肥胖小鼠及脂肪细胞模型中脂质合成所需还原力的来源探究

发布时间：2018-04-18 23:31

本文选题：肥胖 + NADPH　；参考：《江南大学》2017年硕士论文

【摘要】：肥胖是世界范围内的流行性疾病,能够引发心血管疾病、2型糖尿病、血脂异常等一系列慢性疾病。NADPH是脂肪酸合成的还原力来源,主要由氧化磷酸戊糖途径(PPP),异柠檬酸脱氢酶(IDH)、苹果酸酶(ME)及亚甲基四氢叶酸脱氢酶(MTHFD)催化的反应等提供。但是,哪一种还原力产生酶在脂肪形成中发挥重要的作用却不明确。为探究脂肪形成所需NADPH的来源,本课题采用肥胖小鼠模型和脂肪细胞模型,利用荧光定量PCR(RT-qPCR)分析脂肪样本中NADPH产生酶基因的转录水平变化,并对转录水平发生显著上调的关键基因在脂肪细胞中进行基因沉默,确定产NADPH关键基因对细胞脂肪形成的影响,主要结论如下:(1)ob/ob(瘦素缺陷型)和db/db(瘦素受体缺陷型)肥胖小鼠脂肪组织的RTqPCR结果显示,MTHFD2、葡萄糖-6-磷酸脱氢酶(G6PD)、6磷酸葡萄糖酸脱氢酶(PGD)及ME1基因转录水平均发生上调;膳食诱导肥胖小鼠(DIO)中MTHFD2、IDH1、G6PD以及PGD基因转录水平都上调。结果表明,叶酸代谢途径中MTHFD2、PPP途径中G6PD、PGD以及ME1可能是小鼠肥胖形成中NADPH的主要来源。(2)小鼠骨髓基质细胞(op9)诱导分化后的RT-qPCR结果显示,乙醛脱氢酶家族成员4A1(ALDH4A1)、MTHFD2、ME1、ME3、G6PD、PGD基因转录水平均显著上调。脂肪细胞和肥胖小鼠模型RT-qPCR结果共同表明,叶酸代谢途径中MTHFD2、PPP途径中PGD以及ME1可能是脂肪形成所需NADPH的重要来源。(3)对筛选到的关键基因进行基因沉默,发现MTHFD2沉默组脂肪细胞脂滴积累明显减少,总脂肪酸含量及NADPH含量显著减少、脂肪生成相关因子转录水平下降。因此,MTHFD2可能是脂肪形成所需的NADPH来源,叶酸代谢途径可能在脂质合成中具有重要作用。进一步采用抑制剂验证叶酸代谢途径对脂肪细胞分化的作用,结果显示,叶酸合成抑制剂(DAHP)和叶酸代谢平衡抑制剂(TMP)均对脂肪细胞的增殖和分化具有重要作用。结果验证了MTHFD2是脂肪形成所需的NADPH来源,并证实叶酸代谢途径对细胞的脂肪形成具有重要影响。
[Abstract]:Obesity is a worldwide epidemic disease, which can cause cardiovascular disease type 2 diabetes, dyslipidemia and a series of chronic diseases. NADPH is the source of fatty acid synthesis.It is mainly provided by the oxidation of pentose phosphate pathway, isocitrate dehydrogenase (IDH), malate enzyme (MEE) and methylene tetrahydrofolate dehydrogenase (MTHFDs) catalyzed reactions.However, it is not clear which reductase plays an important role in fat formation.In order to explore the source of NADPH needed for fat formation, we used fat mouse model and adipocyte model to analyze the transcriptional level of NADPH producing enzyme gene in fat samples by fluorescence quantitative PCR RT-qPCR.Gene silencing of the key genes, which significantly up-regulated transcription level, was carried out in adipocytes to determine the effect of key genes producing NADPH on cell adipogenesis.The main conclusions were as follows: the RTqPCR results of adipose tissue of obese mice with 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 2 and / or / or / and / or / or / or db- / dbb of obese mice showed that the levels of MTHFD2, G6PDGD6 and ME1 gene transcription were up-regulated in adipose tissues of obese mice.The G6PD and PGD gene transcription levels were up-regulated in Dietary induced obese mice.The results showed that G6PD-PGD and ME1 in the MTHFD2PPP pathway of folic acid metabolism pathway may be the main source of NADPH in obese mice.The transcriptional level of MTHFD2ME1ME3ME3G6PDD gene was significantly up-regulated in ALDH4A1 family member of acetaldehyde dehydrogenase family.The RT-qPCR results of adipocytes and obese mice model showed that PGD and ME1 in the MTHFD2PPP pathway of folic acid metabolism pathway may be an important source of NADPH for adipogenesis.It was found that in MTHFD2 silencing group, lipid droplet accumulation, total fatty acid content and NADPH content were significantly decreased, and the transcription level of adipogenic related factors was decreased.Therefore, MTHFD2 may be the source of NADPH for fat formation, and folic acid metabolism may play an important role in lipid synthesis.The effect of folic acid metabolism pathway on adipocyte differentiation was further verified by inhibitors. The results showed that both folic acid synthesis inhibitor (DAHP) and folic acid metabolic balance inhibitor (TMP) played an important role in the proliferation and differentiation of adipocytes.The results demonstrated that MTHFD2 was the NADPH source for adipogenesis and that folic acid metabolism pathway had an important effect on the adipogenesis of cells.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2

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