糖克煎剂对2型糖尿病大鼠肾功能保护作用的实验研究

【作者】 卞镝；

【导师】 李敬林；

【作者基本信息】 辽宁中医学院 ， 中西医结合临床， 2005， 博士

【摘要】 糖尿病肾病（DN）是糖尿病（DM）主要的慢性微血管并发症之一,是导致慢性肾衰的常见原因,据统计,在终末期肾病病人中约三分之一是由DN引起的。本论文通过理论研究对中西医防治DN的进展进行了较为全面的概述,并通过动物实验,从整体、器官、细胞分子等不同水平对中药复方糖克煎剂防治DN的机理进行了较为深入的探讨。 1.理论研究 由五部分组成,具体包括DN中医药研究概况、祛痰活血法治疗DN、DN现代医学研究进展、转化生长因子β（TGF-β）及其受体与DN、细胞凋亡与DN。在理论研究及文献综述中,参照古、今中医有关文献对糖尿病肾病的中医病名、病因病机、辨证论治方法、临床及实验研究进展和中医研究中存在的问题及对策进行了探讨,总结了导师李敬林教授辨治糖尿病肾病的方法和学术观点,强调从痰瘀论治糖尿病肾病,并对现代医学在糖尿病肾病研究中所取得的进展进行了总结,进一步明确了糖尿病肾病的病理特征、发病机制、分期和治疗,并具体探讨了现代医学关于细胞凋亡和TGF-β及其受体在DN发病机制中的作用。 2.实验研究 采用高糖高脂饲料喂养结合尾静脉注射链脲佐菌素（STZ）的造模方法复制出糖尿病模型,此模型首先采用高糖高脂饲料喂养一个月,诱发动物肥胖,引起胰岛素抵抗,再以小剂量STZ35mg/kg尾静脉注射造成胰岛素代偿性分泌障碍,从而引发高血糖,并出现微量白蛋白尿,系膜基质增生、基底膜增厚等糖尿病肾病典型的早期病理形态学改变,符合2型DN的发病机制。 本实验分正常对照组、中药糖克煎剂预防组、高低剂量组、模型组和西药洛汀新对照组,预防组大鼠灌胃给药12周,其余各治疗组均给药8周。观察糖克煎剂对DN大鼠血糖、糖化血红蛋白、血脂、体重、肾重、尿蛋白排泄、肾功能的影响;通过光镜、电镜观察各组实验性DM大鼠肾组织病理形念学变化;采用逆转录聚合酶链式反应（RT-PCR法）、蛋白质印迹杂交（Western Blot）及免疫组化法,进一步观察糖克煎剂对DN大鼠肾小球细胞外基质（ECM）的主要成分IV型胶原（ColⅣ）和纤维连接蛋白（FN）、转化生长因子（TGF-β₁）及其受体（TGF-BR）mRNA及蛋白表达的影响;采用原位缺口标记技术（TUNEL）以及免疫组化法检测糖尿病大鼠肾组织中细胞凋亡及BCL-2及BAX等凋亡相关基因的表达,以进一步探讨DN发病机制,了解bcl-2、bax在糖尿病肾病发生、发展中的作用,以及中药糖克煎剂对DN细胞凋亡及其相关基因bcl-2/bax表达的影响。更多还原

【Abstract】 Diabetic nephropathy(DN) is one of the chronic complications of diabetes mellitus(DM) and the common reason leading to chronic renal failure ,one third of which is caused by DN. Systematic research on prevention and treatment of DN by TangKeJianJi (TangKe decoction) has been performed from the whole ,organ, cell and molecule levels in this essay which was composed of the reference review and experimental study.1. Theory reviewThe theoretical review including five sections mainly discussed the recent research progresses in prevention and treatment of DN both from the aspects of traditional Chinese medicine (TCM) and modern medicine. In detail, the five sections were made up of the skeleton review of TCM and modern medicine on DN, the relationship between transforming growth factor-P (TGF- β ) , transforming growth factor- P receptor (TGF- βR) and DN , the relationship between apoptosis and DN. In the theoretical research, we have referred to the related ancient and modern TCM literature in order to explore the source of disease name of TCM for DN, etiology and pathogenesis of TCM, selection of therapeutic methods by different stages or according to the differential syndrome diagnosis, advance on the clinical and experimental research as well as the problems lying in the research of TCM and corresponding strategies. In the meanwhile ,we also have summarized the tutor’s ( professor Li Jingling) academic point of view on the differentiation and treatment of DN, moreover further exploited the pathologic characteristics ,pathogenesis , pathologic stages and treatment of DN as well as the function of TGF- P and apoptosis on DN.2. Experimental studyThe experimental rat models at first were fed by a diet enriched with sucrose ,cholesterol and cholic acid for a month in order to induce the models’ obesity and result in insulin resistance, and then injected the low dosage of 35mg/kg streptozotocin(STZ) through vein of the rat’s tail to develop hyperglycemia. This kind of model closely simulated the metabolic abnormalities of the human type 2 diabetes mellitus and its renal structural lesion with microalbuminuria, mild mesangial matrix expansion, thickening of glomerular basement membrane and so on. The experimental rats were randomly divided into 6 groups: normal contrast group, pattern group, prevention group, high dosage group, low dosage group and Lotension group. Drug was delivered daily by gavage for 8 weeks in the therapeutic groups except for the prevention group for 12 weeks. We have detected, BUN, Scr, hemoglobin Aic (HbAic), body weight, kidney weight, urinary albumin excretion (UAE) and renal pathologic lesion through light microscopy and electron microscopy and further observed mRNA and protein expression of TGF- β 1, TGF- β R, Collagen IV and fibronection through RT-PCR, western blot and immunohistochemistry methods to investigate the effects of TangKe decoction on the experimental rat models of DN. The results showed that TangKe decoction possessed the following functions: (1)decreasing blood glucose (BG), ameliorating glucose and lipid metabolic disorders, improving nonenzymatic glycation, attenuating urinary albumin excretion and protecting renal function. (2)ameliorating glomerular hypertrophy, mesangial matrix expansion and thickening of glomerular basement membrane.(3)inhibitingmRNA and protein expression of TGF-3 i, TGF-P R, Collagen IV and fibronection and synthesis of extracellular matrix(ECM),decreasing abnomal accumulation of ECM and preventing the glomerular sclerosis.It has been reported that the number of apoptotic cells increased obviously in the renal cortex of the DN rats and glomerular apoptosis was concerned with deterioration of renal function. Apdptotic cells in renal cortex were evaluated by terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL) and the protein expression of BAX and BCL-2 relating to apoptosis was detected by means of immunohistochemistry to investigate the effects of bax and bcl-2 on DN as well as the influence of them on apoptosis. The e更多还原