本文選題：糖尿病腎病　切入點(diǎn)：細(xì)胞凋亡　出處：《遵義醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景及目的:糖尿病腎病(DN)發(fā)生發(fā)展與腎臟微血管異常和慢性缺氧密切相關(guān),而腎臟固有細(xì)胞凋亡在其進(jìn)程中扮演重要作用。前期實(shí)驗(yàn)已證實(shí)促血管生成素-1(Ang-1)和抑制低氧誘導(dǎo)因子(HIF)降解可減輕糖尿病大鼠腎臟血管生成和缺氧相關(guān)因子異常表達(dá),改善糖尿病腎臟微血管病變和缺氧狀態(tài)。本研究旨在從細(xì)胞凋亡角度評(píng)估Ang-1、含羞草素(L-mimosine,HIF降解抑制劑)對(duì)糖尿病腎臟保護(hù)性作用。方法:構(gòu)建Ang-1基因重組腺病毒載體(Ad-Ang-1)。采用腹腔一次性注射鏈脲菌素誘導(dǎo)SD大鼠糖尿病模型。設(shè)置正常對(duì)照組(N組)、DN組(D組)、空載腺病毒組(K組)、Ang-1干預(yù)組(A組)、L-mimosine干預(yù)組(L組)及Ang-1+L-mimosine聯(lián)合干預(yù)組(AL組)。成模8W后,K組大鼠經(jīng)陰莖靜脈注射空白腺病毒載體(3×108 pfu/只),A組大鼠經(jīng)陰莖靜脈注射Ad-Ang-1(3×108 pfu/只),L組大鼠經(jīng)腹腔注射L-mimosine(50mg/kg,隔日一次),AL組聯(lián)用Ad-Ang-1及L-mimosine。在12W、16W末收集大鼠血、尿標(biāo)本常規(guī)生化檢測(cè),同時(shí)處死大鼠,取出腎臟組織,HE及PAS染色觀察腎臟病理,Western blot測(cè)定凋亡相關(guān)蛋白(cleaved CASP3、CASP3、BAX、BCL-2)的表達(dá)變化,TUNEL法觀察腎臟固有細(xì)胞凋亡情況。結(jié)果:1.血糖、24h尿蛋白:5個(gè)糖尿病模型組血糖、24h尿蛋白均顯著高于N組(P0.05),其中AL、L組24h尿蛋白低于D、A組(P0.05)。2.血肌酐、尿素:5個(gè)糖尿病模型組血肌酐、尿素水平較N組不同程度上升,且3個(gè)藥物干預(yù)組之間無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05),其中血肌酐水平與D組亦無(wú)明顯差異,而12w時(shí)血尿素水平卻較D組升高(P0.01)。3.腎臟病理:5個(gè)糖尿病模型組可見(jiàn)不同程度的腎小球變形增大、系膜基質(zhì)積聚、細(xì)胞總數(shù)增多、部分球囊擴(kuò)張、腎小管管腔擴(kuò)張及腎小管間質(zhì)炎癥細(xì)胞浸潤(rùn),3個(gè)藥物干預(yù)組病理改變相對(duì)減輕。4.腎組織細(xì)胞凋亡相關(guān)蛋白:(1)與N組相比,5個(gè)糖尿病模型組cleaved CASP3蛋白表達(dá)均明顯上調(diào)(P0.01),而CASP3蛋白表達(dá)均明顯下調(diào)(P0.01)。3個(gè)藥物干預(yù)組cleaved CASP3蛋白表達(dá)顯著低于D組(P0.01),而CASP3蛋白表達(dá)顯著高于D組(P0.01),且3個(gè)藥物干預(yù)組間cleaved CASP3、CASP3蛋白表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。(2)5個(gè)糖尿病模型組BAX蛋白表達(dá)較N組均明顯上調(diào)(P0.01),其中3個(gè)藥物干預(yù)組BAX蛋白表達(dá)顯著低于D組(P0.01),且3個(gè)藥物干預(yù)組間BAX蛋白表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。(3)5個(gè)糖尿病模型組BCL-2蛋白表達(dá)較N組均顯著下調(diào)(P0.05),其中3個(gè)藥物干預(yù)組BCL-2蛋白表達(dá)顯著高于D組(P0.05),且3個(gè)藥物干預(yù)組間BCL-2蛋白表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。(4)5個(gè)糖尿病模型組BAX/BCL-2比值較N組均明顯上調(diào)(P0.01),其中3個(gè)藥物干預(yù)組BAX/BCL-2比值低于D組(P0.01),且3個(gè)藥物干預(yù)組間BAX/BCL-2比值無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。5.腎組織細(xì)胞凋亡率:細(xì)胞凋亡主要表現(xiàn)在腎小管上皮細(xì)胞,其次系膜細(xì)胞;5個(gè)糖尿病模型組細(xì)胞凋亡率較N組均明顯升高(P0.01),其中3個(gè)藥物干預(yù)組細(xì)胞凋亡率顯著低于D組(P0.01),且3個(gè)藥物干預(yù)組間細(xì)胞凋亡率無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。6.Pearson相關(guān)性分析:Cleaved CASP3、BAX的表達(dá)及BAX/BCL-2比值與腎臟固有細(xì)胞凋亡率呈顯著正相關(guān)(r=0.963、0.927、0.962,P0.01),而CASP3、BCL-2的表達(dá)與腎臟固有細(xì)胞凋亡率呈顯著負(fù)相關(guān)(r=-0.967、-0.940,P0.01)。結(jié)論:Ang-1和或L-mimosine可降低糖尿病大鼠腎臟固有細(xì)胞凋亡,且與改善細(xì)胞凋亡相關(guān)蛋白的表達(dá)相關(guān)。Ang-1和L-mimosine單用或聯(lián)用無(wú)明顯差異。
[Abstract]:Background and objective: diabetic nephropathy (DN) and the occurrence and development of renal microvascular abnormalities and chronic hypoxia is closely related to apoptosis in renal cell and play an important role in the process. Previous studies have demonstrated that Ang -1 (Ang-1) and inhibition of hypoxia inducible factor (HIF) degradation can reduce renal vascular generation in diabetic rats and the abnormal expression of hypoxia related factors, improve the renal microvascular disease in diabetic and hypoxic condition. The purpose of this study was to evaluate Ang-1 from the aspect of apoptosis, mimosine (L-mimosine, HIF degradation inhibitor) on diabetic kidney protective effect. Methods: to construct the recombinant adenovirus vector containing Ang-1 gene (Ad-Ang-1) by intraperitoneal injection of streptozotocin. Streptozotocin induced diabetic rat model of SD. The normal control group (N group), DN group (D group), empty adenovirus group (K group), Ang-1 group (A group), L-mimosine group (group L) and Ang-1+L-mim osine鑱斿悎騫查緇，

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