本文關(guān)鍵詞： 幾丁寡糖 高脂飲食 脂肪酸 脂代謝紊亂 炎癥　出處：《西南醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討幾丁寡糖(Chitin oligosaccharide,NACOS)對機(jī)體脂代謝紊亂的抑制作用及其潛在的分子機(jī)制。方法:體外細(xì)胞學(xué)實驗:(1)MTT法篩選用藥濃度:分別采用濃度梯度的PA(0、25、50、100和200μM)預(yù)先作用24 h,MTT法用酶標(biāo)儀檢測HepG2細(xì)胞增殖的OD值,計算細(xì)胞存活率,判斷最適PA濃度。將HepG2細(xì)胞經(jīng)NACOS(0、50、100μg/ml)預(yù)處理12 h后,加入PA處理24 h,MTT法檢測細(xì)胞存活率,判斷最適NACOS濃度。(2)油紅O染色法:HepG2細(xì)胞經(jīng)NACOS(0、50、100μg/ml)預(yù)處理12 h后,再用PA刺激24 h。倒置顯微鏡觀察HepG2細(xì)胞形態(tài)學(xué),以此評價NACOS對PA誘導(dǎo)所致的HepG2細(xì)胞中脂質(zhì)沉淀的影響以及NACOSA抑制HepG2細(xì)胞活化的最適濃度。(3)實時熒光定量PCR(Quantitative Real-time PCR)檢測HepG2細(xì)胞經(jīng)PA刺激不同時間后過氧化物酶體增殖體受體共激活因子-1α(PGC1α)、炎癥因子IL-1β、乙酰輔酶A1(ACC1)、細(xì)胞色素氧化酶亞單位-5b(Cox5b)、中鏈�；o酶A脫氧酶(Mcad)的轉(zhuǎn)錄表達(dá)水平,以評價PA對HepG2細(xì)胞的最適處理時間;將HepG2細(xì)胞經(jīng)NACOS(100μg/m L)預(yù)處理12 h后,加入PA處理24 h,檢測過PGC1α、Il-1β、ACC1、Cox5b、Mcad的轉(zhuǎn)錄表達(dá)水平,以評價NACOS對PA誘導(dǎo)HepG2細(xì)胞脂代謝紊亂的抑制作用。(4)免疫印跡法(Western b Lot)檢測HepG2細(xì)胞經(jīng)PA不同時間刺激后MAPKs及PI3K/Akt通路中相關(guān)蛋白激酶p-p38、ERK1/2、Akt的蛋白磷酸化水平的變化,以評價PA對HepG2細(xì)胞的最適處理時間;將HepG2細(xì)胞經(jīng)NACOS(0、50、100μg/ml)預(yù)處理12 h后,再用PA刺激0.5 h,檢測MAPKs及PI3K/Akt通路中相關(guān)蛋白激酶p-p38、p-ERK1/2、p-Akt的蛋白水平的變化,以評價NACOS對PA誘導(dǎo)HepG2細(xì)胞脂代謝紊亂的抑制作用。體內(nèi)實驗雄性C57BL/6小鼠隨機(jī)分為4組(n=5),即正常對照(normal control group,NCD)組、高脂飲食(high fat diet,HFD)組、NACOS組、NACOS+HFD組,造模20周,乙醚麻醉殺死小鼠,提取肝臟組織進(jìn)行檢查,用于制備組織勻漿。主要檢測方法如下:(1)考察高脂模型C57BL/6小鼠在給予NACOS后各項生理指標(biāo)的的變化,如體重、飲水、飲食上的變化。(2)RT-PCR方法檢測肝臟組織中過氧化物酶體增殖體受體共激活因子-1α、白介素因子1-β、乙酰輔酶A1、細(xì)胞色素氧化酶亞單位-5b、中鏈�；o酶A脫氧酶的轉(zhuǎn)錄表達(dá)水平的變化。(3)Western blotting方法檢測MAPKs及PI3K/Akt通路中相關(guān)蛋白激酶p-p38、p-ERK1/2、p-Akt的蛋白水平的變化。結(jié)果:體外細(xì)胞學(xué)實驗:(1)MTT細(xì)胞活性實驗表明,PA在25-200μM范圍內(nèi)對HepG2細(xì)胞的活力沒有明顯的抑制作用,表明PA在該濃度下沒有細(xì)胞毒性,可在此濃度以下進(jìn)行體外藥效學(xué)實驗;NACOS 100μg/m L可略微增加HepG2的活力,但沒有統(tǒng)計學(xué)差異,且NACOS與PA聯(lián)合進(jìn)行藥物處理時亦沒有表現(xiàn)出細(xì)胞毒作用。(2)油紅O染色實驗顯示,PA處理后則細(xì)胞漿中的紅色脂肪顆粒呈顯著聚集趨勢。與PA單相比,NACOS預(yù)處理可明顯抑制PA誘導(dǎo)所致的HepG2細(xì)胞中脂滴的形成。(3)RT-PCR實驗結(jié)果表明,HepG2細(xì)胞經(jīng)PA 100μM處理3-24 h后,PGC1α、IL-1β、ACC1、Cox5b、Mcad均表現(xiàn)出不同程度的升高(P0.05或0.01),并在作用6 h后達(dá)峰值。(4)Western blotting結(jié)果顯示,HepG2細(xì)胞經(jīng)PA 100μM刺激后,絲裂原活化蛋白激酶(Mitogen activated protein kinases,MAPKs)及磷脂酰肌醇3-激酶/蛋白激酶B(Phosphoinositide 3-kinase,PI3K;Proteinkinase B,Akt)通路中的p38、ERK1/2、Akt激酶迅速激活,其磷酸化水平在0-1 h內(nèi)呈時間依賴性增加。其中,p-ERK1/2及p-Akt在0.5 h時達(dá)峰值(P0.05,vs對照組),而p-p38則在1 h時達(dá)最高水平(P0.01,vs對照組)。體內(nèi)實驗結(jié)果表明:(1)與給予基礎(chǔ)飼料的正常對照組(NCD)小鼠比較,高脂模型組(HFD)小鼠的平均體重顯著增加(P0.01)。相反,當(dāng)高脂小鼠同時給予NACOS(1 mg/ml)處理時,可顯著抑制其體重的增加(P0.05或0.01)。高脂飼料和或NACOS均對小鼠的采食量及飲水量沒有明顯影響。(2)RT-PCR實驗結(jié)果表明,HFD組小鼠較NCD組小鼠肝臟組織中PGC1α、ACC1及Mcad顯著升高(P0.05或0.01),炎癥因子IL-1β亦增加明顯(P0.05),但HFD處理對Cox5b的轉(zhuǎn)錄表達(dá)無顯著影響。與HFD組比較,小鼠經(jīng)NACOS處理后,其肝臟組織中脂代謝調(diào)控因子PGC1α、Mcad及ACC1的轉(zhuǎn)錄水平均不同程度地得到抑制(P0.05或0.01),炎癥因子IL-1β亦顯著降低(P0.05或0.01),Cox5b轉(zhuǎn)錄表達(dá)水平變化不明顯。(3)Western blotting結(jié)果顯示,與NCD組小鼠相比,HFD組小鼠肝臟的p-p38、p-Akt蛋白的表達(dá)水平顯著增高(P0.05),而p-ERK1/2則無明顯差別。與HFD組相比,MACOS可顯著抑制高脂飲食喂養(yǎng)所致p-p38及p-Akt的激活(P0.01或0.05)。此外,NACOS亦可降低p-ERK1/2的蛋白表達(dá)水平(P0.05)。結(jié)論:(1)體外細(xì)胞學(xué)實驗結(jié)果表明:NACOS與PA聯(lián)合進(jìn)行藥物處理時沒有表現(xiàn)出細(xì)胞毒作用NACOS預(yù)處理明顯抑制PA誘導(dǎo)所致的HepG2細(xì)胞中脂滴的形成;NACOS抑制PA刺激引起的HepG2細(xì)胞脂代謝的紊亂及相關(guān)炎癥因子的過表達(dá);NACOS預(yù)處理顯著抑制p-p38、p-ERK1/2及p-Akt的表達(dá)水平。(2)體內(nèi)實驗結(jié)果表明:高脂小鼠同時給予NACOS(1 mg/ml)處理時,可顯著抑制小鼠體重的增加,高脂飼料和或NACOS對小鼠的采食量及飲水量沒有明顯影響;NACOS處理可有效逆轉(zhuǎn)高脂飲食喂養(yǎng)所致的小鼠肝臟脂代謝紊亂;NACOS抑制高脂飲食造成的脂代謝紊亂可能是通過抑制MAPKs通路的激活,下調(diào)炎癥反應(yīng)的發(fā)生,從而預(yù)防脂代謝紊亂的發(fā)生。
[Abstract]:Objective: To investigate the effect of chitooligosaccharide (Chitin oligosaccharide, NACOS) inhibitory effects on lipid metabolism and its underlying molecular mechanisms. Methods: in vitro experiments: (1) MTT method for screening drug concentration: the concentration gradient of PA (0,25,50100 and 200 M) pre 24 h OD value by MTT method ELISA detection of HepG2 cell proliferation, cell survival rate was calculated, the optimum PA concentration of HepG2 judgment. Cells were treated with NACOS (0,50100 g/ml) pretreatment of 12 h after treated with PA 24 h, the survival rate of cells was detected by MTT, the optimal concentration of NACOS. (2) by oil red O staining: NACOS HepG2 cells (0,50100 g/ml) pretreatment 12 h after stimulation with PA 24 h. inverted microscope was used to observe the morphology of HepG2 cells, in order to evaluate the optimal concentration of lipid precipitation of NACOS on PA induced HepG2 in NACOSA cells and inhibit the activation of HepG2 cells. (3) real time fluorescence quantitative PCR (Qu Antitative Real-time PCR) detection of HepG2 cells stimulated by PA in different time after peroxisome proliferator receptor coactivator -1 alpha (PGC1 alpha), inflammatory factor beta IL-1, acetyl coenzyme A1 (ACC1), cytochrome oxidase subunit -5b (Cox5b), medium chain acyl coenzyme A oxygenase (Mcad) expression level of transcription, to evaluate PA on HepG2 cells and the optimal treatment time; HepG2 cells were treated with NACOS (100 g/m L) pretreatment of 12 h after treated with PA 24 h, detection of PGC1 alpha, Il-1 beta, ACC1, Cox5b, the expression level of Mcad transcription, to evaluate the inhibitory effect of NACOS on PA induced HepG2 cells lipid metabolism disorder. (4) immunoblotting (Western B Lot) related protein kinase p-p38, HepG2 cells were detected by PA in different time after stimulation of MAPKs and PI3K/Akt in ERK1/2 pathway, changes in protein phosphorylation levels of Akt, PA on HepG2 cells to evaluate the optimal treatment time; HepG2 cells by NACOS ( 0,50100 g/ml) pretreatment 12 h after stimulation with PA 0.5 h, detection of protein kinase MAPKs and PI3K/Akt pathway in p-p38, p-ERK1/2, changes of p-Akt protein level, to evaluate the inhibitory effect of NACOS on PA induced HepG2 cell lipid metabolism in vivo. Male C57BL/6 mice were randomly divided into 4 groups (n=5) normal control (normal, control, group, NCD) group, high fat diet (high fat, diet, HFD) group, NACOS group, NACOS+HFD group, at 20 weeks, the ether killed mice, extracted from liver tissue were examined for tissue homogenate was prepared. The following main detection methods: (1) effects of high fat the model of C57BL/6 mice treated with NACOS changes in various physiological indexes such as body weight, drinking, diet change. (2) factor -1 alpha CO activation of peroxisome proliferation in liver tissue was determined in RT-PCR receptor, interleukin beta factor 1-, acetyl coenzyme A1, cytochrome oxidase Subunit -5b, the expression of transcription chain acyl coenzyme A dehydrogenase. (3) associated protein kinase p-p38, Western MAPKs and PI3K/Akt blotting method to detect the pathway of p-ERK1/2, changes of p-Akt protein level. Results: in vitro experiments: (1) MTT cell activity experiment showed that PA in 25-200 the range of M on the viability of HepG2 cells had no obvious inhibitory effect, PA showed no cytotoxicity in the concentration, the concentration of the following in vitro pharmacodynamic experiment; NACOS 100 g/m L can slightly increase the activity of HepG2, but the difference was not statistically significant, also showed no cytotoxicity and NACOS were combined with PA drug treatment (2). Oil red O staining showed red fat particles in cytoplasm after PA treatment showed a significant accumulation trend. Compared with PA, NACOS pretreatment could significantly inhibit the formation of PA induced by HepG2 cells in lipid droplets. .(3)RT-PCR瀹為獙緇撴灉琛ㄦ槑,HepG2緇嗚優(yōu)緇廝A 100渭M澶勭悊3-24 h鍚，

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