艾塞那肽对NAFLD细胞模型中PGC-1α表达的影响及其机制

发布时间：2018-01-22 01:39

  本文关键词： 胰高血糖素样肽-1 非酒精性脂肪性肝病 PGC-1α　出处：《郑州大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)是由于肝细胞内脂质过度蓄积所引起的临床病理综合征,除外酒精和病毒性肝炎、自身免疫性肝病等其他明确的可能导致肝脏脂肪变性的因素,目前已经成为全球普遍流行的慢性肝脏病。大量的基础及临床研究用来揭示NAFLD的发病机制,但是有关肝细胞中脂质过多蓄积及其疾病进展中的异常代谢机制仍有待进一步的阐明。过氧化物酶体增殖物激活受体γ辅激活因子1α(Peroxisome proliferators-activated receptor-γcoactivator-1α,PGC-1α)最初是在棕色脂肪组织中作为过氧化物酶体增殖物激活受体γ的共激活因子来调节适应性产热而被发现的,随着研究的深入,人们发现PGC-1α在高能量需求的组织中表达丰富,包括棕色脂肪组织、心肌、骨骼肌、肝脏及肾脏等。肝脏中PGC-1α的表达在产后期的较早阶段达到高峰,其能够协调肝脏中复杂的代谢改变如糖异生,酮体生成,血红素的合成及胆汁酸代谢。越来越多的研究表明,PGC-1α在胰岛素抵抗、脂质代谢紊乱及线粒体功能失调等与NAFLD发病相关的因素中有着重要调节作用。PGC-1α的表达受到多种信号通路的调节如AMP活化的蛋白激酶(AMP-activated protein kinase,AMPK)及促分裂原活化的蛋白激酶p38(mitogen-activeted protein kinase p38,p38MAPK)等。艾塞那肽作为一种胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)受体激动剂,具有调节胰岛素敏感性的作用,目前被普遍应用于2型糖尿病的治疗。近年来的研究发现,GLP-1不仅能够调节血糖、减轻体重,其在改善肝细胞脂肪变方面也起着重要作用。AMPK是调节细胞内能量代谢的重要因子,Shani BS等研究发现,GLP-1可通过激活AMPK来减轻肝细胞内的脂质堆积[1]。目前关于GLP-1对人肝细胞LO2细胞中PGC-1α表达的影响及相关分子机制的研究较少,因此,探讨GLP-1能否通过AMPK通路调节PGC-1α的表达,进而改善肝细胞中的脂质堆积,有可能为GLP-1治疗NAFLD提供进一步的理论依据。的(1)体外培养人正常肝细胞LO2系,用游离脂肪酸(FFA)诱导以建立NAFLD细胞模型。(2)观察艾塞那肽干预对NAFLD细胞模型中PGC-1α表达及脂质堆积的影响,以及PGC-1α的表达与肝细胞中脂质堆积的关系,探讨艾塞那肽用于治疗NAFLD的作用机制。方法用含10%胎牛血清(FBS)的RPMI1640培养基培养人肝细胞系LO2细胞。实验分为两个部分。第一部分共分6组:(1)正常组,即未加FFA;(2)FFA 0.5m M组;(3)FFA 0.75m M组;(4)FFA 1m M组;(5)FFA 1.25m M组;(6)FFA 1.5m M组。各组均培养24h后,用CCK-8法检测细胞增殖活力,判断最适FFA浓度。根据结果,选取FFA 1m M作为模型组,干预24h后,进行油红O染色,细胞内甘油三酯(triglyceride,TG)测定来评价细胞内脂质蓄积程度,判断造模是否成功。第二部分共分为5组:(1)正常对照组:培养基中未加FFA;(2)高脂组:培养基中FFA浓度为1mmol/L;(3)高脂+艾塞那肽10nmol/L组:FFA浓度为1mmol/L,艾塞那肽浓度为10nmol/L;(4)高脂+艾塞那肽100 nmol/L组:FFA浓度为1mmol/L,艾塞那肽浓度为100nmol/L;(5)高脂+艾塞那肽100nmol/L+AMPK抑制剂组:培养基中FFA浓度为1mmol/L,艾塞那肽浓度为100nmol/L,AMPK抑制剂Compound C浓度为15μmol/L。各组均在相同环境下干预24h后,采用油红O染色观察细胞内脂滴生成情况,细胞内TG水平测定评价细胞内脂肪沉积,实时荧光定量PCR(Real-time quantitative reverse transcription PCR,RT-q PCR)检测各组细胞内PGC-1α及乙酰辅酶A羧化酶(acetyl Co A carboxylase,ACC)的m RNA表达水平,Western blotting检测各组细胞内PGC-1α蛋白的表达水平。结果(1)(1)CCK-8结果显示:与正常组相比,0.5mmol/L、0.75mmol/L和1mmol/L FFA干预24h对LO2细胞活力无明显影响(P0.05),1.25mmol/L FFA和1.5mmol/L FFA干预24h后LO2细胞活力下降(P0.05),提示可选用1mmol/L PA干预进行造模,从而避免对细胞活力的影响。(2)油红O染色结果显示模型组细胞内可见聚集成片的红染脂滴,而正常组则未见明显脂滴形成。(3)细胞内TG水平测定同样显示模型组细胞内TG水平较正常组明显增高(P0.05)。(2)与正常组相比,高脂组细胞内红染脂滴明显增多,细胞内TG水平增高(P0.05),ACC m RNA的表达明显增加(P0.05),PGC-1αm RNA与蛋白的表达明显下降(P0.05);与高脂组相比,给予艾塞那肽10nmol/L及100nmol/L干预后,细胞内脂滴的数量,细胞内TG含量和ACC m RNA的表达水平均下降,并且艾塞那肽100nmol/L干预组较艾塞那肽10nmol/L干预组降低的水平更多,差异有统计学意义(P0.05),而PGC-1αm RNA与蛋白的表达水平增高,且艾塞那肽100nmol/L干预组较艾塞那肽10nmol/L干预组水平增高的更多,差异有统计学意义(P0.05);与高脂+艾塞那肽100nmol/L组相比,高脂+艾塞那肽100 nmol/L+AMPK抑制剂组细胞内红染脂滴数量,细胞内TG水平和ACC m RNA的表达水平均增高,差异有统计学意义(P0.05),而PGC-1αm RNA与蛋白的表达水平降低,差异有统计学意义(P0.05);相关性分析结果显示,LO2细胞中PGC-1αm RNA和蛋白的表达水平与细胞内TG的水平呈负相关(r=-0.799,r=-0.826,P0.01)。结论GLP-1受体激动剂艾塞那肽可减轻肝细胞内的脂质堆积,且有一定的剂量依赖性,其机制可能是通过AMPK通路上调PGC-1α基因的表达进而改善肝细胞内的脂代谢紊乱。
[Abstract]:The background of nonalcoholic fatty liver disease (nonalcoholic fatty liver disease, NAFLD) is due to the clinical pathological hepatic lipid accumulation caused by the excessive syndrome, alcohol and viral hepatitis, autoimmune liver disease and other clear may lead to hepatic steatosis of factors, has become a global epidemic of chronic liver disease. And a large number of clinical studies to reveal the pathogenesis of basic NAFLD, but the lipid in liver cells of the excessive accumulation of abnormal metabolism and disease progression remains to be further elucidated. Peroxisome proliferator activated receptor gamma coactivator 1 alpha (Peroxisome proliferators-activated receptor- coactivator-1 PGC-1 gamma alpha, alpha) the first is in the brown adipose tissue as peroxisome proliferator activated receptor gamma coactivator to regulate thermogenesis by hair Now, with the in-depth study, it is found that PGC-1 alpha in high energy demand tissues rich, including brown adipose tissue, myocardium, skeletal muscle, liver and kidney. The expression of PGC-1 in liver and reached the peak at an early stage in the postpartum period, which can coordinate the complex metabolic changes such as liver gluconeogenesis students, ketoplasia, heme synthesis and bile acid metabolism. More and more studies show that PGC-1 alpha in insulin resistance, lipid disorders and metabolic factors of mitochondrial function disorders associated with the onset of NAFLD plays an important role in regulating the.PGC-1 expression by various signaling pathway such as AMP activated protein kinase (AMP-activated protein kinase, AMPK) and mitogen activated protein kinase p38 (mitogen-activeted protein kinase p38, p38MAPK). Exenatide as a glucagon like peptide -1 (glucagon-like peptid E-1, GLP-1) receptor agonist, can regulate insulin sensitivity, is currently widely used in treatment of type 2 diabetes. Recent studies have found that GLP-1 can not only regulate blood sugar, reduce body weight, improve the hepatic steatosis also plays an important role in.AMPK is an important regulator of cellular energy metabolism study on Shani, BS found that GLP-1 can reduce the accumulation of [1]. in current research on the effect of GLP-1 on the expression of PGC-1 in human liver cells LO2 cells and the related molecular mechanism is less, therefore, lipid in liver cells through activation of AMPK to investigate the expression of GLP-1 can regulate PGC-1 alpha through AMPK pathway, thereby improving lipid in liver cells the accumulation may provide further theoretical basis for treatment of GLP-1 NAFLD. (1) normal human liver cell line LO2 cultured in vitro, with free fatty acid (FFA) induced by NAFLD cell model (2) view. 瀵熻壘濉為偅鑲藉共棰勫�筃AFLD缁嗚優妯″瀷涓璓GC-1伪琛ㄨ揪鍙婅剛璐ㄥ爢绉�鐨勫奖鍝�,浠ュ強PGC-1伪鐨勮〃杈句笌鑲濈粏鑳炰腑鑴傝川鍫嗙Н鐨勫叧绯，

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