【作者】 刘伟；

【导师】 王俭勤；

【作者基本信息】 兰州大学 ， 内科学， 2009， 硕士

【摘要】 糖尿病肾病（Diabetic nephropathy,DN）是糖尿病严重的微血管并发症,是导致终末期肾病的重要原因。其发病机理尚未完全阐明,一般认为它与高血糖、多元醇-肌醇代谢异常、氧化应激、血流动力学障碍、凝血机制障碍、蛋白的非酶促糖基化等多种因素协同作用有关。糖尿病肾病重要的病理改变之一就是细胞外基质（Extracellular matrix,ECM）的堆积。转化生长因子-β₁（Transformin growthfactorβ₁,TGF-β₁）和结缔组织生长因子（Connective tissue growth factor,CTGF）目前己被公认为是包括糖尿病肾小球硬化在内的多种纤维化病变发病机制中的驱动因子,是抗纤维化治疗的有效靶点。研究证实,转化生长因子-β₁可调节炎症反应,促进细胞外基质的合成和沉积,减少细胞外基质的降解,其过度表达可引起肾小球硬化和肾间质纤维化。结缔组织生长因子（CTGF）是TGF-β₁的下游效应介质。研究发现CTGF不介导TGF-β₁的抗增殖和抗炎作用,仅介导TGF-β₁过度表达时促细胞外基质合成的作用。它可以通过自分泌与旁分泌的形式,刺激肾小球系膜细胞和其他肾脏固有细胞的增殖,促进细胞外基质的堆积,参与着肾小球肥大、肾小球硬化以及间质纤维化。基质金属蛋白酶（Matrix metalloproteinase,MMPs）主要起到降解细胞外基质的作用。基质金属蛋白酶-9（Matrix metalloproteinase-9,MMP-9）又称明胶酶B,是MMPs家族的主要成员之一,其作用底物主要是Ⅳ、Ⅴ、Ⅶ、Ⅹ型胶原、明胶以及弹性纤维。MMP-9以酶原形式分泌,必须激活后才能降解胶原及其它基质蛋白。普遍认为是纤溶酶原在纤溶酶原激活物的作用下活化为纤溶酶,后者以级联放大的瀑布效应激活MMP-9而导致基质降解。腹腔注射链脲佐菌素（Streptozotocin,STZ）可在短时间内建立糖尿病大鼠模型,并出现糖尿病肾脏损害。本文以此模型为基础,探讨糖尿病肾病早期结缔组织生长因子和基质金属蛋白酶-9的表达以及自拟中药宝肾方治疗糖尿病肾病的机制。目的:本研究以自拟中药宝肾方汤剂干预实验性糖尿病大鼠,应用免疫组化方法检测对照组（C组）、糖尿病组（D组）、糖尿病宝肾方治疗组（DB组）肾组织CTGF和MMP-9表达水平,并检测各组大鼠的生化指标,探讨自拟中药宝肾方汤剂保护肾脏的作用机理,为临床防治糖尿病肾病提供实验性理论依据。方法:将实验动物Wistar大鼠随机分为对照组（C组）、糖尿病组（D组）、糖尿病宝肾方治疗组（DB组）。对照组大鼠腹腔注射枸橼酸缓冲液（假造模）,糖尿病组大鼠腹腔注射链脲佐菌素（STZ）建立糖尿病大鼠模型,糖尿病宝肾方治疗组是在建立糖尿病模型基础上给予宝肾方汤剂灌胃。在实验8周后,各组大鼠留取血、尿以及肾脏标本,观察三组大鼠肾重（Weight of kidney）、体重（Bodyweight）、24小时尿蛋白（24hUpro）、血糖（Glu）、胆固醇（CHO）、尿素氮（BUN）、血肌酐（Scr）以及肾组织病理学变化;并通过免疫组织化学方法检测各组大鼠肾组织中CTGF和MMP-9的表达情况。结果:和对照组相比,糖尿病组肾组织CTGF蛋白表达明显升高[（0.234±0.009）vs（0.146±0.007）,P＜0.01],而MMP-9蛋白表达明显降低[（0.146±0.006）vs（0.236±0.007）,P＜0.01],肾脏肥大指数、24小时尿蛋白、血糖、胆固醇、尿素氮、血肌酐明显升高（P＜0.01）;和糖尿病组相比,宝肾方治疗组肾组织CTGF蛋白表达降低[（0.194±0.008）vs（0.234±0.009）,P＜0.01],而MMP-9蛋白表达升高[（0.175±0.007）vs（0.146±0.006）,P＜0.01],肾脏肥大指数降低（P＜0.05）,24小时尿蛋白、胆固醇、尿素氮、血肌酐也降低（P＜0.01）。结论:自拟宝肾方对糖尿病大鼠肾脏具有保护作用,其机制可能与影响肾组织CTGF和MMP-9的表达有关。更多还原

【Abstract】 Diabetic nephropathy is a serious cap vascular complication of diabetes.It is a very important cause bringing about the terminal stage of nephropathy.The pathogenesis of diabetic nephropathy is not clear.It is generally considered that it is interrelated with the synergy of Hyperglycemia,metabolic disturbance of polylol-phaseomannite,oxidizes stress,abnormality of hemodynamics,abnormality of zymoplastic mechanism,nonenzymatic promoted glycosylation.The accumulation of extracellular matrix is one of the important pathologic changes of diabetic nephropathy.Now,TGF-β₁ and CTGF are considered to be one of the important driver molecules for ECM accumulation in the pathogenesis of diverse forms renal fibrosis including glomerulosclerosis in diabetic nephropathy,and may constitute a more specific target for futuramic anti-fibrosis therapies.It is very important to keep the function of inflammatory reaction.It can influence the formation of extracellular matrix（ECM）,which includes inducing synthesis of various matrix proteins,inhibiting expression of ECM degradation enzyme,disturbing synthesis and decomposition of ECM,and leading to excessive accumulation of ECM,which result in glomerulosclerosis at last.It was found that CTGF played a key role in the occurrence and development of diabetic nephropathy.CTGF is the downstream medium of transforming growth factor-β₁（TGF-β₁）,but CTGF does not involve in the resisting proliferation and inflammation function of TGF-β₁,and only involve in accelerating cell matrix synthesis function of TGF-β₁.CTGF can induce mesangial cells and other renal resident cells producing ECM by autocrine and paracrine,thereafter accelerate the glomerular hypertrophy and glomerulosclerosis inevitably.Matrix metalloproteinases are mainly play degradation of extracellular matrix. Matrix metalloproteinases-9 say again gelatinases B,which is one of the main members of the family of MMPs.Ⅳ,Ⅴ,Ⅶ,Ⅹtype collagen,gelatin and elastic fiber are the substrate.MMP-9 in the form of enzyme secretion,must be activated to degrade matrix collagen and other proteins.Generally considered fibrinolytic enzyme in fibrinolytic enzyme activator activation is under the action of fibrinolytic enzyme, the latter to cascade amplification waterfall effect MMP-9 to activate substrate biodegradation.The intraperitoneal injection of streptozotocin was conducted to made models of diabetic rats within a short period of time and impairment of diabetic nephropathy occurred after that.This article aims at discuss the expressions of CTGF and MMP-9 at the early stage of diabetic nephropathy and the mechanism of the curative effects of self-made Bao’shen recipe on diabetic nephropathy.Objective:The study use Bao’shen recipe to intervent diabetic rats and detect the expression level of CTGF and MMP-9 of renal tissue in control group,diabetic rat group and Bao’shen recipe treating diabetic rat group by immumohistochemistry way. In addition,in order to provide theoretical basis for the treatment of DN and the possible mechanism of protecting kidney,we have detected the clinical and metabolic parameters of this three groups.Methods:Wistar rats were randomized divided into control group,diabetic rat group and Bao’shen recipe treatment group.The control group received intraperitoneal injection with citric acid buffer solution.STZ was injected through abdominal cavity to make models of diabetic rats.Bao’shen recipe treatment group was set up based on models of diabetic rats with feeding Bao’shen recipe.Blood, urine and renal tissue were obtained after 8 weeks.Blood glucose,cholesterol,urea nitrogen,serum creatinine,24 hour-urinary albumin,weight of kidney,body weight and the pathological changes of kidney tissues were measured.Technique of immunohistochemistry was also taken to measure the expression of CTGF and MMP-9 in this three groups.Result:Compared with group C,expressions of CTGF were higher[（0.234±0.009） vs（0.146±0.007）,P＜0.01],while the expressions of MMP-9 were lower[（0.146±0.006） vs（0.236±0.007）,P＜0.01]in renal tissue of group D. Kidney hypertrophy index,24-hour urinary protein,blood glucose,cholesterol,urea nitrogen and serum creatinine increased significantly in the group D.The level of MMP-9 expression was advanced[（0.175±0.007） vs（0.146±0.006）,P＜0.01],while the level of CTGF expression was reduced[（0.194±0.008） vs（0.234±0.009）,P＜0.01] by using Bao’shen recipe.In group DB,kidney hypertrophy index,24-hour urinary protein,cholesterol,urea nitrogen and serum creatinine obviously lower than diabetic rat group.Conclusion:Self-made Bao’shen recipe can depress the expressions of CTGF and increase the expressions of MMP-9 in the kidney of diabetic rats,as a result, which can be served as a protective effect to prevent progress of diabetic nephropathy.更多还原