發(fā)布時間：2018-07-15 20:14

【摘要】：目的:觀察他克莫司(FK506)對2型糖尿病(T2DM)大鼠足細(xì)胞氧化應(yīng)激作用的影響。方法:將40只正常雄性wistar大鼠隨機(jī)分為正常對照組(NC組)10只,其余30只采用高脂高糖飼養(yǎng)(10.0%豬油,20.0%蔗糖,2.5%膽固醇,1.0%膽酸鹽,66.5%常規(guī)飼料)。8周后,聯(lián)合小劑量鏈脲佐菌素STZ(30mg/kg)一次性腹腔注射,建立2型糖尿病大鼠模型。造模成功的21只隨機(jī)分為糖尿病組(DM組)10只,FK506治療組(FK組)11只,FK組給予FK506 0.5mg/(kg·d)灌胃,NC組與DM組給予等量枸櫞酸緩沖液灌胃。各組大鼠分別于藥物干預(yù)前1天及干預(yù)8周后測定空腹血糖(FBG)、內(nèi)生肌酐清除率(Ccr)、總膽固醇(TG)、甘油三酯(TC)、尿肌酐(UCr)、腎質(zhì)量/體質(zhì)量(KW/BW)、血壓(SBP)、谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)、血白細(xì)胞(WBC)。通過酶聯(lián)免疫吸附測定尿白蛋白(UAL),尿白蛋白排泄率(UAER)以UAL(mg/24h)/UCr(mg/24h)比率表示,根據(jù)Ucr、Scr計算內(nèi)生肌酐清除率(Ccr)。在光學(xué)顯微鏡和電子顯微鏡下觀察腎組織病理學(xué)改變,通過Western-blot免疫印跡檢測腎組織中NOX4蛋白的表達(dá)。結(jié)果:30只wistar大鼠造模,成功21只,造模成功率為70%。(1)與NC組比較,DM大鼠空腹血糖(FBG)、鼠尾收縮壓血壓(SBP)、內(nèi)生肌酐清除率(Ccr)、總膽固醇(TG)、甘油三酯(TC)、24h尿白蛋白排泄率(UAER)、腎肥大脂數(shù)(KW/BW)均明顯升高(P0.05);與DM組比較,FK組UAER、KW/BW明顯降低(P0.05),各組血清肌酐(SCr)、丙氨酸氨基轉(zhuǎn)移酶(ALT)、天冬氨酸氨基轉(zhuǎn)移酶(AST)及血白細(xì)胞(WBC)間差異無統(tǒng)計學(xué)意義(P0.05);(2)光鏡下,NC組腎小球無明顯異常,DM組腎小球體積明顯增大,系膜區(qū)增寬,基底膜增厚;FK組病理改變較DM組明顯減輕;(3)電鏡下,DM組與NC組相比,腎小球基底膜增厚,足突排列紊亂、融合率顯著升高,FK組上述病變有所減輕;(4)免疫組化結(jié)果:DM組Nephrin表達(dá)較NC組明顯減少,而FK506干預(yù)后Nephrin表達(dá)上調(diào);(5)Western blot結(jié)果顯示,DM組腎組織NOX4表達(dá)較NC組明顯增多(P0.05),FK組中NOX4的表達(dá)較DM組明顯減少(P0.05)。(6)Pearson相關(guān)性分析提示,NOX4/β-actin蛋白與Nephrin半定量含量成負(fù)相關(guān)(r=㧟0.65,p0.01)。結(jié)論:他克莫司減少DKD大鼠蛋白尿,其機(jī)制可能是通過減少NOX4的表達(dá),抑制腎組織氧化應(yīng)激,減少足細(xì)胞損傷,從而起到延緩DKD進(jìn)程的作用。
[Abstract]:Aim: to observe the effect of tacrolimus (FK506) on oxidative stress of podocytes in type 2 diabetes mellitus (T2DM) rats. Methods: forty normal male wistar rats were randomly divided into normal control group (NC group) and control group (NC group). The remaining 30 rats were fed with high fat and high sugar (10.0% lard 20.0% sucrose 2.5% cholesterol 1.0% cholate 1.0% conventional diet) for 8 weeks. A rat model of type 2 diabetes was established by intraperitoneal injection of low dose streptozotocin (30mg/kg). The 21 successful rats were randomly divided into diabetic group (DM group), 10 FK506 treatment group (FK group), 11 FK506 treated group (FK group), and FK506 0.5mg/ (kg d) intragastric perfusion group (NC group) and DM group (DM group) with the same amount of citric acid buffer intragastric perfusion. Fasting blood glucose (FBG), endogenous creatinine clearance (CCR), total cholesterol (TG), triglyceride (TC), urinary creatinine (UCr), renal mass / body weight (KW / BW), blood pressure (SBP), alanine transaminase (alt), aspartate aminotransferase (AST) and white blood cell (WBC) were measured 1 day before and 8 weeks after the intervention. Urinary albumin (UAL) was measured by enzyme-linked immunosorbent assay (Elisa). Urinary albumin excretion rate (UAER) was expressed as UAL (mg/24h) / UCr (mg/24h) ratio, and endogenous creatinine clearance (CCR) was calculated by Ucr-SCR. The pathological changes of renal tissue were observed under optical microscope and electron microscope, and the expression of NOX4 protein in renal tissue was detected by Western-blot Western blot. Results of the 30 wistar rats, 21 were successful. (1) compared with NC group, fasting blood glucose (FBG), tail systolic blood pressure (SBP), endogenous creatinine clearance (CCR), total cholesterol (TG), urinary albumin excretion rate (UAER) and renal hypertrophic fat (KW / BW) in DM rats were significantly higher than those in DM group (P0.05). Compared with FK group, serum creatinine (SCR), alanine aminotransferase (alt), aspartate aminotransferase (AST) and white blood cell (WBC) were significantly decreased (P0.05). Compared with NC group, the glomerular basement membrane thickened and the foot process was disordered in the FK group, and the pathological changes in the FK group were significantly less than those in the DM group, (3) compared with the NC group, the glomerular basement membrane was thickened and the basement membrane was thickened in the FK group. The expression of Nephrin in FK group was significantly lower than that in NC group, and the expression of Nephrin in FK group was lower than that in NC group. (5) the expression of NOX4 in renal tissue in DM group was significantly higher than that in NC group (P0.05) and the expression of NOX4 in FK group was significantly lower than that in DM group (P0.05). (6) Pearson correlation analysis showed that NOX4 / 尾 -actin protein was negatively correlated with the semi-quantitative content of Nephrin in DM group (P 0.05 p0.01). Conclusion: tacrolimus reduces proteinuria in DKD rats by reducing the expression of NOX4, inhibiting oxidative stress in renal tissue and reducing damage of podocytes, which may delay the progression of DKD.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R692.9

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