發(fā)布時(shí)間：2018-04-05 23:08

  本文選題：非酒精性脂肪肝　切入點(diǎn)：波動(dòng)性高糖　出處：《浙江大學(xué)》2014年博士論文

【摘要】：非酒精性脂肪肝(NAFLD)是一種無過量飲酒史,但病理學(xué)改變類似酒精性脂肪肝,以肝細(xì)胞脂肪變性和脂質(zhì)貯積為特征的臨床綜合征。臨床研究發(fā)現(xiàn),2型糖尿病(T2DM)患者發(fā)生NAFLD風(fēng)險(xiǎn)增加,且糖尿病患者更易從單純性非酒精性脂肪肝進(jìn)展為非酒精性脂肪性肝炎(NASH)及肝纖維化,但機(jī)制不明。氧化應(yīng)激和肝細(xì)胞凋亡是NAFLD進(jìn)展的核心環(huán)節(jié),氧化應(yīng)激可導(dǎo)致炎癥因子釋放增多,多種促凋亡因子表達(dá)增強(qiáng),肝細(xì)胞發(fā)生炎癥、凋亡或壞死,使病程向NASH及肝纖維化進(jìn)展。而線粒體通透性轉(zhuǎn)換(MPT)是調(diào)控細(xì)胞凋亡的“生命閘門”。 血糖波動(dòng)異常是糖代謝紊亂的重要標(biāo)志之一。多項(xiàng)臨床研究表明,血糖波動(dòng)指數(shù)升高是引起脂質(zhì)過氧化物水平升高的最主要因素,改善血糖水平或減少血糖波動(dòng)幅度都有助于減少氧化應(yīng)激引起的損害。那么在T2DM患者中,糖尿病是否可能通過波動(dòng)性高血糖的形式誘發(fā)氧化應(yīng)激促進(jìn)肝細(xì)胞凋亡從而促進(jìn)NAFLD的進(jìn)展呢 本研究擬建立肝細(xì)胞脂肪變性模型和糖尿病合并NAFLD小鼠模型,并在體內(nèi)外模擬血糖波動(dòng),以透射電鏡技術(shù)、流式細(xì)胞術(shù)、TUNEL技術(shù)、病理染色、熒光探針及蛋白免疫印跡等技術(shù),觀察波動(dòng)性高血糖對(duì)肝細(xì)胞凋亡、肝組織炎癥性壞死以及纖維化的作用；并探討波動(dòng)性高糖誘發(fā)的氧化應(yīng)激是否通過誘導(dǎo)MPT發(fā)生損傷線粒體形態(tài)和呼吸鏈功能,并啟動(dòng)細(xì)胞色素C介導(dǎo)的線粒體凋亡途徑來促進(jìn)肝細(xì)胞凋亡,以期能揭示糖尿病促進(jìn)NAFLD進(jìn)展的機(jī)制。 第一部分非酒精性脂肪肝小鼠模型中血糖波動(dòng)對(duì)于肝細(xì)胞的凋亡的作用研究 目的： 建立2型糖尿病(T2DM)合并非酒精性脂肪肝(NAFLD)小鼠模型,體內(nèi)模擬血糖波動(dòng),觀察波動(dòng)性高血糖(IHG)對(duì)肝組織壞死性炎癥、纖維化和肝細(xì)胞凋亡的作用,并探討IHG誘發(fā)的氧化應(yīng)激是否通過損傷線粒體功能,并啟動(dòng)細(xì)胞色素C(Cytc)介導(dǎo)的線粒體凋亡途徑來促進(jìn)肝細(xì)胞凋亡。 方法： 利用高脂飲食(HFD)誘導(dǎo)C57BL/6J小鼠建立T2DM合并NAFLD小鼠模型,并通過每日2次腹腔葡萄糖注射(3g/kg)或同體積生理鹽水(NS)注射建立高脂飲食波動(dòng)性高血糖(HFD+F)和持續(xù)性高血糖小鼠模型(HFD+NS).首先監(jiān)測對(duì)照組小鼠(普通飼料喂養(yǎng)并NS注射,STD+NS)、HFD+NS組和HFD+F組小鼠體重、血糖、胰島素及肝酶譜改變；其次,利用病理染色觀察IHG對(duì)小鼠肝臟脂質(zhì)沉積、肝臟纖維化,肝臟脂質(zhì)過氧化水平的影響,再者,利用TUNEL和Western Blotting技術(shù)觀察IHG對(duì)于肝細(xì)胞凋亡的影響,最后,利用Western Blotting及生化法觀察IHG對(duì)于線粒體功能,如Cyt c的釋放、ATP的產(chǎn)生的影響。 結(jié)果： 1.HFD喂養(yǎng)較普通飲食(STD)喂養(yǎng)可顯著增加小鼠體重、誘導(dǎo)糖耐量異常、胰島素抵抗,并增加肝臟脂質(zhì)沉積、肝酶水平以及肝臟纖維化(P0.01)。但上述指標(biāo)在HFD+F與]HFD+NS小鼠間并無差異(P0.05)。 2.HFD喂養(yǎng)較STD喂養(yǎng)可增加小鼠肝細(xì)胞凋亡比率及凋亡相關(guān)蛋白,如Bax、Cleaved caspase9和Cleaved caspase3的表達(dá)(P0.01),而HFD+F小鼠肝細(xì)胞凋亡水平顯著高于HFD+NS小鼠(P0.01)。 3.HFD喂養(yǎng)較STD喂養(yǎng)可增加小鼠脂質(zhì)過氧化水平,丙二醛(MDA)、壬烯(HNE)含量顯著升高(P0.01),而IHG可進(jìn)一步升高小鼠脂質(zhì)過氧化水平(P0.05vs.HFD+NS)。 4.HFD+NS小鼠較STD+NS小鼠胞漿Cyt c含量及肝臟ATP產(chǎn)生顯著升高(P0.01),HFD+F組小鼠胞漿Cyt c含量進(jìn)一步更高(P0.01),而肝臟ATP合成則較HFD+NS下降(P0.01)。 結(jié)論： 1.HFD喂養(yǎng)的小鼠糖耐量、胰島素敏感性、肝臟脂質(zhì)沉積、肝酶水平、肝臟纖維化程度、肝細(xì)胞凋亡、脂質(zhì)過氧化水平如MDA、HNE含量、線粒體Cyt c釋放及ATP產(chǎn)生較STD喂養(yǎng)小鼠明顯增加。 2.盡管予HFD喂養(yǎng)小鼠IHG后,小鼠糖耐量、胰島素敏感性、肝臟脂質(zhì)沉積、肝酶水平以及肝臟纖維化程度上與單純HFD喂養(yǎng)小鼠比較并無差異,但I(xiàn)HG小鼠肝細(xì)胞凋亡,脂質(zhì)過氧化水平,以及線粒體Cyt c釋放明顯增高,而ATP的產(chǎn)生下降。表明在T2DM伴NAFLD的小鼠模型中,IHG可通過增加肝臟氧化應(yīng)激水平、能量合成障礙、線粒體功能受損而增加肝細(xì)胞凋亡,而IHG增加肝細(xì)胞凋亡的具體機(jī)制有待體外實(shí)驗(yàn)進(jìn)一步研究。 第二部分血糖波動(dòng)誘導(dǎo)線粒體通透性轉(zhuǎn)換促進(jìn)脂肪變性肝細(xì)胞凋亡的作用研究 目的： 建立肝細(xì)胞脂肪變性模型,并在體外模擬血糖波動(dòng)。觀察波動(dòng)性高糖(IHG)對(duì)棕櫚酸(PA)誘導(dǎo)肝細(xì)胞凋亡的作用；并探討IHG誘發(fā)的氧化應(yīng)激是否通過誘導(dǎo)線粒體通透性轉(zhuǎn)換(MPT)發(fā)揮作用。 方法： L02細(xì)胞分別用：①正常血糖組(NG,10%FBS1640培養(yǎng)液+葡萄糖5.5mmol/l);②正常血糖脂肪變性組(NG+P,10%FBS1640培養(yǎng)液+0.125mmM棕櫚酸)；③持續(xù)高糖組(SHG,10%FBS1640培養(yǎng)液+葡萄糖30.0mmol/l);④持續(xù)高糖脂肪變性組(SHG+P,10%FBS1640培養(yǎng)液+0.125mmM棕櫚酸)；⑤波動(dòng)性高糖組(IHG,10%FBS1640培養(yǎng)液+葡萄糖5.5mmol/l和30.0mmol/l,每間隔12h更換1次)；⑥波動(dòng)性高糖脂肪變性組(IHG+P,10%FBS1640培養(yǎng)液+0.125mM棕櫚酸+葡萄糖5.5mmol/l和30.0mmol/l,每間隔12h更換1次)培養(yǎng)72h。利用油紅染色及熒光探針觀察細(xì)胞脂肪變性情況,利用流式細(xì)胞計(jì)數(shù)和Western blotting檢測L02細(xì)胞凋亡、ROS產(chǎn)生,線粒體膜電位(△ψm)及細(xì)胞色素C (Cyt c)釋放情況,并利用電鏡觀察細(xì)胞超微結(jié)構(gòu)的改變。予環(huán)孢霉素A (CsA)抑制MPT,觀察關(guān)閉MPT孔是否能逆轉(zhuǎn)肝細(xì)胞的氧化應(yīng)激及凋亡。 結(jié)果： 1.經(jīng)過72h培養(yǎng),PA處理組L02細(xì)胞內(nèi)脂滴明顯增加,但不同糖濃度間脂滴數(shù)量和大小并無顯著差異。 2.在PA存在時(shí),與NG組比較,SHG可誘導(dǎo)肝細(xì)胞凋亡比率及凋亡相關(guān)蛋白,如Cleaved caspase9、Cleaved caspase3及Cleaved PARP表達(dá)增加(P0.05),而IHG可進(jìn)一步惡化肝細(xì)胞凋亡(P0.05vs. SHG+P組)；但單純SHG或IHG對(duì)L02細(xì)胞凋亡并無誘導(dǎo)作用(P0.05)。 3.在PA存在時(shí),與NG組比較,SHG處理后ROS產(chǎn)生及線粒體氧化應(yīng)激增加(P0.05),且IHG較SHG更易誘發(fā)氧化應(yīng)激(P0.05)；但單純SHG或IHG對(duì)L02細(xì)胞ROS產(chǎn)生及線粒體氧化應(yīng)激并無明顯誘導(dǎo)作用(P0.05)。 4. SHG+P組較NG+P組電鏡下線粒體微結(jié)構(gòu)異常(線粒體內(nèi)嵴減少、基質(zhì)腫脹并出現(xiàn)空泡),提示MPT孔開放；肝細(xì)胞線粒體功能障礙更為顯著,表現(xiàn)為△ψm下降、線粒體Cyt c釋放增加、而IHG+P對(duì)線粒體形態(tài)和功能損傷作用較SHG更強(qiáng)。但在不存在PA時(shí),不同糖濃度對(duì)線粒體功能無明顯影響。 5.予CsA預(yù)處理關(guān)閉MPT孔則能阻斷SHG和IHG對(duì)脂肪變性肝細(xì)胞ROS產(chǎn)生、線粒體形態(tài)和功能異常及肝細(xì)胞凋亡的誘導(dǎo)作用。 結(jié)論： 在脂毒性的基礎(chǔ)上,IHG較SHG更易誘導(dǎo)肝臟氧化應(yīng)激和線粒體損傷,并啟動(dòng)Cyt c介導(dǎo)的線粒體凋亡途徑從而促進(jìn)肝細(xì)胞凋亡；而MPT在其中起重要介導(dǎo)作用,特異性關(guān)閉MPT能夠阻斷PA與IHG誘導(dǎo)的ROS產(chǎn)生、線粒體形態(tài)和功能改變及肝細(xì)胞凋亡。
[Abstract]:Nonalcoholic fatty liver disease (NAFLD) is a kind of no history of excessive alcohol consumption, but the pathological changes similar to alcoholic fatty liver, clinical liver steatosis and lipid storage characteristics of syndrome. The clinical study found that type 2 diabetes mellitus (T2DM) patients with increased risk of NAFLD, and more easily from simple diabetic patients progress of non alcoholic fatty liver for nonalcoholic steatohepatitis (NASH) and hepatic fibrosis, but the mechanism is not clear. Oxidative stress and apoptosis of liver cells is the core part of the development of NAFLD, oxidative stress can lead to increased release of various inflammatory factors, apoptosis factor expression, liver cell inflammation, apoptosis or necrosis. The progression to NASH and hepatic fibrosis. The mitochondrial permeability transition (MPT) is the regulation of apoptosis "life gate".
The blood glucose fluctuation is one of the important signs of abnormal glucose metabolism disorder. Number of clinical studies have shown that elevated blood glucose fluctuation index is the main factor for causing increased lipid peroxide levels, improve blood glucose levels or reduced blood glucose fluctuations will help to reduce the damage caused by oxidative stress. So in T2DM patients, whether diabetes may form through induced oxidation the fluctuation of stress hyperglycemia and promoting the progress of liver cell apoptosis so as to promote NAFLD.
This study intends to establish a model of hepatocyte steatosis and diabetic NAFLD mice model, and simulate the fluctuation of blood glucose in vivo by transmission electron microscopy, flow cytometry, TUNEL staining, fluorescence probe technique and Western blot technique, observation of high blood glucose fluctuation on hepatic cell apoptosis, inflammatory necrosis of liver tissue the role of oxidative stress and fibrosis; and to investigate whether intermittent high glucose induced by inducing MPT damage of mitochondrial morphology and function of the respiratory chain, and activate the mitochondrial apoptosis pathway mediated by cytochrome C to promote apoptosis of liver cells, in order to reveal the mechanism of diabetes promote the progression of NAFLD.
The effect of blood glucose fluctuation on the apoptosis of hepatocyte in the first part of nonalcoholic fatty liver model
Objective:
The establishment of type 2 diabetes mellitus (T2DM) combined with nonalcoholic fatty liver disease (NAFLD) mice model in vivo to simulate blood glucose fluctuation, the observation of high blood glucose fluctuation (IHG) of liver tissue inflammation, fibrosis and liver cell apoptosis, and to explore whether IHG induced oxidative stress injury through mitochondrial function, and activate cells cytochrome C (Cytc) mediated mitochondrial apoptotic pathway to promote liver cell apoptosis.
Method錛，

本文編號(hào)：1716886