本文關(guān)鍵詞：n-3、n-6多不飽和脂肪酸對3T3-L1脂肪細(xì)胞脂聯(lián)素表達(dá)的影響及PPARγ介導(dǎo)的機(jī)制，由筆耕文化傳播整理發(fā)布。

        背景：如今,肥胖、2型糖尿病、心血管疾病等肥胖相關(guān)疾病的患病人數(shù)急劇增加,正給人類健康及社會發(fā)展帶來巨大負(fù)擔(dān),因此越來越多人開始研究此類疾病的預(yù)防和治療措施。近十年來的研究表明,脂肪組織不僅可以儲存能量,還具有內(nèi)分泌的功能。脂聯(lián)素(adiponectin)作為脂肪組織合成和分泌的脂肪細(xì)胞因子具有減輕體重、調(diào)節(jié)糖脂代謝、調(diào)節(jié)生育功能、改善胰島素抵抗、抗炎抗動脈粥樣硬化、抗腫瘤等作用。多不飽和脂肪酸如n-3系的二十二碳六烯酸(Docosahe--xaenoic acid,DHA)、二十碳五烯酸(Eicosapentaenoic Acid,EPA)和n-6系的亞油酸(Linoleic acid, LA)、花生四烯酸(Arachidonic acid, AA),在調(diào)節(jié)糖脂代謝和脂肪細(xì)胞分化中發(fā)揮重要作用,但其調(diào)節(jié)脂肪細(xì)胞脂聯(lián)素表達(dá)的作用機(jī)制還不完全清楚。目的：以體外培養(yǎng)的3T3-L1脂肪細(xì)胞為研究對象,以不同濃度n-3、n-6多不飽和脂肪酸(Polyunsaturated fatty acid, PUFA)為處理因素,檢測其對3T3-L1脂肪細(xì)胞脂聯(lián)素mRNA和PPARy mRNA表達(dá)的影響,并初步探討n-3、n-6PUFAs通過PPARγ調(diào)節(jié)脂聯(lián)素表達(dá)的可能機(jī)制,對防治代謝綜合癥和2型糖尿病等肥胖相關(guān)疾病提供新思路和理論依據(jù)。方法和內(nèi)容：選用濃度為0μmol/L、25μmol/L、50μmol/L、100μmol/L、200μmol/L、400μmol/L的DNA、EPA、 LA處理體外培養(yǎng)成熟的3T3-L1脂肪細(xì)胞24h,實時熒光定量PCR (real-time PCR,RT-PCR)檢測處理前后脂聯(lián)素和PPARy mRNA的表達(dá)水平。并選用100μmol/L的DNA、EPA、LA分別加與不加過氧化物酶體增殖物激活受體γ (PPARγ)拮抗劑GW9662處理分化成熟的脂肪細(xì)胞24h,實時熒光定量PCR和蛋白免疫印跡反應(yīng)(western-blotting)分別測定脂聯(lián)素mRNA表達(dá)和蛋白分泌水平�？紤]到多不飽和脂肪酸受自由基等有害物質(zhì)攻擊而發(fā)生脂質(zhì)過氧化,檢測高濃度多不飽和脂肪酸下調(diào)脂聯(lián)素表達(dá)和分泌是否與脂質(zhì)過氧化有關(guān),收集24h細(xì)胞培養(yǎng)液,測定其中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽過氧化物轉(zhuǎn)移酶(GSH)濃度。所得數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(χ±s)表示,采用統(tǒng)計軟件SPSS13.0分析,運(yùn)用單樣本t檢驗進(jìn)行樣本均數(shù)與已知總體均數(shù)的比較。運(yùn)用方差分析進(jìn)行多個樣本均數(shù)比較,進(jìn)行方差分析之前先進(jìn)行方差齊性檢驗,如果方差齊,運(yùn)用方差分析和LSD法分別進(jìn)行多組比較和兩兩比較；如果方差不齊,運(yùn)用Welch法和Dunnett’s T3法分別進(jìn)行多組比較和兩兩比較。統(tǒng)計結(jié)果給出方差是否齊結(jié)果,多組比較的F值和P值,兩兩比較的P值。用Pearson;相關(guān)分析兩變量之間是否存在相關(guān)關(guān)系,統(tǒng)計結(jié)果給出Pearson相關(guān)系數(shù)r值和P值,P<0.05為差異顯著。結(jié)果：(1) n-3PUFAs對脂聯(lián)素和PPARy mRNA的表達(dá)水平的影響：不同濃度DHA對脂聯(lián)素和PPARy mRNA表達(dá)水平影響的差異有統(tǒng)計學(xué)意義(F=59.944,P<0.001; F=41.051,P<0.001)。與對照組相比,當(dāng)DHA濃度為50μmol/L至50μmol/L時,脂聯(lián)素和PPARy mRNA表達(dá)水平均增加,其中DHA濃度為100μmol/L時,脂聯(lián)素mRNA表達(dá)量最高(P<0.001),隨著濃度的增加脂聯(lián)素表達(dá)和PPARy mRNA均降低,當(dāng)DHA濃度為400μmol/L時,脂聯(lián)素和PPARy mRNA表達(dá)量最低,Pearson相關(guān)分析得r=0.775,P<0.001；不同濃度EPA對脂聯(lián)素和PPARy mRNA表達(dá)水平影響的差異有統(tǒng)計學(xué)意義(F=140.564,P<0.001;F=27.601,P<0.001),當(dāng)EPA濃度為25μmol/L時,脂聯(lián)素和PPARy mRNA表達(dá)水平最高(P<0.001),當(dāng)EPA濃度在50μmo1/L至100μmol/L時,脂聯(lián)素和PPARy mRNA均增加,但隨著濃度增加脂聯(lián)素和PPARy mRNA表達(dá)逐漸降低,當(dāng)EPA濃度為400μmol/L時,脂聯(lián)素和PPARγ mRNA表達(dá)量最低,但無顯著性差異。Pearson相關(guān)分析得r=0.678,P=0.001。(2) n-6PUFAs對脂聯(lián)素和PPARy mRNA的表達(dá)水平的影響：不同濃度LA對脂聯(lián)素和PPARy mRNA表達(dá)水平影響的差異有統(tǒng)計學(xué)意義(F=47.752,P<0.001; F=76.943,P<0.001),與對照組相比,當(dāng)LA濃度為50μmol,100μmol/L時,脂聯(lián)素和PPARy mRNA表達(dá)水平均增加(P=0.060、0.276),其中脂聯(lián)素在50μmol時表達(dá)量最高,隨著濃度的增加二者表達(dá)水平均降低,當(dāng)LA濃度為400μmol時,脂聯(lián)素和PPARγ mRNA表達(dá)水平最低,Pearson相關(guān)分析得r=0.798,P<0.003。(3)n-3、n-6PUFAs中加入PPARy拮抗劑GW9662對脂聯(lián)素和PPARγ mRNA的影響：n-3、n-6PUFAs中加入PPARγ拮抗劑GW9662時脂聯(lián)素和PPARγ mRNA表達(dá)水平影響的差異有統(tǒng)計學(xué)意義(F=29.033.P=0.002; F=130.858,P<0.001),與對照組相比,在100μmol/LDHA, EPA、LA中加入100μmol/L GW9662后,脂聯(lián)素和PPARymRNA均顯著降低。(4) n-3、n-6PUFAs對脂聯(lián)素蛋白分泌的影響：與對照組相比,100μmol/L的LA、DHA、EPA處理后,脂肪細(xì)胞內(nèi)脂聯(lián)素蛋白分別增加109.56%(P=0.030)、221.89%(P=0.020)、110.62%(P=0.008),差異具有顯著性：在PUFAs中加入GW9662處理后,脂肪細(xì)胞內(nèi)脂聯(lián)素蛋白分別降低94.83%(P<0.001)、83.04%(P<0.001)、88.46%(P<0.001),差異具有顯著性。(5)各濃度DHA、 EPA、 LA細(xì)胞培養(yǎng)液脂質(zhì)過氧化程度：當(dāng)各組脂肪酸濃度為100μmol/L時,MDA含量最低,隨著濃度增加,MDA含量逐漸提升,差異具有顯著性；SOD活力和GSH水平在高濃度PUFAs作用下有提升。結(jié)論：(1)在一定濃度范圍內(nèi),n-3、n-6PUFAs對脂聯(lián)素表達(dá)的影響呈劑量依賴關(guān)系。DHA、EPA、LA分別在100μmo1/L、25μmol/L、50μmol/L濃度時,脂聯(lián)素mRNA表達(dá)量最高,具有統(tǒng)計學(xué)意義。生理濃度下(100μmol/L), n-3PUFAs比n-6PUFAs更能促進(jìn)脂肪細(xì)胞脂聯(lián)素的表達(dá)和分泌。超過生理濃度2倍以上時,n-3、n-6PUFAs均顯著降低脂肪細(xì)胞脂聯(lián)素的表達(dá)。(2)生理濃度下n-3、n-6PUFAs與PPARy激動劑Pioglitazone一樣,可以同時提高脂聯(lián)素表達(dá)和分泌水平,但給予PPARγ拮抗劑GW9662可以顯著阻斷其對脂聯(lián)素表達(dá)和分泌的促進(jìn)作用,故推測PUFAs可能是通過PPARγ途徑調(diào)控脂肪細(xì)胞的脂聯(lián)素表達(dá)。(3)高濃度多不飽和脂肪酸降低脂聯(lián)素表達(dá)可能與高濃度時多不飽和脂肪酸更易發(fā)生脂質(zhì)過氧化有關(guān)。創(chuàng)新點(diǎn)：脂聯(lián)素是目前倍受關(guān)注的防治肥胖,糖尿病等代謝綜合征極具潛力的分子靶點(diǎn),目前的研究一致認(rèn)為膳食結(jié)構(gòu)和生活方式的改變是導(dǎo)致肥胖以及與之相關(guān)的胰島素抵抗、糖尿病等主要環(huán)境因素,高脂肪飲食及脂肪酸構(gòu)成不合理可能是其中的重要誘因之一,國內(nèi)外研究鮮有報道n-3、n-6多不飽和脂肪酸對脂聯(lián)素表達(dá)的影響,本實驗則通過體外細(xì)胞培養(yǎng)實驗,側(cè)重研究多不飽和脂肪酸對脂聯(lián)素表達(dá)的影響,并深入探討多不飽和脂肪酸調(diào)節(jié)脂聯(lián)素表達(dá)從而影響肥胖和胰島素抵抗的分子機(jī)制。

    Background:In recent years there has been a marked rise in over-nutrition, obesity, and obesity-associated diseases such as type2diabetes and cardiovascular disease (CVD). An increase in the prevalence of such conditions has led to much research aimed at establishing the underlying causes. Adipose tissue can not only store energy, but also has the function of endocrine. Adipokines secreted from adipose tissue are thought to loss weight, adjust glucolipid metabolism, regulate fertility function, improve insulin resistance, anti-inflammatory, anti-atherosclerosis, antitumor and so on. Polyunsa--turated acids, including n-3PUFAs, such as Docosahexaenoic acid (DHA), Eicosapentaenoic Acid (EPA) and n-6PUFAs such as Linoleic acid (LA), Arachidonic acid (AA), paly an important role in regulating glucolipid metabolism and the differentiation of adipocytes, but the mechanism of PUFAs adjusting adiponectin expression has not entirely clear.Objective:To study the effects of n-3、n-6polyunsaturated fatty acids (PUFAs) on adiponectin and PPARγ mRNA,as well as to research it’s mechanism in3T3-L1adipocytes to provide a new thought and theory basis of the prevention and cure of metabolic syndrome、type2diabetes and obesity related disease. Method:3T3-L1adipocytes were incubated with25μmol/L、50μmoLT、100μmol/L200μmol/L、400μmol/L DHA、EPA、LA or with Bull Serum Albumin (BSA) alone (control) for24h. Adipocytes were also incubated for24h with DHA plus GW-9662, a PPARy antagonist, or with PUFAs alone. The mRNA expression of adiponectin and PPARy was assessed by real-time fluorescence quantitative PCR. The secreted adiponectin protein was analysed by western blotting. PUFAs can be attacked easily by harmful substances such as free radicals and happened to be lipid peroxidation. To ensure whether the high concentration PUFAs cutting adiponectin expression and secretion is relevant to lipid peroxidation,24h cell cultures were collected and examined the concentration of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxide shift enzyme (GSH).The data were shown with the mean±tandard deviation (X±s) by SPSS13.0software.The differences between sample mean and overall mean were compared by one-sample T test. Use One-Way ANOVA analysis on multiple sample mean, before which the variance analysis of homogeneity was made for all inspection, if variance was homogeneous, use the analysis of variance and LSD method for two groups of comparisons and more than two;if variance was not homogeneous, use Welch method and Dunnett’s T3method for two groups of comparisons and more than two comparison. The paper shows the statistical variance result, F and P value in all of the groups. Pearson correlation analysis tested if the two variables were correlated. Pearson correlation coefficient r and P were given in statistical results, P<0.05means significantly different.Result:(1) The effects of n-3PUFAs on adiponectin and PPARy mRNA:Effects of the different concentrations of DHA on adiponectin and PPAR y mRNA expression have a statistical significance(F=59.944, P<0.001; F=41.051, P<0.001). Both50μmol/L and100μmol/L DHA increased the expression of adiponectin and PPARy mRNA compared with the control, the highest was in group100μmol/L (P<0.001). With the increasing of the concentration of adiponectin and PPAR y mRNA expression was lower, especially in group400μmol/L. Pearson correlation analysis was r=0.775,P<0.001; Effects of the different concentrations of EPA on adiponectin and PPAR y mRNA expression have a statistical significance (F=140.564,P<0.001; F=27.601, P<0.001). When the EPA concentration was25μ mol/L, adiponectin and PPAR y mRNA express reached the highest level (P<0.001),when the EPA concentration was in50μmol/L to100μ mol/L, adiponectin and PPAR y mRNA increased, adiponectin and PPAR y mRNA expression gradually reduced with the further concentrations increasing. When the EPA concentration was400μ mol/L, adiponectin and PPAR y mRNA expression was the lowest, Pearson correlation analysis was r=0.678, P=0.001.(2) The effects of n-6PUFAs on adiponectin and PPAR y mRNA expression: Effects of the different concentrations of LA on adiponectin and PPAR y mRNA expression have a statistical significance(F=47.752, P<0.001; F=76.943, P<0.001). Compared with the control, when the LA concentration were50μmol/L、100μmol/L, adiponectin and PPAR y mRNA expression increased (P=0.060; P=0.276), the highest express quantity in50μmol/L group. Adiponectin and PPAR y mRNA expression gradually reduced with the further concentrations increasing. When LA concentration was400μmol/L, adiponectin and PPAR y mRNA expression caught the bottom (P<0.001), Pearson correlation analysis was r=0.972, P=0.003.(3) Effects of antagonist (GW9662) on adiponectin and PPARy mRNA:Effects of diferent n-3、n-6PUFAs with PPAR y antagonist GW9662on adiponectin and PPARy mRNA expression have a statistical significance (F=29.033, P=0.002; F=130.858, P<0.001). Compared with the control,100μmol/L DHA, EPA, LA plused with100μmol/L GW9662respectively, adiponectin and PPAR y mRNA decreased significatively.(4) Effects of n-3、n-6PUFAs on adiponectin secretion:compared with the control,100μmol/L of DHA、EPA and LA enhanced adiponectin secretion by109.56%(P=0.030),221.89%(P=0.020) and110.62%(P=0.008) respectively; when adipocytes were incubated with GW9662, adiponectin secretion was reduced by94.83%,83.04%, and88.46%respectively (P<0.001).(5) Effects of DHA、EPA、LA on lipid peroxidation:MDA content was the lowest in the group100μmol/L and gradually enhanced with increased concentration (P=0.018; P=0.011; P=0.004);The high concentration PUFAs increased SOD vitality and GSH level to a greater extent.Conclusion:(1) Our results demonstrate that n-3、n-6PUFAs affects the expression of adiponectin mRNA in3T3-L1adipocytes concentration-dependently.When DHA, EPA, LA are in the concentration of100μmol/L,25μmol/L,50μmol/L respect--tively,adiponectin mRNA expression reaches the peak with statistical significance. N-3PUFAs can promote adiponectin expression and secretion more than n-6PUFAs in physiological concentrations(100μmol/L). N-3, n-6PUFAs can significantly reduce adiponectin expression in more than two times of physiological concentrations.(2) As the same as PPAR y agonists Pioglitazone, n-3,n-6PUFAs can also improve adiponectin expression and secretion levels in physiological concentrations, but PPAR y antagonists GW9662can significantly block the PUFAs promoting function of adiponectin expression and secretion, so that PUFAs may regulate adiponectin expression through PPAR y in3T3-L1adipocytes. (3) High levels of PUFAs can reduce adiponectin expression that may be relative to lipid peroxidation.Innovation points:Interest in obesity and the metabolic syndrome has prompted renewed research into the physiology of adipose tissue. Adiponectin is the potential molecular target to the prevention and control of the current obesity, diabetes and metabolic syndrome. The present studys consider that the change of dietary structure and lifestyle are linked to obesity, related insulin resistance and diabetes. High fat diet and fatty acid composition may be one of the important factors. The domestic and foreign researchs have few reports about the effects of fatty acid on adiponectin expression. This experiment, in vitro cell culture experiment, focused on polyunsaturated fatty acids on adiponectin express, and further discussed the molecular mechanism of polyunsaturated fatty acids adjust adiponectin expression which affect obesity and insulin resistance.

        n-3、n-6多不飽和脂肪酸對3T3-L1脂肪細(xì)胞脂聯(lián)素表達(dá)的影響及PPARγ介導(dǎo)的機(jī)制

摘要3-7ABSTRACT7-11第1章 前言14-18第2章 材料和方法18-29    1 主要材料18-19    2 主要儀器19    3 主要方法19-28    4 統(tǒng)計學(xué)處理28-29第3章 結(jié)果29-44    1 3T3-L1前脂肪細(xì)胞的分化與鑒定29    2 實時熒光定量PCR熔解曲線29-31    3 n-3 PUFAs對脂聯(lián)素和PPARγmRNA表達(dá)水平的影響31-34    4 n-6 PUFAs對脂聯(lián)素和PPARγmRNA表達(dá)水平的影響34-36    5 PPARγ拮抗劑GW9662對PUFAs上調(diào)脂聯(lián)素表達(dá)的阻斷作用36-39    6 n-3、n-6 PUFAs對3T3-L1脂肪細(xì)胞細(xì)胞內(nèi)脂聯(lián)素蛋白表達(dá)的影響39-41    7 n-3、n-6 PUFAs對3T3-L1脂肪細(xì)胞脂質(zhì)過氧化的影響41-44第4章 討論44-48    n-3、n-6多不飽和脂肪酸對3T3-L1脂肪細(xì)胞脂聯(lián)素表達(dá)與分泌的影響44-46    n-3、n-6多不飽和脂肪酸調(diào)節(jié)3T3-L1脂肪細(xì)胞脂聯(lián)素表達(dá)與分泌的機(jī)制46-48第5章 結(jié)論48-49參考文獻(xiàn)49-53綜述53-64    參考文獻(xiàn)60-64碩士研究生期間參加科研活動及論文發(fā)表情況64-66    1 論文發(fā)表情況64    2 參加學(xué)術(shù)會議64-66致謝66-69統(tǒng)計學(xué)證明69

  本文關(guān)鍵詞：n-3、n-6多不飽和脂肪酸對3T3-L1脂肪細(xì)胞脂聯(lián)素表達(dá)的影響及PPARγ介導(dǎo)的機(jī)制，，由筆耕文化傳播整理發(fā)布。