本文選題：宮內發(fā)育遲緩　切入點：生長追趕　出處：《華中科技大學》2015年博士論文

【摘要】：第一部分 生長追趕IUGR大鼠的PGC-1α啟動子甲基化水平及轉錄活性 目的：研究宮內生長發(fā)育受限而生后出現追趕生長(CG-IUGR)的大鼠成年時PGC-1α啟動子的DNA甲基化水平及PGC-1a基因的轉錄活性,同時測定其核心體溫及自發(fā)運動量是否發(fā)生改變。 方法：將受孕后的SD大鼠隨機分為限食組和正常飲食組,給予限食組母鼠約相當于正常食量的30%,并于新生鼠出生后減少每窩數量以促進生長追趕。于大鼠8周齡時處死并取其肝臟及骨骼肌。用熒光定量PCR (realtime PCR)方法檢測PGC-1α的轉錄活性。用亞硫酸氫鹽轉化DNA,再用PCR擴增PGC-1α啟動子目的序列,最后用焦磷酸測序(pyrosequencing)的方法檢測PGC-1α啟動子上的CpG位點-803、-787、-582、-326、-215、-186、-178的甲基化水平。并用數字探頭溫度計測定大鼠直腸溫度,用運動籠及AniLab軟件檢測其自發(fā)運動量。 結果：IUGR大鼠出生體重及BMI均低于正常對照組(AGA組),并于3周齡前追趕上AGA組。8周齡CG-IUGR大鼠的體重、BMI及PGC-1α啟動子的CpG位點-803和-787的DNA甲基化程度均高于AGA組(p0.05),而PGC-1α的轉錄活性低于正常組(p0.05)。兩組大鼠的直腸溫度及自發(fā)運動量無明顯差異(p0.05)。 結論：宮內發(fā)育受限及生后追趕生長可能共同介導了PGC-1α啟動子DNA甲基化水平及PGC-1α轉錄活性的改變,這可能是CG-IUGR大鼠成年期胰島素抵抗的重要機制之一。 第二部分 生長追趕IUGR大鼠線粒體含量及相關PGC-1α應答基因的轉錄活性 目的：研究宮內發(fā)育受限而生后出現追趕生長(CG-IUGR)的大鼠肝臟和骨骼肌線粒體含量及涉及到氧化磷酸化和脂肪酸氧化的PGC-1α下游相關靶基因轉錄活性的改變,并檢測胰島素信號通路關鍵分子(包括磷脂酰肌醇3-激酶PI3K和蛋白激酶Akt2)的轉錄后活性的改變以進一步確定PGC-1α啟動子甲基化介導代謝編程。 方法:將受孕后的SD大鼠隨機分為限食組和正常飲食組,給予限食組母鼠約相當于正常食量的30%,并于新生鼠出生后減少每窩數量以促進生長追趕。于大鼠8周齡時處死并取血、肝臟及骨骼肌。用熒光定量PCR (real-time PCR)方法檢測肝臟和骨骼肌的線粒體含量(線粒體／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應答基因的轉錄活性；用Western blot的方法檢測胰島素信號通路的PI3K和Akt2蛋白表達及磷酸化水平：用ELISA方法檢測血漿胰島素水平；用比色法檢測肝臟和骨骼肌的甘油三酯水平。 結果：8周齡CG-IUGR大鼠線粒體含量(mtDNA/nDNA)低于AGA組,肝臟和骨骼肌線粒體含量分別和PGC-1αmRNA水平呈正相關及與PGC-1α甲基化含量呈負相關。CG-IUGR大鼠中涉及到氧化磷酸化的PGC-1α應答基因(包括肝臟中的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應答基因(包括PPAR-α以及肝臟組織的CD36及PPAR-γ)的轉錄活性以及胰島素信號通路的關鍵蛋白PI3Kp85和磷酸化的Akt2水平低于正常對照組(p0.05)。CG-IUGR大鼠肝臟甘油三酯含量及血漿胰島素水平較AGA組增高(p0.05)。并且,胰島素水平和PGC-1α啟動子CpG位點-787及-803的甲基化水平呈正相關(p0.05)。 結論：PGC-1α啟動子甲基化水平及其轉錄活性的改變可能通過介導線粒體含量的減少與涉及到脂肪酸氧化和氧化磷酸化的PGC-1α應答基因的轉錄活性的降低,以及肝臟甘油三酯的積聚增多來編程CG-IUGR大鼠遠期胰島素抵抗相關代謝疾病的風險。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R714.5

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