR mRNA expression and AR activity of serum and brain tissue in diabetic group were significantly higher than that in control group.The increased AR expression of brain tissue homogenate had close correlation with pathological changes in brain,and it indicated high AR expression was a main reason of brain tissue damage. AR expression of brain tissue in diabetic H group was lower than in L group,which hinted AR didn’t have linear relationship with rising of blood glucose.5.Antineutrophil cytoplasmic antibody（ANCA） was detected neither in human nor in rat control group,but there was one probable positive case diabetic rat group.and four positive case in diabetic human group,and it manifested that ANCA did not take the principal role in capillary inflammation,but it might be one of the etiological factors of diabetes.There were evident oxidative stress and aldose reductase alteration in diabetic rat brain tissue,and they had close correlation with vasculopathy and neuropathy in brain tissue.Oxidative stress and aldose reductase promoted diabetic brain damage in common.The relationship of oxyradical,antioxidase and aldose reductase with blood glucose concentration showed different regularities,and it might be the key reasons for altered curing effects of anti-oxidized therapia and aldose reductase inhibitor on diabetic neuropathy.更多还原