生长追赶IUGR大鼠的PGC-1α启动子甲基化与胰岛素抵抗机制研究

发布时间：2018-04-09 06:43

本文选题：宫内发育迟缓　切入点：生长追赶　出处：《华中科技大学》2015年博士论文

【摘要】：第一部分生长追赶IUGR大鼠的PGC-1α启动子甲基化水平及转录活性目的：研究宫内生长发育受限而生后出现追赶生长(CG-IUGR)的大鼠成年时PGC-1α启动子的DNA甲基化水平及PGC-1a基因的转录活性,同时测定其核心体温及自发运动量是否发生改变。方法：将受孕后的SD大鼠随机分为限食组和正常饮食组,给予限食组母鼠约相当于正常食量的30%,并于新生鼠出生后减少每窝数量以促进生长追赶。于大鼠8周龄时处死并取其肝脏及骨骼肌。用荧光定量PCR (realtime PCR)方法检测PGC-1α的转录活性。用亚硫酸氢盐转化DNA,再用PCR扩增PGC-1α启动子目的序列,最后用焦磷酸测序(pyrosequencing)的方法检测PGC-1α启动子上的CpG位点-803、-787、-582、-326、-215、-186、-178的甲基化水平。并用数字探头温度计测定大鼠直肠温度,用运动笼及AniLab软件检测其自发运动量。结果：IUGR大鼠出生体重及BMI均低于正常对照组(AGA组),并于3周龄前追赶上AGA组。8周龄CG-IUGR大鼠的体重、BMI及PGC-1α启动子的CpG位点-803和-787的DNA甲基化程度均高于AGA组(p0.05),而PGC-1α的转录活性低于正常组(p0.05)。两组大鼠的直肠温度及自发运动量无明显差异(p0.05)。结论：宫内发育受限及生后追赶生长可能共同介导了PGC-1α启动子DNA甲基化水平及PGC-1α转录活性的改变,这可能是CG-IUGR大鼠成年期胰岛素抵抗的重要机制之一。第二部分生长追赶IUGR大鼠线粒体含量及相关PGC-1α应答基因的转录活性目的：研究宫内发育受限而生后出现追赶生长(CG-IUGR)的大鼠肝脏和骨骼肌线粒体含量及涉及到氧化磷酸化和脂肪酸氧化的PGC-1α下游相关靶基因转录活性的改变,并检测胰岛素信号通路关键分子(包括磷脂酰肌醇3-激酶PI3K和蛋白激酶Akt2)的转录后活性的改变以进一步确定PGC-1α启动子甲基化介导代谢编程。方法:将受孕后的SD大鼠随机分为限食组和正常饮食组,给予限食组母鼠约相当于正常食量的30%,并于新生鼠出生后减少每窝数量以促进生长追赶。于大鼠8周龄时处死并取血、肝脏及骨骼肌。用荧光定量PCR (real-time PCR)方法检测肝脏和骨骼肌的线粒体含量(线粒体／核DNA比率,mtDNA/nDNA)以及涉及到氧化磷酸化和脂肪酸氧化的PGC-1α应答基因的转录活性；用Western blot的方法检测胰岛素信号通路的PI3K和Akt2蛋白表达及磷酸化水平：用ELISA方法检测血浆胰岛素水平；用比色法检测肝脏和骨骼肌的甘油三酯水平。结果：8周龄CG-IUGR大鼠线粒体含量(mtDNA/nDNA)低于AGA组,肝脏和骨骼肌线粒体含量分别和PGC-1αmRNA水平呈正相关及与PGC-1α甲基化含量呈负相关。CG-IUGR大鼠中涉及到氧化磷酸化的PGC-1α应答基因(包括肝脏中的Cycs、 Sdhd和Atp5b以及骨骼肌的Sdhd and Uqcrcl)以及涉及到脂肪酸氧化的PGC-1α应答基因(包括PPAR-α以及肝脏组织的CD36及PPAR-γ)的转录活性以及胰岛素信号通路的关键蛋白PI3Kp85和磷酸化的Akt2水平低于正常对照组(p0.05)。CG-IUGR大鼠肝脏甘油三酯含量及血浆胰岛素水平较AGA组增高(p0.05)。并且,胰岛素水平和PGC-1α启动子CpG位点-787及-803的甲基化水平呈正相关(p0.05)。结论：PGC-1α启动子甲基化水平及其转录活性的改变可能通过介导线粒体含量的减少与涉及到脂肪酸氧化和氧化磷酸化的PGC-1α应答基因的转录活性的降低,以及肝脏甘油三酯的积聚增多来编程CG-IUGR大鼠远期胰岛素抵抗相关代谢疾病的风险。
[Abstract]:The first part
Catch up growth of IUGR rat PGC-1 promoter methylation and transcriptional activity
Objective: To study intrauterine growth restriction and after catch-up growth (CG-IUGR) the transcriptional activity of DNA methylation and PGC-1a gene promoter of PGC-1 rats in adulthood, whether spontaneous movement and determination of its core temperature change.
Methods: SD rats were randomly divided into pregnancy after restricted feeding group and normal diet group, food restriction group were given equivalent to about 30% of the normal intake, and reduce the number of neonatal rats in each litter after birth. In order to promote the catch-up growth in rats aged 8 weeks were sacrificed and the liver and skeletal muscle. Fluorescence quantitative PCR (realtime PCR) method to detect the transcription activity of PGC-1 alpha. DNA transformed with bisulfite, then PCR amplification of PGC-1 promoter to sequence, finally using pyrosequencing (pyrosequencing) method to detect PGC-1 alpha CpG loci on the -803 promoter, -787, -582, -326, -215, -186. The methylation level of -178. And the rectal temperature of the rats were measured using a digital thermometer probe, detecting the spontaneous movement by movement of cage and AniLab software.
Results: IUGR rats birth weight and BMI were lower than the normal control group (group AGA), and at 3 weeks ago after the AGA group on the.8 week old CG-IUGR rats weight, CpG locus BMI and PGC-1 alpha promoter DNA methylation of -803 and -787 were higher than that of group AGA (P0.05), and the transcriptional activity of PGC-1 alpha is lower than the normal group (P0.05). No significant difference between the rectal temperature of the two groups of rats and spontaneous motor activity (P0.05).
Conclusion: intrauterine growth retardation and postnatal catch-up growth may be mediated by the PGC-1 promoter methylation level of DNA and PGC-1 transcriptional activity changes, which may be one of the important mechanisms of CG-IUGR in adult rats during insulin resistance.
The second part
The transcriptional activity of mitochondrial catch-up growth in IUGR rats and PGC-1 alpha response gene
Objective: To study the effects of intrauterine growth restriction and catch-up growth after birth (CG-IUGR) in rat liver and skeletal muscle mitochondrial content and involves PGC-1 alpha downstream related target gene transcription activity of oxidative phosphorylation and fatty acid oxidation and change detection of insulin signaling key molecules (including phosphatidylinositol 3- kinase and PI3K protein kinase Akt2) post transcriptional activity changes to further determine the PGC-1 promoter methylation mediated metabolic programming.
Methods: SD rats were randomly divided into pregnancy after restricted feeding group and normal diet group, food restriction group were given equivalent to about 30% of the normal intake, and reduce the number of neonatal rats in each litter after birth. In order to promote the catch-up growth in rats aged 8 weeks were sacrificed and blood, liver and skeletal muscle. Using fluorescence quantitative PCR (real-time PCR) method to detect the content of mitochondria in liver and skeletal muscle (mitochondrial nuclear / DNA ratio, mtDNA/nDNA) as well as related to the transcriptional activity of PGC-1 alpha gene in response to oxidative phosphorylation and fatty acid oxidation; method of using Western blot to detect the insulin signal pathway of PI3K and Akt2 protein expression and phosphorylation the level of plasma insulin level: ELISA detection method; colorimetric detection of liver and skeletal muscle triglyceride levels.
Results: 8 week old CG-IUGR rats mitochondrial content (mtDNA/nDNA) than in the AGA group, the content of mitochondria in liver and skeletal muscle were related to alpha mRNA and PGC-1 level was positively and PGC-1 methylation content were related to alpha PGC-1 alpha gene response to oxidative phosphorylation is negatively related to.CG-IUGR rats (including liver Cycs, Sdhd Atp5b and Sdhd and Uqcrcl and skeletal muscle) as well as related to PGC-1 alpha response genes of fatty acid oxidation (including PPAR- alpha and liver tissue CD36 and PPAR- gamma) transcription activity and key protein PI3Kp85 and phosphorylation of the insulin signaling pathway Akt2 levels lower than the normal control group (P0.05) and hepatic triglyceride content and insulin levels the plasma levels of.CG-IUGR rats were significantly higher than AGA group (P0.05) and insulin levels and PGC-1 alpha promoter CpG loci -787 and -803 methylation levels were positively correlated (P0.05).
Conclusion: the level of PGC-1 alpha promoter and transcription activity of the promoter may be mediated by changes of mitochondrial content decreased with the dielectric involves reduced transcriptional activity of PGC-1 alpha gene in response to fatty acid oxidation and oxidative phosphorylation of hepatic triglyceride accumulation and increased risk to program CG-IUGR rats long-term insulin resistance related metabolic diseases.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R714.5

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