糖肾合剂对糖尿病肾病大鼠模型足细胞相关分子nephrin和podocin保护作用机制研究

发布时间：2018-01-26 07:20

本文关键词： 糖尿病肾病糖肾合剂 nephrin podocin　出处：《南京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的:糖尿病肾病是临床上糖尿病最常见的并发症,具有进展快、治疗难度大的特点,且近年来患病率逐步攀升。蛋白尿是糖尿病肾病的主要临床表现,从早期微量白蛋白尿到中晚期大量蛋白尿,目前已经证实蛋白尿是影响糖尿病肾病进展的独立危险因素,因此探求蛋白尿的发病机制及寻找降低蛋白尿保护肾功能的方法显得尤为迫切。以往研究者对糖尿病肾病病理的研究主要集中在肾小球肥大、基底膜增厚、系膜区细胞增生及基质增多、小球硬化等方面,然而这些病理改变仅能解释部分临床表现,更多的病因仍不明确,近些年,人们将研究方向转向了肾小球足细胞上面,因为足细胞是肾脏滤过屏障的最后一层,不仅与机械屏障相关更涉及到电荷屏障,所以一时成为热点[1]。足细胞在维持肾小球滤过及阻止蛋白尿漏出方面起着至关重要的作用[2-3]。任何形式的损伤无论是自身免疫反应还是药物毒性作用均可导致足细胞受损,进而影响其功能。足细胞损伤主要表现为细胞肥大、足突消失、数量减少甚至脱落凋亡。相邻足突之间有着类似于拉链状结构的一层隔膜,隔膜是由多种分子相互结合而成的复合体,裂孔隔膜是滤过膜的最后一层,如果其完整性遭到破坏将会导致蛋白尿的漏出,体内高血糖、AGEs、氧化应激作用均会引起足细胞的损伤裂孔隔膜受损。有实验表明在糖肾鼠模型中初期足细胞的数量就有所减少,隔膜上的分子Nephrin、Podocin蛋白含量也会下降,到后期这种变化更加明显。足细胞损伤与肾小球硬化亦有关系[4]。Donblier等[5]在糖肾患者病理中观察到在早期Nephrin的表达就可出现异常,并且与蛋白尿的发生和肾小球滤过有明显相关性,因此研究足细胞损伤与糖尿病肾病的关系,为临床上防治糖尿病肾病提供新的依据。吾师姚源璋教授从事临床诊疗工作近四十年,擅长肾脏病的防治尤其对糖尿病肾病有其独特见解。在结合多年临床经验及糖尿病肾病发病特点的基础上创制出了糖肾合剂,该方切合糖肾病机,兼顾标本虚实,本以气阴两虚为主,标实以血瘀、痰湿、浊毒为甚,所以该方具有益气生津、通络活血之功,在临床上取得了满意疗效。前期实验中我们证实了该方具有调节JAK/STAT通路[6]、调节肾组织SOCS—1、TGF—β 1及VEGF[7]的表达来保护肾脏的作用。糖肾合剂方降低蛋白尿的作用机制是否跟调节足细胞分子nephrin和podocin的表达有关呢?该实验通过动物模型的方式将分为模型组、治疗组、对照组合正常组,最后通过测定各组肾组织中nephrin和podocin的含量,来分析糖肾合剂方对糖尿病大鼠肾脏的保护作用,及其可能机制。研究方法:糖尿病大鼠模型的建立:给予SD大鼠高糖高脂饲料5周后腹腔注射小剂量链尿佐菌素(35mg/kg);成模标准:大鼠非空腹血糖16.7mmol/L。1.分组:将大鼠分为正常组,成模后再分为模型组、对照组及糖肾合剂高、低组共5组。2.给药方法:按照15ml/Kg/d的剂量以灌胃的方式分别给予各组相应的药液。给药干预共8周。3.疗效评价方法:给药8周期后,在处死大鼠前以尾部采血的方式测定每只大鼠血糖、称重、收集并测定大鼠24小时尿蛋白定量;麻醉大鼠后用一次性采血针腹主动脉采血并检测相关生化指标(血肌酐、血清白蛋白、尿素氮);对大鼠肾组织做病理切片并HE染色观察肾组织结构病变;采用免疫印迹(Western blotting)法和聚合酶链反应(PCR)方法检测糖尿病大鼠肾组织中nephrin、podocin蛋白的表达和nephrinmRNA、podocinmRNA的含量并应用图像分析软件(Image—Pro Plus)进行半定量分析,SPSS17.0对数据进行分析处理,P0.05为有统计学意义,P0.01为有显著统计学意义。结果:1.24h尿蛋白定量:模型组蛋白定量明显高于正常组,糖肾高、低组及氯沙坦对照组蛋白定量低于模型组且有统计学意义;2.肾功能:模型组肌酐、尿素氮水平明显高于正常组,糖肾高、低组及氯沙坦对照组肌酐、尿素氮水平低于模型组且有统计学意义;3.血清白蛋白:模型组血清蛋白明显低于正常组,糖肾高、低组及氯沙坦对照组血清蛋白高于模型组且有统计学意义;4.模型组肾组织中nephrin、podocin蛋白含量与正常组相比明显减少(p0.01),糖肾高、低剂量组及药物对照组与模型组相比肾组织中nephrin、podocin表达含量增加(P0.01),与正常组比较nephrin、podocin表达含量减少(p0.01)。5.病理变化:光镜下可见正常组肾小球形态规整清晰可见,无明显细胞增生,系膜区无明显增宽,毛细血管襻无压迫,无球囊粘连,小管间质未见纤维化。模型组光镜下可见肾小球肥大,系膜区明显增宽、基底膜增厚,部分球囊粘连及纤维化,偶见间质炎症细胞浸润。糖肾高低组及氯沙坦组镜下改变较模型组减轻,其中糖肾高组和氯沙坦对照组肾脏病理改变最为明显。结论:糖肾合剂可降低模型鼠尿蛋白定量、升高血清蛋白含量、对肌酐尿素氮有不同程度的降低并可减轻肾脏组织病理变化。糖肾合剂对肾脏的保护作用机制可能与上调肾组织局部nephrin、podocinmRNA及蛋白的表达有关。
[Abstract]:Background and objective: diabetic nephropathy is the most common complication of diabetes mellitus, with characteristics of rapid development, the treatment is very difficult, and in recent years the prevalence of proteinuria is gradually rising. The main clinical manifestations of diabetic nephropathy, from early microalbuminuria late in the macroalbuminuria, now it has been confirmed that proteinuria is an independent risk factor affect the progression of diabetic nephropathy, and explore the pathogenesis of proteinuria and to find ways to reduce proteinuria and protect renal function is particularly urgent. Previous research on diabetic nephropathy mainly concentrated in glomerular hypertrophy, basement membrane thickening, mesangial hyperplasia and stromal cells increased, glomerular sclerosis etc. However, these pathological change can only explain some clinical manifestations, more the etiology remains unclear, in recent years, the research direction of the podocyte foot on the surface, because the fine The cell is the last layer of kidney filtration barrier and mechanical barrier, not only related but also involves the charge barrier, so it became plays in maintaining the glomerular filtration and prevent leakage of hot [1]. proteinuria podocyte crucial role for [2-3]. in any form of damage whether autoimmune reaction or toxic effects of drugs can cause podocyte damage. Then influence its function. The main features of podocyte injury to cell hypertrophy, podocyte disappear, reducing the number of anoikis. Even between adjacent foot processes is similar to a membrane zipper like structure, the diaphragm is complex by a variety of molecules with each other and the slit diaphragm is filtration membrane in the last layer, if the complete destruction will lead to the leakage of urine protein, high blood glucose, AGEs, oxidative stress can cause podocyte injury in slit diaphragm damage. The experiment shows that the The number of initial foot cells is decreased in kidney in rat model of sugar, Nephrin molecules on the membrane, the protein content of Podocin will fall to late this change is more obvious. Podocyte injury and glomerulosclerosis are related to [4].Donblier and [5] were observed in the early expression of Nephrin can be abnormal in patients with renal pathological sugar and, with the occurrence and development of proteinuria and glomerular filtration have significant correlation, so the research on the relationship between podocyte injury and diabetic nephropathy, and provide a new basis for clinical prevention and treatment of diabetic nephropathy. Professor Yao Yuanzhang has engaged in clinical work in the past forty years, especially good at prevention of kidney disease has its unique insights on diabetic nephropathy. On the basis of the characteristics of the incidence of diabetic nephropathy and clinical experience for many years on the created TangShenHeJi, the party with the sugar nephropathy, taking into account the specimens of the actual situation, the Qi and yin deficiency two Lord, while blood stasis, phlegm turbid poison, why, so the party with Yiqi Tongluo Huoxue, power, and achieved satisfactory curative effect in clinic. In previous experiments we confirmed the regulation of JAK/STAT [6] pathway, regulating the renal tissue of SOCS - 1, TGF - beta 1 and VEGF[7]. To protect the kidney function. TangShenHeJi can reduce the mechanism of proteinuria is associated with the regulation of expression of podocyte molecules nephrin and podocin? The experimental animal model by the way divided into model group, treatment group, control group normal group, the content of nephrin and podocin were determined in each group in renal tissue, to analyze the TangShenHeJi Decoction on renal protective effect in diabetic rats and its possible mechanism. Methods: to establish the model of diabetic rats: SD rats were given high-fat diet for 5 weeks after intraperitoneal injection of small dose of streptozotocin (35mg/kg) model; Standard: non fasting blood glucose 16.7mmol/L.1. packet in rats: the rats were divided into normal group, model were divided into model group, control group and TangShenHeJi high, low group 5 group.2. administration method: according to the dose of 15ml/Kg/d by intragastric administration was given to the corresponding solution groups. Drug intervention a total of 8 weeks.3. evaluation method: administered after 8 cycles, the rats were killed prior to the tail blood glucose determination method, each rat weighing, collection and determination of rat urine protein in 24 hours; after rats were anesthetized with disposable blood taking needle abdominal aortic blood and related biochemical indicators (serum creatinine. Serum albumin, urea nitrogen); pathological section of renal tissue in rats and observe the structure of renal tissue lesions HE staining; by immunoblotting (Western blotting) and polymerase chain reaction (PCR) nephrin in renal tissue of diabetic rats detected by nephrinmRN and the expression of podocin protein. The content of A, and application of podocinmRNA image analysis software (Image Pro Plus) semi quantitative analysis of SPSS17.0 for data analysis and processing, P0.05 has statistical significance, P0.01 was statistically significant. Results: 1.24h urinary protein: protein in model group significantly higher than the normal group, renal sugar high, low group the control group and the Losartan protein lower than model group and there was statistical significance; 2. renal creatinine: model group, urea nitrogen levels were significantly higher than the normal group, renal sugar high, low group and losartan group creatinine, urea nitrogen level is lower than the model group and the difference was statistically significant; 3. serum albumin serum protein was significantly lower than that of model group normal group, renal sugar high, low group and losartan group serum protein was higher than in the model group and there was statistical significance; 4. nephrin of renal tissue in the model group, podocin protein content decreased significantly compared with the normal group (P0.01), high sugar kidney, Low dose group and drug control group and model group compared to nephrin in renal tissue, the expression of podocin was increased (P0.01), compared with the normal group nephrin, the expression of podocin was decreased (P0.01).5. pathological changes under light microscope: normal glomerular morphology regularity is clearly visible, no obvious cell hyperplasia, mesangial region no obvious increase wide, capillary loops without compression, no balloon adhesions, tubulointerstitial fibrosis. No model group under light microscope, glomerular hypertrophy, mesangial area widened obviously, basement membrane thickening, partial balloon adhesions and fibrosis, occasionally interstitial inflammatory cells infiltration. The renal sugar level group and losartan group compared with model changes under microscope the high sugar group, kidney group and losartan group renal pathological changes were most obvious. Conclusion: Tangshen mixture can reduce rat urine protein, serum protein content, and reduced in different degrees of reduction of serum creatinine and urea nitrogen The protective mechanism of sugar kidney mixture on kidney may be related to the up regulation of the expression of nephrin, podocinmRNA and protein in renal tissue.

【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5;R-332

【参考文献】