本文關(guān)鍵詞： 糖尿病腎病 糖腎合劑 nephrin podocin　出處：《南京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的:糖尿病腎病是臨床上糖尿病最常見的并發(fā)癥,具有進(jìn)展快、治療難度大的特點,且近年來患病率逐步攀升。蛋白尿是糖尿病腎病的主要臨床表現(xiàn),從早期微量白蛋白尿到中晚期大量蛋白尿,目前已經(jīng)證實蛋白尿是影響糖尿病腎病進(jìn)展的獨立危險因素,因此探求蛋白尿的發(fā)病機(jī)制及尋找降低蛋白尿保護(hù)腎功能的方法顯得尤為迫切。以往研究者對糖尿病腎病病理的研究主要集中在腎小球肥大、基底膜增厚、系膜區(qū)細(xì)胞增生及基質(zhì)增多、小球硬化等方面,然而這些病理改變僅能解釋部分臨床表現(xiàn),更多的病因仍不明確,近些年,人們將研究方向轉(zhuǎn)向了腎小球足細(xì)胞上面,因為足細(xì)胞是腎臟濾過屏障的最后一層,不僅與機(jī)械屏障相關(guān)更涉及到電荷屏障,所以一時成為熱點[1]。足細(xì)胞在維持腎小球濾過及阻止蛋白尿漏出方面起著至關(guān)重要的作用[2-3]。任何形式的損傷無論是自身免疫反應(yīng)還是藥物毒性作用均可導(dǎo)致足細(xì)胞受損,進(jìn)而影響其功能。足細(xì)胞損傷主要表現(xiàn)為細(xì)胞肥大、足突消失、數(shù)量減少甚至脫落凋亡。相鄰足突之間有著類似于拉鏈狀結(jié)構(gòu)的一層隔膜,隔膜是由多種分子相互結(jié)合而成的復(fù)合體,裂孔隔膜是濾過膜的最后一層,如果其完整性遭到破壞將會導(dǎo)致蛋白尿的漏出,體內(nèi)高血糖、AGEs、氧化應(yīng)激作用均會引起足細(xì)胞的損傷裂孔隔膜受損。有實驗表明在糖腎鼠模型中初期足細(xì)胞的數(shù)量就有所減少,隔膜上的分子Nephrin、Podocin蛋白含量也會下降,到后期這種變化更加明顯。足細(xì)胞損傷與腎小球硬化亦有關(guān)系[4]。Donblier等[5]在糖腎患者病理中觀察到在早期Nephrin的表達(dá)就可出現(xiàn)異常,并且與蛋白尿的發(fā)生和腎小球濾過有明顯相關(guān)性,因此研究足細(xì)胞損傷與糖尿病腎病的關(guān)系,為臨床上防治糖尿病腎病提供新的依據(jù)。吾師姚源璋教授從事臨床診療工作近四十年,擅長腎臟病的防治尤其對糖尿病腎病有其獨特見解。在結(jié)合多年臨床經(jīng)驗及糖尿病腎病發(fā)病特點的基礎(chǔ)上創(chuàng)制出了糖腎合劑,該方切合糖腎病機(jī),兼顧標(biāo)本虛實,本以氣陰兩虛為主,標(biāo)實以血瘀、痰濕、濁毒為甚,所以該方具有益氣生津、通絡(luò)活血之功,在臨床上取得了滿意療效。前期實驗中我們證實了該方具有調(diào)節(jié)JAK/STAT通路[6]、調(diào)節(jié)腎組織SOCS—1、TGF—β 1及VEGF[7]的表達(dá)來保護(hù)腎臟的作用。糖腎合劑方降低蛋白尿的作用機(jī)制是否跟調(diào)節(jié)足細(xì)胞分子nephrin和podocin的表達(dá)有關(guān)呢?該實驗通過動物模型的方式將分為模型組、治療組、對照組合正常組,最后通過測定各組腎組織中nephrin和podocin的含量,來分析糖腎合劑方對糖尿病大鼠腎臟的保護(hù)作用,及其可能機(jī)制。研究方法:糖尿病大鼠模型的建立:給予SD大鼠高糖高脂飼料5周后腹腔注射小劑量鏈尿佐菌素(35mg/kg);成模標(biāo)準(zhǔn):大鼠非空腹血糖16.7mmol/L。1.分組:將大鼠分為正常組,成模后再分為模型組、對照組及糖腎合劑高、低組共5組。2.給藥方法:按照15ml/Kg/d的劑量以灌胃的方式分別給予各組相應(yīng)的藥液。給藥干預(yù)共8周。3.療效評價方法:給藥8周期后,在處死大鼠前以尾部采血的方式測定每只大鼠血糖、稱重、收集并測定大鼠24小時尿蛋白定量;麻醉大鼠后用一次性采血針腹主動脈采血并檢測相關(guān)生化指標(biāo)(血肌酐、血清白蛋白、尿素氮);對大鼠腎組織做病理切片并HE染色觀察腎組織結(jié)構(gòu)病變;采用免疫印跡(Western blotting)法和聚合酶鏈反應(yīng)(PCR)方法檢測糖尿病大鼠腎組織中nephrin、podocin蛋白的表達(dá)和nephrinmRNA、podocinmRNA的含量并應(yīng)用圖像分析軟件(Image—Pro Plus)進(jìn)行半定量分析,SPSS17.0對數(shù)據(jù)進(jìn)行分析處理,P0.05為有統(tǒng)計學(xué)意義,P0.01為有顯著統(tǒng)計學(xué)意義。結(jié)果:1.24h尿蛋白定量:模型組蛋白定量明顯高于正常組,糖腎高、低組及氯沙坦對照組蛋白定量低于模型組且有統(tǒng)計學(xué)意義;2.腎功能:模型組肌酐、尿素氮水平明顯高于正常組,糖腎高、低組及氯沙坦對照組肌酐、尿素氮水平低于模型組且有統(tǒng)計學(xué)意義;3.血清白蛋白:模型組血清蛋白明顯低于正常組,糖腎高、低組及氯沙坦對照組血清蛋白高于模型組且有統(tǒng)計學(xué)意義;4.模型組腎組織中nephrin、podocin蛋白含量與正常組相比明顯減少(p0.01),糖腎高、低劑量組及藥物對照組與模型組相比腎組織中nephrin、podocin表達(dá)含量增加(P0.01),與正常組比較nephrin、podocin表達(dá)含量減少(p0.01)。5.病理變化:光鏡下可見正常組腎小球形態(tài)規(guī)整清晰可見,無明顯細(xì)胞增生,系膜區(qū)無明顯增寬,毛細(xì)血管襻無壓迫,無球囊粘連,小管間質(zhì)未見纖維化。模型組光鏡下可見腎小球肥大,系膜區(qū)明顯增寬、基底膜增厚,部分球囊粘連及纖維化,偶見間質(zhì)炎癥細(xì)胞浸潤。糖腎高低組及氯沙坦組鏡下改變較模型組減輕,其中糖腎高組和氯沙坦對照組腎臟病理改變最為明顯。結(jié)論:糖腎合劑可降低模型鼠尿蛋白定量、升高血清蛋白含量、對肌酐尿素氮有不同程度的降低并可減輕腎臟組織病理變化。糖腎合劑對腎臟的保護(hù)作用機(jī)制可能與上調(diào)腎組織局部nephrin、podocinmRNA及蛋白的表達(dá)有關(guān)。
[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.

【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5;R-332

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