【作者】 李永智；

【导师】 史本康；

【作者基本信息】 山东大学 ， 外科学， 2008， 硕士

【摘要】 研究目的探讨糖尿病膀胱发生纤维化的机制以及氧化应激在糖尿病膀胱进程中的作用,同时观察生长因子抑制剂（太得恩）在改善糖尿病膀胱病变进程中的作用。研究材料、方法一建立糖尿病大鼠模型取30只3月龄（200±10g）雄性Wistar大鼠随机分为3组,分笼饲养。分别为对照组（n=10）;糖尿病组（n=10）;治疗组（n=10）。建模成功后饲养4周,然后对治疗组大鼠给予太得恩药物(100mg.Kg^-1.d^-1,溶于2ml花生油中,胃管注入)治疗4周,对照组及糖尿病组大鼠给予同等剂量花生油赋形剂。以上动物共饲养8周。8周后处死完整切取膀胱组织。二糖尿病膀胱中纤维化的检测1采用碱水解法检测膀胱组织中羟脯氨酸的含量采用碱水解法检测各组膀胱组织中羟脯氨酸的含量,用方差分析比较各组之间羟脯氨酸的表达差异,探讨糖尿病病变时膀胱纤维化的改变。2采用免疫组化、RT-PCR及ELISA方法检测膀胱组织中TGFβ1、bFGF的表达分别采用免疫组化、RT-PCR及ELISA方法检测各组膀胱组织中TGFβ1、bFGF的表达水平,用方差分析比较各组之间TGFβ1、bFGF的表达差异,探讨糖尿病病变时膀胱组织中TGFβ1、bFGF表达的变化。三糖尿病膀胱中氧化应激状态的检测1采用生化方法检测各组膀胱组织中过氧化氢酶（CAT）活性、超氧化物歧化酶（SOD）活性、丙二醛（MDA）水平2采用免疫组化方法检测膀胱组织中可诱导一氧化氮合酶（iNOS）水平采用免疫组化方法检测各组膀胱组织中的iNOS表达水平,用方差分析比较各组之间iNOS的表达差异。结果1各组大鼠体重及血糖变化糖尿病组大鼠和治疗组大鼠体重（240.82±10.32g,245.76±9.87g）明显低于对照组（340.34±10.12g）（P＜0.01）;血糖（25.46±4.45mmol/L,25.41±4.45mmol/L）明显高于对照组（4.36±3.46mmol/L）（P＜0.01）,说明糖尿病大鼠模型诱导成功。2羟脯氨酸结果糖尿病组羟脯氨酸含量（2.7917±1.1642μg/mg膀胱重量）比对照组（1.5326±0.5405μg/mg膀胱重量）显著升高（P＜0.05）,治疗组羟脯氨酸含量（1.1745±0.7043μg/mg膀胱重量）比糖尿病组显著降低（P＜0.05）。3 TGFβ1及bFGF免疫组化结果免疫组化结果显示TGFβ1和bFGF阳性表达主要分布在平滑肌细胞胞浆,糖尿病组阳性TGFβ1积分光密度值（48.81±5.05）比对照组（3.73±2.26）显著升高（P＜0.05）,治疗组阳性TGFβ1积分光密度值（18.65±4.60）比糖尿病组（48.81±5.05）显著降低（P＜0.05）;糖尿病组阳性bFGF积分光密度值（58.40±7.04）比对照组（4.83±3.16）显著升高（P＜0.05）,治疗组阳性bFGF积分光密度值（7.75±4.10）比糖尿病组（58.40±7.04）显著降低（P＜0.05）。4 TGFβ1及bFGF的表达糖尿病组TGFβmRNA表达中位数（0.422±0.127）及蛋白表达（21.436±1.65pg./μgprotein）比对照组（0.1 45±0.052）及（7.159±1.956pg./μgprotein）显著升高（P＜0.05）,治疗组TGFβmRNA表达中位数（0.281±0.066）及蛋白表达（14.641±1.938pg./μgprotein）比糖尿病组（0.422±0.127）及（21.436±1.65 Pg./μg protein）显著降低（P＜0.05）;糖尿病组bFGFmRNA表达中位数（0.489±0.096）及蛋白表达（25.968±1.648pg./μg protein）比对照组（0.118±0.021）及（7.952±0.854 pg./μg protein）显著升高（P＜0.05）,治疗组bFGFmRNA表达中位数（0.097±0.011）及蛋白表达（12.911±0.984pg./μgprotein）比糖尿病组（0.489±0.096）及（25.968±1.648 pg./μg protein）显著降低（P＜0.05）。5氧化应激水平糖尿病组膀胱组织中的CAT活性（11.458±1.688 U/mgprotein）和SOD活性（7.159±2.956 U/mgprotein）与对照组（16.099±1.767 U/mgprotein,21.436±5.645U/mgprotein）相比显著降低（P＜0.05）,糖尿病组中MDA水平（15.968±1.648nmol/mgprotein）与对照组（7.952±0.854 nmol/mgprotein）相比显著升高（P＜0.05）,免疫组化研究显示糖尿病组膀胱组织中iNOS水平（67.5±10.6）与对照组（0.0±0.0）相比显著升高（P＜0.05）。治疗组膀胱组织中的CAT活性（14.472±1.529 U/mgprotein）和SOD活性（16.641±4.938 U/mgprotein）与糖尿病组（11.458±1.688 U/mgprotein,7.159±2.956 U/mgprotein）相比显著升高（P＜0.05）;治疗组中MDA水平（10.911±0.984 nmol/mgprotein）与糖尿病组（15.968±1.648 nmol/mgprotein）相比显著降低（P＜0.05）;免疫组化研究显示治疗组膀胱组织中iNOS水平（13.2±5.4）与糖尿病组（67.5±10.6）相比显著降低（P＜0.05）.结论1糖尿病膀胱发生明显纤维化。2 TGFβ1及bFGF在糖尿病膀胱中表达明显增加,可能是导致糖尿病膀胱病变的一个重要因素。3 CAT、SOD活性明显降低,MDA水平及iNOS表达明显增加,氧化应激状态的异常可能是导致糖尿病膀胱病变的一个重要因素。4生长因子抑制剂能改善糖尿病膀胱纤维化及氧化应激状态。更多还原

【Abstract】 Objective:To discuss the pathogenesis of bladder fibrosis and investigate oxidative stress in the bladder 8 weeks after diabetes induction and evaluate the effect of Tadenan on improving bladder fibrosis and oxidative stress status due to diabetes.Materials and Methods:1.To set up the model of diabetic cystopathy in Wistar rats:30 Wistar rats（3months, 200±10g）were divided into three groups:control（n=10）,streptozotocin-induced diabetic group（n=10）,treatment(n=10;diabetic rats were fed with Tadenan100 mg.Kg^-1.day^-1).8 weeks later,the bladders were dissected.2.Measurement of hydroxyproline using a method based on alkaline hydrolysis, Variance analysis compares the difference of results of each group.3.Measurement of the expression of TGFβ1 and bFGF by immunohistochemistry, RT-PCR and ELISA in the bladder,Variance analysis compares the difference of results of each group.4.Measurement of catalase（CAT）,superoxide dismutase（SOD）,mark for lipid peroxidation,maleic dialdehyde（MDA）by chemical methods and the levels of inducible nitric oxide synthase（iNOS）using immunohistochemistry.Results:1.Bodyweight and blood glucose in each group:bodyweight and blood glucose of control group,diabetic group and treatment group are 340.34±10.12g, 4.36±3.46mmol/L,240.82±10.32g,25.46±4.45mmol/L,245.76±9.87g, 25.41±4.45mmol/L,which illustrated the rats in diabetic and treatment group had successfully induced into diabetic rats.2.The content of hydroxyproline in control,diabetic and treatment group are 1.5326±0.5405μg/mg bladder weight,2.7917±1.1642μg/mg bladder weight, 1.1745±0.7043μg/mg bladder weight.There is great difference in diabetic group vs control（P＜0.05）and in treatment vs diabetic group（P＜0.05）.3.Immunohistochemical results in each group:the IOD of TGF and bFGF in control, diabetic and treatment group are 3.73±2.26,4.83±3.16;48.81±5.05,58.40±7.04; 18.65±4.60,7.75±4.10.There is great difference in diabetic group vs control（P＜0.05） and in treatment group vs diabetic group（P＜0.05）.4.Expression of TGF and bFGF in each group:the median expression of TGF mRNA and protein in diabetic group（0.422±0.127;21.436±1.65 pg./μgprotein）are significantly higher than that in control（0.145±0.052;7.159±1.956 pg./μgprotein） （P＜0.05）,the median expression of TGF mRNA and protein in treatment group（0.281±0.066;14.641±1.938 pg./μgprotein）are significantly lower than that in diabetic group（0.422±0.127;21.436±1.65 pg./μgprotein）（P＜0.05）.The median expression of bFGF mRNA and protein in diabetic group（0.489±0.096;25.968±1.648 pg./μg protein）are significantly higher than that in control（0.118±0.021;7.952±0.854 pg./μg protein）（P＜0.05）,the median expression of bFGF mRNA and protein in treatment group（0.097±0.011;12.911±0.984 pg./μgprotein）are significantly lower than that in diabetic group（0.489±0.096;25.968±1.648 pg./μg protein）（P＜0.05）.5.The CAT and SOD activity（11.458±1.688 U/mgprotein,7.159±2.956 U/mgprotein） significantly decreased from diabetic group compared with control group（16.099±1.767 U/mgprotein,21.436±5.645 U/mgprotein）（P＜0.05）,MDA levels（15.968±1.648 nmol/mgprotein）significantly increased from diabetic group compared with control group（7.952±0.854 nmol/mgprotein）（P＜0.05）, Immunohistochemical studies showed a statistically significant increased number of iNOS-positive cells in diabetic group（67.5±10.6）compared with control group（0.0±0.0）（P＜0.05）.The CAT and SOD activity（14.472±1.529 U/mgprotein, 16.641±4.938 U/mgprotein）significantly increased from treatment group compared with diabetic group（11.458±1.688 U/mgprotein,7.159±2.956 U/mgprotein）（P＜0.05）, MDA levels（10.911±0.984 nmol/mgprotein）significantly decreased from treatment group compared with diabetic group（15.968±1.648 nmol/mgprotein）（P＜0.05）, Immunohistochemical studies showed a statistically significant decreased number of iNOS-positive cells in treatment group（13.2±5.4）compared with diabetic group（67.5±10.6）（P＜0.05）.Conclusions:1.Bladder fibrosis occurred easily during diabetic cystopathy.2.Expression of TGFβ1 and bFGF were increased significantly in diabetic bladder, which maybe an important factor in diabetic cystopathy.3.Activities of CAT and SOD were decreased significantly and the level of MDA and expression of iNOS were increased significantly in diabetic bladder,which indicated oxidative stress played an important role in diabetic cystopathy.4.Tadenan could effectively slow down the process of bladder fibrosis and improve oxidative stress status due to diabetes.更多还原