本文選題：糖尿病腎病 + 瑞格列奈�。� 參考：《河北醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:糖尿病腎病(Diabetic Nephropathy,DN)是糖尿病(Diabetes Mellituse,DM)最常見(jiàn)的嚴(yán)重微血管并發(fā)癥之一。近年來(lái)研究發(fā)現(xiàn),氧化應(yīng)激、炎癥反應(yīng)和纖維化在糖尿病腎病發(fā)生發(fā)展過(guò)程中發(fā)揮了重要作用。糖尿病時(shí)機(jī)體處于氧化應(yīng)激狀態(tài),通過(guò)產(chǎn)生過(guò)多的活性氧簇(Reactive Oxygen Species,ROS)直接損傷機(jī)體蛋白質(zhì)和核酸等生物大分子,還可通過(guò)激活多功能轉(zhuǎn)錄調(diào)節(jié)因子NF-κB(Nuclear Transcription FactorκB),促進(jìn)多種炎癥因子、巨噬細(xì)胞及促腎臟纖維化關(guān)鍵因子TGF-β等的過(guò)度表達(dá),共同導(dǎo)致腎臟病變。如何阻斷或抑制氧化應(yīng)激及炎癥反應(yīng),減緩腎臟纖維化成為預(yù)防和治療DN的關(guān)鍵。瑞格列奈(Repaglinide,RE)是一種非磺酰脲類促胰島素分泌劑,是Ⅱ型糖尿病患者臨床一線口服降糖藥物,可有效控制餐后血糖。有研究報(bào)道,RE還可減緩DN進(jìn)展,但確切機(jī)制尚不明了。本課題擬通過(guò)建立鏈脲佐菌素(Streptozotocin,STZ)誘導(dǎo)的DM大鼠模型,測(cè)定大鼠腎臟組織中氧化應(yīng)激指標(biāo)丙二醛(MDA)含量及總抗氧化能力(T-AOC)活性,應(yīng)用免疫組織化學(xué)、實(shí)時(shí)定量熒光PCR(real-time FQ-PCR)等方法檢測(cè)腎組織中NF-κB、TGF-β表達(dá)情況,并予瑞格列奈(Repaglinide,RE)2mg/Kg進(jìn)行干預(yù),觀察DM大鼠腎臟組織內(nèi)氧化應(yīng)激、炎癥反應(yīng)及纖維化狀況,并探討瑞格列奈對(duì)其存在的干預(yù)作用及機(jī)制,為人類糖DN的防治提供理論依據(jù)。方法:選擇健康雄性清潔級(jí)SD大鼠36只,體重約200±20g,(購(gòu)自河北醫(yī)科大學(xué)動(dòng)物中心),隨機(jī)分為正常對(duì)照(NC組,n=12)、糖尿病模型組(DM組,n=12)、糖尿病瑞格列奈干預(yù)組(RT組,n=12)。適應(yīng)性喂養(yǎng)1周后,空腹12小時(shí),糖尿病模型組大鼠一次性腹腔注射STZ(65mg/kg),72h后尾靜脈取血檢測(cè)血糖≥16.7mmol/L,尿糖+++~++++,視為造模成功。NC組一次性腹腔注射等劑量檸檬酸緩沖液。試驗(yàn)期間所有大鼠均自由進(jìn)食水,動(dòng)物房溫度(20℃~26℃)、濕度(40%~70%)相對(duì)恒定。RT組大鼠給予瑞格列奈(RE)2mg/kg,1/日,植物油溶劑灌胃,NC組、DM組每天予等量植物油灌胃。分別于造模成功后8周、12周末,每組任取6只大鼠,測(cè)定24h尿蛋白(24 h UP)、血糖、尿素氮、血肌酐。并收集大鼠腎組織標(biāo)本,制備組織切片,行蘇木素-伊紅染色(HE染色)及糖原染色(PAS),光學(xué)顯微鏡下觀察大鼠腎臟病理變化;檢測(cè)腎組織MDA含量、T-AOC活性,免疫組織化學(xué)及real-time PCR方法檢測(cè)TGF-β、NF-κB的表達(dá)。實(shí)驗(yàn)所得數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(x±S)表示,應(yīng)用SPSS 16.0統(tǒng)計(jì)分析軟件對(duì)數(shù)據(jù)進(jìn)行單因素方差分析,不滿足正態(tài)性和方差齊性的數(shù)據(jù)資料采用非參數(shù)檢驗(yàn),P0.05有統(tǒng)計(jì)學(xué)意義。結(jié)果:1一般情況DM組、RT組大鼠腹腔注射STZ后逐漸出現(xiàn)多飲、多食、多尿,反應(yīng)遲緩,毛發(fā)失去光澤,體重逐漸下降,其中DM組癥狀較RT組明顯加重,而NC組大鼠飲食正常,反應(yīng)靈敏,生長(zhǎng)發(fā)育良好,體重明顯增長(zhǎng)。2生化指標(biāo)2.124小時(shí)尿蛋白定量與同期NC組相比,8周末、12周末,DM組、RT組大鼠24小時(shí)尿蛋白水平均升高(P0.05);與同期DM組相比,RT組大鼠24小時(shí)尿蛋白水平降低(P0.05)。2.2血糖與同期NC組相比,8周末、12周末,DM組、RT組大鼠血糖水平均明顯升高(P0.05);與同期DM組相比,RT組血糖變化不明顯(P0.05)。2.3血肌酐、尿素氮與同期NC組相比,8周末、12周末,DM組、RT組大鼠血肌酐、尿素水平升高(P0.01);與同期DM組相比,RT組血肌酐、尿素氮水平下降(P0.05)。3氧化應(yīng)激指標(biāo)3.1MDA測(cè)定與同期NC組相比,8周末、12周末,DM組、RT組大鼠腎組織MDA水平顯著升高(P0.05);與同期DM組相比,RT組MDA水平均有不同程度的下降(P0.05)。3.2 T-AOC測(cè)定與同期NC組相比,8周末、12周末,DM組、RT組大鼠腎組織內(nèi)的T-AOC活性顯著降低(P0.05);與同期DM組相比,RT組T-AOC活性升高(P0.05)。4腎臟組織變化光鏡顯示,NC組大鼠腎臟組織結(jié)構(gòu)清晰,腎小管上皮細(xì)胞排列整齊,基底膜染色均勻一致,無(wú)系膜細(xì)胞及系膜基質(zhì)增多等異常改變。與NC組相比,DM組腎小球體積增大,腎小球系膜基質(zhì)彌漫增多,系膜細(xì)胞增多,基底膜增厚,12周末時(shí)病理改變更明顯。RT組腎臟組織結(jié)構(gòu)較DM組得到改善。5免疫組織化學(xué)檢測(cè)與同期NC組相比,8周末、12周末,DM組、RT組NF-κB、TGF-β表達(dá)均增多(P0.05);與同期DM組相比,RT組NF-κB、TGF-β表達(dá)水平不同程度下降(P0.05)。6 Real-time FQ-PCR檢測(cè)與同期NC組相比,8周末、12周末時(shí),DM組、RT組NF-κB m RNA、TGF-βm RNA表達(dá)均增多(P0.05);與同期DM組相比,RT組NF-κB m RNA、TGF-βm RNA表達(dá)水平不同程度下降(P0.05)。結(jié)論:1 DM大鼠腎臟組織MDA表達(dá)增高,T-AOC活性降低,提示處于氧化應(yīng)激狀態(tài),同時(shí)NF-κB、TGF-β表達(dá)升高,表明有炎癥反應(yīng)及纖維化機(jī)制參與。2瑞格列奈可減少DM大鼠蛋白尿的產(chǎn)生、改善腎臟功能;可降低腎臟組織MDA含量,提高T-AOC活性并減少NF-κB、TGF-β表達(dá),從而減輕氧化應(yīng)激、炎癥反應(yīng),延緩腎臟纖維化,起到腎臟保護(hù)作用。
[Abstract]:Objective: Diabetic Nephropathy (DN) is one of the most common severe microvascular complications of Diabetes Mellituse (DM). In recent years, it is found that oxidative stress, inflammatory reaction and fibrosis play a important role in the development of diabetic nephropathy. Too much Reactive Oxygen Species (ROS) directly damages biological macromolecules, such as protein and nucleic acid, and also activates the multifunction transcription regulator NF- kappa B (Nuclear Transcription Factor kappa B) to promote a variety of inflammatory factors, macrophages and the overexpression of the key factor of renal fibrosis, TGF- beta, etc. Renal lesions. How to block or inhibit oxidative stress and inflammatory response and reduce renal fibrosis is the key to the prevention and treatment of DN. Repaglinide (RE) is a non sulfonylurea secretory agent, an oral hypoglycemic agent in patients with type 2 diabetes, which can effectively control postprandial blood glucose. Research reports, RE also It can slow down the progress of DN, but the exact mechanism is still unknown. By establishing the DM rat model induced by Streptozotocin (STZ), this subject is to determine the content of malondialdehyde (MDA) and the total antioxidant capacity (T-AOC) activity of oxidative stress in kidney tissue of rats, and the application of immunohistochemistry and real-time quantitative fluorescent PCR (real-time FQ-PCR). The expression of NF- kappa B, TGF- beta in renal tissue and the intervention of Repaglinide (RE) 2mg/Kg were used to observe the oxidative stress, inflammatory reaction and fibrosis in the renal tissue of DM rats, and to explore the intervention and mechanism of reaglinide on its existence, and to provide a theoretical basis for the prevention and control of human sugar DN. 36 SD rats, weight about 200 + 20g, were randomly divided into normal control group (group NC, n=12), diabetes model group (DM group, n=12), diabetic regerel intervention group (RT group, n=12). 1 weeks after adaptive feeding, 12 hours after adaptive feeding, rats in diabetic model group were intraperitoneally injected STZ (65mg/kg), 72h rear tail. Blood blood blood glucose more than 16.7mmol/L, urine sugar + + + + + +, considered as a model successful.NC group of one-time intraperitoneal injection of citric acid buffer. All rats during the test were free to eat water, animal room temperature (20 C ~26 C), humidity (40%~70%) relative constant.RT group rats to Yo Ray Glenn Nai (RE) 2mg/kg, 1/ day, vegetable oil solvent gavage, NC group, DM The group was given the same amount of vegetable oil every day. After 8 weeks and 12 weeks after the success of the model, 6 rats in each group were taken to determine 24h urine protein (24 h UP), blood sugar, urea nitrogen and blood creatinine. The specimens of rat kidney tissue were collected and the tissue sections were collected. The pathological changes of kidney pathology were observed under optical microscope. Changes; detection of renal tissue MDA content, T-AOC activity, immunohistochemistry and real-time PCR method to detect the expression of TGF- beta, NF- kappa B. The experimental data were expressed with mean standard deviation (x + S). The data were analyzed by the statistical analysis software of SPSS 16, and the data which did not satisfy the normality and homogeneity of variance were non parameters. Test, P0.05 has statistical significance. Results: 1 general situation DM group, group RT rats after intraperitoneal injection of STZ gradually polydrink, polydipsia, polyuria, slow reaction, hair loss luster, weight gradually decreased, and the DM group symptoms are significantly worse than the RT group, while the NC group rats have a normal diet, the reaction is sensitive, the growth and development are good, the weight of the body weight obviously increases.2 biochemical index. 2.124 hours urine protein quantitative compared with the same period NC group, 8 weekend, 12 weekend, DM group, group RT rats' 24 hours urine protein level increased (P0.05). Compared with the DM group, the 24 hour urine protein level in the RT group decreased (P0.05).2.2 blood sugar compared with the same period NC group, the 8 weekend, the 12 weekend, DM group, RT group rats blood sugar water (P0.05); with the same period Compared with group M, blood glucose in RT group was not significant (P0.05).2.3 blood creatinine, urea nitrogen was compared with NC group, 8 weekend, 12 weekend, DM group, serum creatinine and urea level in RT group increased (P0.01). Compared with group DM, the level of serum creatinine and urea nitrogen in RT group decreased (P0.05) and.3 oxidative stress index was compared with that of the same period, 8 weekend, 12 weekend The MDA level of renal tissue in T group was significantly increased (P0.05). Compared with the DM group, the MDA level in RT group decreased (P0.05).3.2 T-AOC compared with the NC group in the same period. The activity of renal tissue in the renal tissue of the RT group was significantly lower than that in the 8 weekend, the 12 weekend, the DM group and the kidney tissue of the RT group. The change light microscopy showed that the renal tissue structure of the NC group was clear, the epithelial cells of the renal tubules were arranged neatly, the basal membrane staining was uniform, and the number of mesangial cells and mesangial matrix increased. Compared with the NC group, the glomerular volume increased, the glomerular mesangial matrix increased, the mesangial cells increased, the basement membrane thickened, and the pathology at the 12 weekend was pathological. The renal tissue structure of the.RT group was better than that of the DM group, and the.5 immuno histochemical test was improved on the 8 weekend, the 12 weekend, the DM group, the RT group, NF- kappa B, and the TGF- beta expression increased (P0.05). At the 12 weekend, group DM, group RT, NF- kappa B m RNA, TGF- beta m RNA expression increased (P0.05). .2 reagnonide can reduce the production of proteinuria in DM rats and improve renal function, reduce MDA content in kidney tissue, improve T-AOC activity and reduce NF- kappa B, TGF- beta expression, thus alleviating oxidative stress, inflammatory reaction, postponing renal fibrosis and playing the role of renal protection.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.2

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相關(guān)期刊論文 前1條

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本文編號(hào)：1844118