【作者】 魏明刚；

【导师】 孙伟；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的:肾脏纤维化是慢性肾脏病发展为慢性肾衰竭的主要原因,慢性肾脏病在发展为慢性肾衰竭的过程中有一个共同的病理学特点:从纤维化到硬化。肾脏纤维化的病理特征主要表现为细胞外基质积聚,肾小球正常结构消失,肾脏功能逐渐丧失直至废用。导致这种病理变化并最终出现肾功能下降的因素有以下几个方面:蛋白尿、血脂代谢异常、凝血和血液流变学异常、足细胞损害、肾脏组织及细胞因子的表达和代谢异常。首先,实验研究以阿霉素肾病大鼠作为实验对象,通过对实验动物的蛋白尿、血液生化、血液流变学、足细胞标志蛋白和肾脏组织及细胞因子的动态检测,探讨益肾清利、和络泄浊疗法治疗阿霉素肾病大鼠的机制及疗效。临床研究对已经接受完善降压、纠酸、调脂、改善水电平衡等基础治疗的慢性肾脏病2-3期“肾虚湿瘀证”的患者开展临床研究,以仅接受基础治疗的慢性肾脏病2-3患者作为对照组,评价以“益肾清利、和络泄浊法”为主的中医疗法的疗效,为进一步深入探讨中医辨证治疗在慢性肾脏病临床治疗中的作用寻求依据。方法:实验研究,参考相关文献,应用阿霉素对SD大鼠尾静脉进行一次性注射,造成大鼠肾脏产生类似人类慢性肾炎的病理学改变。按照实验设计将大鼠随机分为空白对照组、模型组、阴性对照组、中药治疗组、西药治疗组、中药+西药治疗组。动态留取大鼠实验开始后24小时小便标本,测定尿蛋白定量和尿N-酰—β—D—葡萄糖酐酶（NAG）酶;不同时间点测定血清肌酐（Scr）、尿素氮（BUN）、白蛋白（ALB）、甘油三脂（TG）、胆固醇（CHO）水平。造模后第8周末处死大鼠,取出肾脏组织分别处理,然后应用光学显微镜观察肾脏组织病理学形态变化,应用免疫组织化学法检测组织纤维化相关细胞因子如纤维连接蛋白（FN）、基质金属蛋白酶（MMP-9）和胶原Ⅳ（CoⅣ）的含量,应用分子生物学研究方法以及图象分析技术,分析各组大鼠肾脏组织中TIMP—1、TGF-β1、MMP-9和Nephrin的表达水平。临床研究,按照美国NKF-K/DOGI工作组制定的《慢性肾脏病及透析的临床实践指南Ⅱ》中的诊断标准和中国国家食品和药品监督管理局制定的《中药新药临床指导原则（慢性肾衰竭）》中的诊断标准和治疗原则,对多种影响肾脏功能的因素进行临床监测。在控制血压、纠正水电解质紊乱等治疗的基础上,应用以益肾清利、和络泄浊法为原则辨证论治的中药方剂,对2—3期慢性肾脏病患者的相关实验室检查项目、中医症候积分动态观察,对比不同疗法的临床疗效。结果:实验研究,阿霉素肾病大鼠的中药治疗组尿蛋白含量和尿NAG酶与阿霉素肾病大鼠模型组和西药治疗组比较减轻明显。实验结果还提示中药配合西药可以调节脂代谢,降低胆固醇水平和改善血液流变学情况,使血浆粘度下降。肾脏的病理组织学观察发现模型组的肾小球、细胞外基质和肾小管间质病变明显。中药治疗组、西药治疗组和中药+西药治疗组与模型组比较可见上述改变出现不同程度的减轻。免疫组织化学中应用光密度积分定量检测肾脏组织的组织纤维化相关因子如FN、MMP-9和CoⅣ的含量情况。通过SPSS统计软件分析表明,各治疗组的表达量与模型组之间有明显差别,提示各组治疗药物可以抑制阿霉素肾病大鼠肾脏细胞外基质增生相关的细胞因子的过度表达。我们应用逆转录聚合酶链式反应（RT-PCR）的方法检测各组大鼠肾脏组织的TIMP—1、TGF-β₁、MMP-9和Nephrin指标的RNA表达情况。各组治疗药物（不含阴性对照组）可以减轻阿霉素肾病大鼠纤维化正相关因子TIMP—1和TGF-β₁的表达。中药+西药治疗组疗效明显优于其他各组,差异有显著性（P＜0.05）。肾脏纤维化负相关因子MMP-9和足细胞相关蛋白Nephrin的RNA表达情况提示各组治疗药物（不含阴性对照组）可以保护阿霉素肾病大鼠肾脏足细胞并提高纤维化负相关因子MMP-9的表达量,中药+西药治疗组疗效明显优于其他各治疗组,差异有显著性。临床研究病例来源于江苏省中医院和苏州大学附属第一医院的门诊和住院患者,共纳入2—3期慢性肾脏病患者90例,其中男性58例,女性32例。收集患者相关的临床数据资料,应用SPSS15.0统计软件分析。统计分析结果提示,以益肾清利、和络泄浊为治疗原则的中药方剂与西药基础治疗相配合的中西药治疗组与单纯西药基础治疗组进行比较。结合相关临床疗效指标提示中西药治疗组相比单纯西药治疗组,可以降低患者肾脏功能的下降速度,改善患者的症候,提高患者临床疗效和依从性。结论:“益肾清利、和络泄浊法”的中药方剂,可以减少阿霉素肾病大鼠的蛋白尿、降低尿液的NAG酶、减轻肾脏组织细胞外基质合成、抑制TIMP、TGF-β1的表达、提高抗组织纤维化因子MMP-9和足细胞标志蛋白（nephrin）的表达。中药方剂和贝那普利合用可以起到协同作用,提高抗肾脏纤维化的治疗效果。临床应用发现中药方剂具有补益脾肾、益气养血等作用。临床研究统计数据表明配合了西药的基础治疗,可以更有效地减少患者蛋白尿、保护患者肾功能,降低肾功能的下降速度。更多还原

【Abstract】 Objective:Fibrotic kidney is a common characteristic during the course from chronic kidney disease to chronic renal failure（CRF）.The pathological feature of fibrotic kidney is progressing extracellular matrix accumulation.The result is the loss of normal structure of the glomerulus,the destroy of nephron and the deprivation of renal function.The treatment of tonifying kidney & regulating and dispersing muddy（TKRDM） is a kind of means which is set up by professor sun wei.The study includes two parts.First we establish an animal model of renal sclerosis by adriamycin（ADM）.Then we use the TKRDM to treat the animal and observe the change of biochemical indicator,the change of histopathology,the change of ECM,the change of cytokine and the marker of cell.Second,we use the TKRDM to treat the patient by 2-3 stage chronic renal disease.Approaches:Research study:1.We did once-intravenous injections to establish an animal model for diffusion mesangioproliferation following with focal segmental glomerulosclerosis.The rats were randomly divided into sham group,adriamycin nepropathy（AN）,TKRDM group, benazepril group,TKRDM and benazepril group.Urinary protein excretion,serum urea nitrogen,serum creatinine,albumin,serum cholesterol and triglyceride were be detected by automatic biochemistry meter.We detected the cytokine and the marker of cell,which includes collagenⅣ（CoⅣ）,fibronectin（FN）,the transforming growth factor-β₁,by the means of immunohistochemistry and molecular biology.2.We would analyse the date by the statistics software.Clinic study:1.We set up the diagnostic criteria and therapeutic principle according to Clinical practice guide for the chronic renal disease and dialysis which was compiled by American NKF-K/DOGI group and Clinical practice guide to the new medicine of TCM which was compiled by the National Food and Medicine Administration Bureau.2.We used the TKRDM to treat the patient by 2-3 stage chronic renal failure and concluded the Urinary protein excretion,the serum urea nitrogen and calculated the date of renal function.3.We analysed the date by the SPSS15.0 statistics software.Result:TKRDM and benazepril have significantly difference in proteinuria extract and urinary NAG enzyme,the serum total cholesterol was significantly distinguished by statistic means. TKRDM can improve the circulating of the blood but the benazepril doesn’t have the effect.We can get a significant conclusion by statistic ways.We observed the tissue of the kidney by the way of pathohistology.We could find that the intercapillary cells and the ECM had obviously proliferation in the group of the AN.There were many foam cells in glomerulus of kidney.The cell of the renal tubule had vacuolar degeneration,analosis and cellular necrosis.There were also many protein cast and RBC cast in the nephric tubule.TKRDM group,benazepril group, TKRDM and benazepril group had different degree lessens for the index from the pathohistology. We found the group of the TKRDM and benazepril had the best conclusion by statistic way.We tested the FN,TGF-β1,MMP-9 and Col-Ⅳwhich were the compositions correlating with the fibrotic tissue by immunohistochemistry.Then we analyzed the compositions in the payhofigure by the semiquantitative analysis.TKRDM group,benazepril group,TKRDM and benazepril group could restrain the overexpression of the cytokine for the AN.In the mechanism of molecular biology,we used the ways of reverse transcription polymerase chain reaction（RT-PCR）.We analyzed the cytokines which included TIMP,TGF-β1,Nephrin.All of the cytokines had a high of the date in the group of AN.The others of experimental groups had a relative lower dates.We used the TKRDM to treat patients.Then we write down the symptoms and the renal function every month.All the symptoms,were selected from Clinical practice guide for the chronic renal disease and dialysis and Clinical practice guide to the new medicine of TCM,which correlate with the chronic renal disease.We analyzed all of the date which come from the symptoms and the renal function by spss15.0 statistic software.Conclusion:From the dates and the analyzing of the experiments,we could get the results. First TKRDM can decrease the proteinuria and protect the function of the renal tubular for AN in rats.Second TKRDM can reduce the proliferation of ECM at different levels for AN in rats.If we use the TKRDM and benazepril,the effects of the therapeutic action will be better than use any other one of them.For the chronic study,TKRDM can tonify spleen and kidney,benefit vital energy and smooth the blood vessel.TKRDM can also elevate the function of the entrails.The experiment hints the two points by analyzing the date of statistic results.First the TKRDM can decrease proteinuria extract and urinary NAG enzyme.Second the TKRDM can delay the aggravate speed of the renal function.更多还原