本文關鍵詞： 胰高血糖素樣肽-1 非酒精性脂肪性肝病 PGC-1α　出處：《鄭州大學》2016年碩士論文　論文類型：學位論文

【摘要】：背景非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)是由于肝細胞內脂質過度蓄積所引起的臨床病理綜合征,除外酒精和病毒性肝炎、自身免疫性肝病等其他明確的可能導致肝臟脂肪變性的因素,目前已經成為全球普遍流行的慢性肝臟病。大量的基礎及臨床研究用來揭示NAFLD的發(fā)病機制,但是有關肝細胞中脂質過多蓄積及其疾病進展中的異常代謝機制仍有待進一步的闡明。過氧化物酶體增殖物激活受體γ輔激活因子1α(Peroxisome proliferators-activated receptor-γcoactivator-1α,PGC-1α)最初是在棕色脂肪組織中作為過氧化物酶體增殖物激活受體γ的共激活因子來調節(jié)適應性產熱而被發(fā)現(xiàn)的,隨著研究的深入,人們發(fā)現(xiàn)PGC-1α在高能量需求的組織中表達豐富,包括棕色脂肪組織、心肌、骨骼肌、肝臟及腎臟等。肝臟中PGC-1α的表達在產后期的較早階段達到高峰,其能夠協(xié)調肝臟中復雜的代謝改變如糖異生,酮體生成,血紅素的合成及膽汁酸代謝。越來越多的研究表明,PGC-1α在胰島素抵抗、脂質代謝紊亂及線粒體功能失調等與NAFLD發(fā)病相關的因素中有著重要調節(jié)作用。PGC-1α的表達受到多種信號通路的調節(jié)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。艾塞那肽作為一種胰高血糖素樣肽-1(glucagon-like peptide-1,GLP-1)受體激動劑,具有調節(jié)胰島素敏感性的作用,目前被普遍應用于2型糖尿病的治療。近年來的研究發(fā)現(xiàn),GLP-1不僅能夠調節(jié)血糖、減輕體重,其在改善肝細胞脂肪變方面也起著重要作用。AMPK是調節(jié)細胞內能量代謝的重要因子,Shani BS等研究發(fā)現(xiàn),GLP-1可通過激活AMPK來減輕肝細胞內的脂質堆積[1]。目前關于GLP-1對人肝細胞LO2細胞中PGC-1α表達的影響及相關分子機制的研究較少,因此,探討GLP-1能否通過AMPK通路調節(jié)PGC-1α的表達,進而改善肝細胞中的脂質堆積,有可能為GLP-1治療NAFLD提供進一步的理論依據。的(1)體外培養(yǎng)人正常肝細胞LO2系,用游離脂肪酸(FFA)誘導以建立NAFLD細胞模型。(2)觀察艾塞那肽干預對NAFLD細胞模型中PGC-1α表達及脂質堆積的影響,以及PGC-1α的表達與肝細胞中脂質堆積的關系,探討艾塞那肽用于治療NAFLD的作用機制。方法用含10%胎牛血清(FBS)的RPMI1640培養(yǎng)基培養(yǎng)人肝細胞系LO2細胞。實驗分為兩個部分。第一部分共分6組:(1)正常組,即未加FFA;(2)FFA 0.5m M組;(3)FFA 0.75m M組;(4)FFA 1m M組;(5)FFA 1.25m M組;(6)FFA 1.5m M組。各組均培養(yǎng)24h后,用CCK-8法檢測細胞增殖活力,判斷最適FFA濃度。根據結果,選取FFA 1m M作為模型組,干預24h后,進行油紅O染色,細胞內甘油三酯(triglyceride,TG)測定來評價細胞內脂質蓄積程度,判斷造模是否成功。第二部分共分為5組:(1)正常對照組:培養(yǎng)基中未加FFA;(2)高脂組:培養(yǎng)基中FFA濃度為1mmol/L;(3)高脂+艾塞那肽10nmol/L組:FFA濃度為1mmol/L,艾塞那肽濃度為10nmol/L;(4)高脂+艾塞那肽100 nmol/L組:FFA濃度為1mmol/L,艾塞那肽濃度為100nmol/L;(5)高脂+艾塞那肽100nmol/L+AMPK抑制劑組:培養(yǎng)基中FFA濃度為1mmol/L,艾塞那肽濃度為100nmol/L,AMPK抑制劑Compound C濃度為15μmol/L。各組均在相同環(huán)境下干預24h后,采用油紅O染色觀察細胞內脂滴生成情況,細胞內TG水平測定評價細胞內脂肪沉積,實時熒光定量PCR(Real-time quantitative reverse transcription PCR,RT-q PCR)檢測各組細胞內PGC-1α及乙酰輔酶A羧化酶(acetyl Co A carboxylase,ACC)的m RNA表達水平,Western blotting檢測各組細胞內PGC-1α蛋白的表達水平。結果(1)(1)CCK-8結果顯示:與正常組相比,0.5mmol/L、0.75mmol/L和1mmol/L FFA干預24h對LO2細胞活力無明顯影響(P0.05),1.25mmol/L FFA和1.5mmol/L FFA干預24h后LO2細胞活力下降(P0.05),提示可選用1mmol/L PA干預進行造模,從而避免對細胞活力的影響。(2)油紅O染色結果顯示模型組細胞內可見聚集成片的紅染脂滴,而正常組則未見明顯脂滴形成。(3)細胞內TG水平測定同樣顯示模型組細胞內TG水平較正常組明顯增高(P0.05)。(2)與正常組相比,高脂組細胞內紅染脂滴明顯增多,細胞內TG水平增高(P0.05),ACC m RNA的表達明顯增加(P0.05),PGC-1αm RNA與蛋白的表達明顯下降(P0.05);與高脂組相比,給予艾塞那肽10nmol/L及100nmol/L干預后,細胞內脂滴的數量,細胞內TG含量和ACC m RNA的表達水平均下降,并且艾塞那肽100nmol/L干預組較艾塞那肽10nmol/L干預組降低的水平更多,差異有統(tǒng)計學意義(P0.05),而PGC-1αm RNA與蛋白的表達水平增高,且艾塞那肽100nmol/L干預組較艾塞那肽10nmol/L干預組水平增高的更多,差異有統(tǒng)計學意義(P0.05);與高脂+艾塞那肽100nmol/L組相比,高脂+艾塞那肽100 nmol/L+AMPK抑制劑組細胞內紅染脂滴數量,細胞內TG水平和ACC m RNA的表達水平均增高,差異有統(tǒng)計學意義(P0.05),而PGC-1αm RNA與蛋白的表達水平降低,差異有統(tǒng)計學意義(P0.05);相關性分析結果顯示,LO2細胞中PGC-1αm RNA和蛋白的表達水平與細胞內TG的水平呈負相關(r=-0.799,r=-0.826,P0.01)。結論GLP-1受體激動劑艾塞那肽可減輕肝細胞內的脂質堆積,且有一定的劑量依賴性,其機制可能是通過AMPK通路上調PGC-1α基因的表達進而改善肝細胞內的脂代謝紊亂。
[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NAFLD緇嗚優(yōu)妯″瀷涓璓GC-1偽琛ㄨ揪鍙婅剛璐ㄥ爢縐殑褰卞搷,浠ュ強PGC-1偽鐨勮〃杈句笌鑲濈粏鑳?yōu)涓剛璐ㄥ爢绉殑鍏尘p，

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