本文選題：糖尿病腎病 + 脂毒性��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：目的 糖尿病腎病(Diabetic nephropathy, DN)是糖尿病的嚴(yán)重并發(fā)癥。在美國(guó)DN引起的終末期腎臟病(ESRD)占終末期腎臟病人總數(shù)的50%,是世界范圍內(nèi)引起ESRD的首位病因。隨著對(duì)DN病理生理機(jī)制研究逐漸深入,除了高血糖的毒性作用,脂代謝異常在DN發(fā)生發(fā)展中的作用也日益受到重視。近期Maeda等對(duì)大量日本2型糖尿病伴DN病人(T2DN),2型糖尿病不伴DN病人(T2DM)進(jìn)行基因分析,發(fā)現(xiàn)乙酰輔酶A羧化酶2(Acetyl-CoA carboxylase2, ACC2)的基因多態(tài)性與糖尿病腎病的發(fā)生呈顯著相關(guān)性。后續(xù)對(duì)其它多種族的研究分析也證實(shí)了這一結(jié)果。ACC2能通過(guò)抑制肉堿棕櫚酰轉(zhuǎn)移酶I(CPTI)抑制脂肪酸β氧化,是脂肪酸代謝的關(guān)鍵酶。這為DN致病機(jī)制的研究指明了新的方向。腎間質(zhì)纖維化被認(rèn)為是糖尿病腎病進(jìn)展到腎功能衰竭必經(jīng)的病理途徑,小管上皮細(xì)胞間充質(zhì)轉(zhuǎn)化(Epithelial mesenchymal transition, EMT)是腎間質(zhì)纖維化的關(guān)鍵環(huán)節(jié)。 本實(shí)驗(yàn)旨在探討下調(diào)ACC2表達(dá),對(duì)高糖培養(yǎng)的入腎小管上皮細(xì)胞(HKC)脂質(zhì)沉積及小管上皮細(xì)胞EMT的影響,探討ACC2、脂代謝異常及腎間質(zhì)纖維化之間的可能關(guān)聯(lián),尋找DN防治可能的新靶點(diǎn)。 方法 構(gòu)建ACC2基因特異性的短發(fā)夾雙鏈RNA(shRNA)'慢病毒感染HKC后按照培養(yǎng)條件分為以下5組：正常糖組(5.5mmol/L)、高糖組(30mmol/L)、高滲對(duì)照組(5mmol/L葡萄糖±25mmol/L甘露醇)、ACC2干擾組(感染含ACC2干擾序列慢病毒)、干擾對(duì)照組(感染攜帶綠色螢光蛋白的陰性對(duì)照慢病毒),分別處理96h：①Western Blot檢測(cè)ACC2干擾效率；②光鏡下觀察細(xì)胞形態(tài)變化；③油紅O染色檢測(cè)細(xì)胞內(nèi)脂質(zhì)沉積；④Western Blot檢測(cè)高糖對(duì)ACC2和P-ACC2表達(dá)的影響；⑤Western Blot檢測(cè)高糖對(duì)E-鈣粘蛋白(E-cad)、a-平滑肌肌動(dòng)蛋白(a-SMA)表達(dá)。 結(jié)果 ①干擾效率檢測(cè)ACC2干擾組相對(duì)于干擾對(duì)照組,ACC2蛋白表達(dá)減少85%,成功地減少了ACC2蛋白表達(dá)； ②HKC細(xì)胞形態(tài)正常的HKC細(xì)胞呈卵圓形,細(xì)胞鋪滿(mǎn)后融合呈鋪路石狀,高糖刺激96h后,HKC細(xì)胞拉長(zhǎng)呈長(zhǎng)梭形,細(xì)胞間連接松散,干擾ACC2基因后相對(duì)高糖組及干擾對(duì)照組HKC細(xì)胞形態(tài)恢復(fù)； ③油紅染色檢測(cè)細(xì)胞內(nèi)脂質(zhì)沉積檢測(cè)高糖刺激96h后,HKC細(xì)胞內(nèi)出現(xiàn)清晰的紅色脂質(zhì)沉積,干擾ACC2基因后相對(duì)高糖組及干擾對(duì)照組HKC細(xì)胞內(nèi)脂質(zhì)沉積減少； ④estern Blot檢測(cè)ACC2和P-ACC2蛋白表達(dá)ACC2磷酸化失活,高糖條件下P-ACC2蛋白減少(P0.05), ACC2蛋白量未見(jiàn)明顯變化,ACC2總體活性增強(qiáng)； ⑤Western Blot檢測(cè)E-cad和a-SMA蛋白表達(dá)高糖組相對(duì)于高滲對(duì)照組上皮細(xì)胞標(biāo)志蛋白E-cad表達(dá)減少(p0.05),間質(zhì)細(xì)胞標(biāo)志蛋白a-SMA表達(dá)增多(P0.05),干擾ACC2基因后,E-cad和a-SMA表達(dá)趨于正常。 結(jié)論 ①高糖刺激使HKC細(xì)胞ACC2活性增加,細(xì)胞內(nèi)脂質(zhì)沉積增多,細(xì)胞發(fā)生EMT； ②下調(diào)ACC2表達(dá),可改善高糖誘導(dǎo)的HKC細(xì)胞內(nèi)脂質(zhì)沉積和小管上皮細(xì)胞EMT, ACC2是DN防治的潛在新靶點(diǎn)。
[Abstract]:PurposeDiabetic nephropathy (DNs) is a serious complication of diabetes mellitus.DN is the leading cause of ESRD in the United States, which accounts for 50% of the total number of end-stage renal patients.In addition to the toxic effect of hyperglycemia, the role of abnormal lipid metabolism in the pathogenesis and development of DN has been paid more and more attention with the deepening of the study on the pathophysiological mechanism of DN.In recent years, Maeda was used to analyze the T2DM gene of a large number of Japanese type 2 diabetes mellitus patients without DN. It was found that the gene polymorphism of acetyl-coenzyme A carboxylase 2(Acetyl-CoA carboxylase2 (ACC2) was significantly correlated with the occurrence of diabetic nephropathy.The analysis of other multiracial studies confirmed that ACC2 could inhibit the oxidation of fatty acid 尾 by inhibiting carnitine palmitoyltransferase Ion CPTI, which was the key enzyme in fatty acid metabolism.This indicates a new direction for the study of pathogenesis of DN.Renal interstitial fibrosis is considered to be a necessary pathological pathway for the progression of diabetic nephropathy to renal failure. Epithelial mesenchymal transition (EMTT) is the key link of renal interstitial fibrosis.The aim of this study was to investigate the effects of down-regulation of ACC2 expression on lipid deposition in cultured tubular epithelial cells and EMT in tubular epithelial cells, and to explore the possible association between ACC2, lipid metabolism abnormalities and renal interstitial fibrosis.Look for possible new targets for DN prevention.MethodAfter constructing ACC2 gene specific short hairpin double-stranded RNAs, lentivirus was divided into the following five groups according to the culture conditions: normal glucose group (5.5 mmol / L), high glucose group (30 mmol / L), hypertonic control group (5 mmol / L 鹵25mmol/L mannitol) interference group (infected with ACC2 interference sequence)Lentivirus and interfering control group (infected with lentivirus containing green fluorescent protein) were treated with 96h:1Western Blot to detect the interference efficiency of ACC2.(2) the morphological changes of cells were observed under light microscope. The effect of high glucose on the expression of ACC2 and P-ACC2 was detected by Western Blot. The expression of E-cadherin E-cada- smooth muscle actin (a-SMA) was detected by high glucose on E-cadherin (E-cadA) smooth muscle actin (SMA).Result1 the expression of ACC2 protein in ACC2 interference group was 85% less than that in control group, and the expression of ACC2 protein was reduced successfully.The HKC cells with normal morphology were oval, the cells were paved and fused, and the cells were elongated and fusiform after 96 h of high glucose stimulation, and the intercellular junctions were loose.After interfering with ACC2 gene, the relative high glucose group and the control group were involved in the morphological recovery of HKC cells.3Oil red staining was used to detect the lipid deposition in the cells. After 96 h of high glucose stimulation, there was a clear red lipid deposition in the cells, and the lipid deposition in the HKC cells decreased after interfering with the ACC2 gene in the high glucose group and the control group.The expression of ACC2 phosphorylation in ACC2 and P-ACC2 protein was inactivated by 4estern Blot, P-ACC2 protein decreased P0.05N under high glucose condition, and the total activity of ACC2 was not significantly changed by 4estern Blot.Compared with the hyperosmotic control group, the expression of E-cad and a-SMA protein in hyperglycemic group was significantly lower than that in hyperosmotic control group (P 0.05), and the expression of a-SMA in interstitial cells was increased (P 0.05). The expression of E-cad and a-SMA tended to be normal after interfering with ACC2 gene.Conclusion(1) High glucose stimulation increased the activity of ACC2, the accumulation of lipid and the occurrence of EMTs in HKC cells.2 down-regulation of ACC2 expression can improve lipid deposition in HKC cells induced by high glucose and tubule epithelial cells. ACC2 is a potential new target for the prevention and treatment of DN.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R587.2;R692

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