n-3、n-6多不饱和脂肪酸对3T3-L1脂肪细胞脂联素表达的影响及PPARγ介导的机制

  本文关键词：n-3、n-6多不饱和脂肪酸对3T3-L1脂肪细胞脂联素表达的影响及PPARγ介导的机制，由笔耕文化传播整理发布。

        背景：如今,肥胖、2型糖尿病、心血管疾病等肥胖相关疾病的患病人数急剧增加,正给人类健康及社会发展带来巨大负担,因此越来越多人开始研究此类疾病的预防和治疗措施。近十年来的研究表明,脂肪组织不仅可以储存能量,还具有内分泌的功能。脂联素(adiponectin)作为脂肪组织合成和分泌的脂肪细胞因子具有减轻体重、调节糖脂代谢、调节生育功能、改善胰岛素抵抗、抗炎抗动脉粥样硬化、抗肿瘤等作用。多不饱和脂肪酸如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系的亚油酸(Linoleic acid, LA)、花生四烯酸(Arachidonic acid, AA),在调节糖脂代谢和脂肪细胞分化中发挥重要作用,但其调节脂肪细胞脂联素表达的作用机制还不完全清楚。目的：以体外培养的3T3-L1脂肪细胞为研究对象,以不同浓度n-3、n-6多不饱和脂肪酸(Polyunsaturated fatty acid, PUFA)为处理因素,检测其对3T3-L1脂肪细胞脂联素mRNA和PPARy mRNA表达的影响,并初步探讨n-3、n-6PUFAs通过PPARγ调节脂联素表达的可能机制,对防治代谢综合症和2型糖尿病等肥胖相关疾病提供新思路和理论依据。方法和内容：选用浓度为0μmol/L、25μmol/L、50μmol/L、100μmol/L、200μmol/L、400μmol/L的DNA、EPA、 LA处理体外培养成熟的3T3-L1脂肪细胞24h,实时荧光定量PCR (real-time PCR,RT-PCR)检测处理前后脂联素和PPARy mRNA的表达水平。并选用100μmol/L的DNA、EPA、LA分别加与不加过氧化物酶体增殖物激活受体γ (PPARγ)拮抗剂GW9662处理分化成熟的脂肪细胞24h,实时荧光定量PCR和蛋白免疫印迹反应(western-blotting)分别测定脂联素mRNA表达和蛋白分泌水平。考虑到多不饱和脂肪酸受自由基等有害物质攻击而发生脂质过氧化,检测高浓度多不饱和脂肪酸下调脂联素表达和分泌是否与脂质过氧化有关,收集24h细胞培养液,测定其中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物转移酶(GSH)浓度。所得数据采用均数±标准差(χ±s)表示,采用统计软件SPSS13.0分析,运用单样本t检验进行样本均数与已知总体均数的比较。运用方差分析进行多个样本均数比较,进行方差分析之前先进行方差齐性检验,如果方差齐,运用方差分析和LSD法分别进行多组比较和两两比较；如果方差不齐,运用Welch法和Dunnett’s T3法分别进行多组比较和两两比较。统计结果给出方差是否齐结果,多组比较的F值和P值,两两比较的P值。用Pearson;相关分析两变量之间是否存在相关关系,统计结果给出Pearson相关系数r值和P值,P<0.05为差异显著。结果：(1) n-3PUFAs对脂联素和PPARy mRNA的表达水平的影响：不同浓度DHA对脂联素和PPARy mRNA表达水平影响的差异有统计学意义(F=59.944,P<0.001; F=41.051,P<0.001)。与对照组相比,当DHA浓度为50μmol/L至50μmol/L时,脂联素和PPARy mRNA表达水平均增加,其中DHA浓度为100μmol/L时,脂联素mRNA表达量最高(P<0.001),随着浓度的增加脂联素表达和PPARy mRNA均降低,当DHA浓度为400μmol/L时,脂联素和PPARy mRNA表达量最低,Pearson相关分析得r=0.775,P<0.001；不同浓度EPA对脂联素和PPARy mRNA表达水平影响的差异有统计学意义(F=140.564,P<0.001;F=27.601,P<0.001),当EPA浓度为25μmol/L时,脂联素和PPARy mRNA表达水平最高(P<0.001),当EPA浓度在50μmo1/L至100μmol/L时,脂联素和PPARy mRNA均增加,但随着浓度增加脂联素和PPARy mRNA表达逐渐降低,当EPA浓度为400μmol/L时,脂联素和PPARγ mRNA表达量最低,但无显著性差异。Pearson相关分析得r=0.678,P=0.001。(2) n-6PUFAs对脂联素和PPARy mRNA的表达水平的影响：不同浓度LA对脂联素和PPARy mRNA表达水平影响的差异有统计学意义(F=47.752,P<0.001; F=76.943,P<0.001),与对照组相比,当LA浓度为50μmol,100μmol/L时,脂联素和PPARy mRNA表达水平均增加(P=0.060、0.276),其中脂联素在50μmol时表达量最高,随着浓度的增加二者表达水平均降低,当LA浓度为400μmol时,脂联素和PPARγ mRNA表达水平最低,Pearson相关分析得r=0.798,P<0.003。(3)n-3、n-6PUFAs中加入PPARy拮抗剂GW9662对脂联素和PPARγ mRNA的影响：n-3、n-6PUFAs中加入PPARγ拮抗剂GW9662时脂联素和PPARγ mRNA表达水平影响的差异有统计学意义(F=29.033.P=0.002; F=130.858,P<0.001),与对照组相比,在100μmol/LDHA, EPA、LA中加入100μmol/L GW9662后,脂联素和PPARymRNA均显著降低。(4) n-3、n-6PUFAs对脂联素蛋白分泌的影响：与对照组相比,100μmol/L的LA、DHA、EPA处理后,脂肪细胞内脂联素蛋白分别增加109.56%(P=0.030)、221.89%(P=0.020)、110.62%(P=0.008),差异具有显著性：在PUFAs中加入GW9662处理后,脂肪细胞内脂联素蛋白分别降低94.83%(P<0.001)、83.04%(P<0.001)、88.46%(P<0.001),差异具有显著性。(5)各浓度DHA、 EPA、 LA细胞培养液脂质过氧化程度：当各组脂肪酸浓度为100μmol/L时,MDA含量最低,随着浓度增加,MDA含量逐渐提升,差异具有显著性；SOD活力和GSH水平在高浓度PUFAs作用下有提升。结论：(1)在一定浓度范围内,n-3、n-6PUFAs对脂联素表达的影响呈剂量依赖关系。DHA、EPA、LA分别在100μmo1/L、25μmol/L、50μmol/L浓度时,脂联素mRNA表达量最高,具有统计学意义。生理浓度下(100μmol/L), n-3PUFAs比n-6PUFAs更能促进脂肪细胞脂联素的表达和分泌。超过生理浓度2倍以上时,n-3、n-6PUFAs均显著降低脂肪细胞脂联素的表达。(2)生理浓度下n-3、n-6PUFAs与PPARy激动剂Pioglitazone一样,可以同时提高脂联素表达和分泌水平,但给予PPARγ拮抗剂GW9662可以显著阻断其对脂联素表达和分泌的促进作用,故推测PUFAs可能是通过PPARγ途径调控脂肪细胞的脂联素表达。(3)高浓度多不饱和脂肪酸降低脂联素表达可能与高浓度时多不饱和脂肪酸更易发生脂质过氧化有关。创新点：脂联素是目前倍受关注的防治肥胖,糖尿病等代谢综合征极具潜力的分子靶点,目前的研究一致认为膳食结构和生活方式的改变是导致肥胖以及与之相关的胰岛素抵抗、糖尿病等主要环境因素,高脂肪饮食及脂肪酸构成不合理可能是其中的重要诱因之一,国内外研究鲜有报道n-3、n-6多不饱和脂肪酸对脂联素表达的影响,本实验则通过体外细胞培养实验,侧重研究多不饱和脂肪酸对脂联素表达的影响,并深入探讨多不饱和脂肪酸调节脂联素表达从而影响肥胖和胰岛素抵抗的分子机制。

    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多不饱和脂肪酸对3T3-L1脂肪细胞脂联素表达的影响及PPARγ介导的机制

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 主要仪器19    3 主要方法19-28    4 统计学处理28-29第3章 结果29-44    1 3T3-L1前脂肪细胞的分化与鉴定29    2 实时荧光定量PCR熔解曲线29-31    3 n-3 PUFAs对脂联素和PPARγmRNA表达水平的影响31-34    4 n-6 PUFAs对脂联素和PPARγmRNA表达水平的影响34-36    5 PPARγ拮抗剂GW9662对PUFAs上调脂联素表达的阻断作用36-39    6 n-3、n-6 PUFAs对3T3-L1脂肪细胞细胞内脂联素蛋白表达的影响39-41    7 n-3、n-6 PUFAs对3T3-L1脂肪细胞脂质过氧化的影响41-44第4章 讨论44-48    n-3、n-6多不饱和脂肪酸对3T3-L1脂肪细胞脂联素表达与分泌的影响44-46    n-3、n-6多不饱和脂肪酸调节3T3-L1脂肪细胞脂联素表达与分泌的机制46-48第5章 结论48-49参考文献49-53综述53-64    参考文献60-64硕士研究生期间参加科研活动及论文发表情况64-66    1 论文发表情况64    2 参加学术会议64-66致谢66-69统计学证明69

  本文关键词：n-3、n-6多不饱和脂肪酸对3T3-L1脂肪细胞脂联素表达的影响及PPARγ介导的机制，，由笔耕文化传播整理发布。