乙酰辅酶A羧化酶2表达下调对高糖培养的人肾小管上皮细胞脂质沉积和间充质转化的影响

发布时间：2018-04-18 03:19

本文选题：糖尿病肾病 + 脂毒性　；参考：《山东大学》2014年硕士论文

【摘要】：目的糖尿病肾病(Diabetic nephropathy, DN)是糖尿病的严重并发症。在美国DN引起的终末期肾脏病(ESRD)占终末期肾脏病人总数的50%,是世界范围内引起ESRD的首位病因。随着对DN病理生理机制研究逐渐深入,除了高血糖的毒性作用,脂代谢异常在DN发生发展中的作用也日益受到重视。近期Maeda等对大量日本2型糖尿病伴DN病人(T2DN),2型糖尿病不伴DN病人(T2DM)进行基因分析,发现乙酰辅酶A羧化酶2(Acetyl-CoA carboxylase2, ACC2)的基因多态性与糖尿病肾病的发生呈显著相关性。后续对其它多种族的研究分析也证实了这一结果。ACC2能通过抑制肉碱棕榈酰转移酶I(CPTI)抑制脂肪酸β氧化,是脂肪酸代谢的关键酶。这为DN致病机制的研究指明了新的方向。肾间质纤维化被认为是糖尿病肾病进展到肾功能衰竭必经的病理途径,小管上皮细胞间充质转化(Epithelial mesenchymal transition, EMT)是肾间质纤维化的关键环节。本实验旨在探讨下调ACC2表达,对高糖培养的入肾小管上皮细胞(HKC)脂质沉积及小管上皮细胞EMT的影响,探讨ACC2、脂代谢异常及肾间质纤维化之间的可能关联,寻找DN防治可能的新靶点。方法构建ACC2基因特异性的短发夹双链RNA(shRNA)'慢病毒感染HKC后按照培养条件分为以下5组：正常糖组(5.5mmol/L)、高糖组(30mmol/L)、高渗对照组(5mmol/L葡萄糖±25mmol/L甘露醇)、ACC2干扰组(感染含ACC2干扰序列慢病毒)、干扰对照组(感染携带绿色萤光蛋白的阴性对照慢病毒),分别处理96h：①Western Blot检测ACC2干扰效率；②光镜下观察细胞形态变化；③油红O染色检测细胞内脂质沉积；④Western Blot检测高糖对ACC2和P-ACC2表达的影响；⑤Western Blot检测高糖对E-钙粘蛋白(E-cad)、a-平滑肌肌动蛋白(a-SMA)表达。结果 ①干扰效率检测ACC2干扰组相对于干扰对照组,ACC2蛋白表达减少85%,成功地减少了ACC2蛋白表达； ②HKC细胞形态正常的HKC细胞呈卵圆形,细胞铺满后融合呈铺路石状,高糖刺激96h后,HKC细胞拉长呈长梭形,细胞间连接松散,干扰ACC2基因后相对高糖组及干扰对照组HKC细胞形态恢复； ③油红染色检测细胞内脂质沉积检测高糖刺激96h后,HKC细胞内出现清晰的红色脂质沉积,干扰ACC2基因后相对高糖组及干扰对照组HKC细胞内脂质沉积减少； ④estern Blot检测ACC2和P-ACC2蛋白表达ACC2磷酸化失活,高糖条件下P-ACC2蛋白减少(P0.05), ACC2蛋白量未见明显变化,ACC2总体活性增强； ⑤Western Blot检测E-cad和a-SMA蛋白表达高糖组相对于高渗对照组上皮细胞标志蛋白E-cad表达减少(p0.05),间质细胞标志蛋白a-SMA表达增多(P0.05),干扰ACC2基因后,E-cad和a-SMA表达趋于正常。结论 ①高糖刺激使HKC细胞ACC2活性增加,细胞内脂质沉积增多,细胞发生EMT； ②下调ACC2表达,可改善高糖诱导的HKC细胞内脂质沉积和小管上皮细胞EMT, ACC2是DN防治的潜在新靶点。
[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 卤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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692

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