胎盘PGC-1α启动子DNA甲基化及脐血betatrophin的水平与妊娠期血糖相关性的研究

发布时间：2018-05-31 01:08

本文选题：妊娠期高血糖 + DNA甲基化　；参考：《华中科技大学》2016年博士论文

【摘要】：目的：宫内暴露于高糖环境可增加子代代谢性疾病的发生风险。胎儿可能通过DNA甲基化编程介导这一发病机制,本部分利用焦磷酸测序的方法测定GDM胎盘及脐血中PPARGC1A (PGC-1α)启动子DNA的甲基化水平。方法：本部分研究共纳入58例在我院行常规剖宫产术的孕母,记录孕妇的年龄、血糖值、体重指数、产次等。剖宫产后立即收集胎盘组织以及脐血组织,提取胎盘以及脐血组织中的基因组DNA,并经过亚硫酸氢盐转化,通过PCR技术扩增PGC-1α启动子的目的序列,最后用焦磷酸测序(pyrosequencing)的方法对PGC-1α启动子的CpG位点-841,-816,-783,-617,-652,-260的甲基化水平进行测定；测定脐血血糖及胰岛素。并利用SPSS软件对PGC-1α甲基化水平进行分析。结果：根据IADPSG标准纳入GDM组24例,正常血糖组34例。GDM组妊娠24-28周血糖水平(空腹血糖、餐后1h血糖、餐后2h血糖)以及脐血血糖水平高于正常对照组(p0.01)。GDM组脐血HOMA-IR略高于正常组(p0.05)。两组的胎盘与脐血中PGC-1α启动子特异性CpG位点的平均甲基化水平无明显差异。同一位点脐血的PGC-1α启动子特异性CpG位点的甲基化水平高于胎盘组织。结论：PGC-1α启动子特异性CpG位点的DNA甲基化水平具有组织特异性,可能不存在具体血糖阈值以评估高糖风险。目的：宫内暴露于高糖环境可增加后代代谢性疾病的发病风险。孕母高血糖所致胎儿编程的潜在机制尚未完全明确,近来的研究报道表明表观遗传修饰可能在这一过程中起重要的作用。过氧化物酶体增殖物激活受体γ的共激活因子-1α (PGC-1α)在多种生物过程中发挥着重要的作用,可介导机体能量平衡和代谢过程,是联系外界环境与能量代谢的枢纽。本部分旨在分析PGC-1α启动子的DNA甲基化水平与孕妇妊娠期血糖水平的相关性。同时测定胎盘组织的线粒体含量和PGC-1α蛋白的表达情况,并分析其与PGC-1α启动子的DNA甲基化水平之间的相关性。为健康和发育起源学说提供实验依据。方法：本研究纳入同济医院58例实行常规剖宫产术的母亲,母孕期血糖浓度在孕24-28周时需经75g OGTT实验测定,并根据孕期血糖的水平将58例孕妇分为GDM组以及正常对照组。并记录孕妇的年龄、血糖值、体重指数、产次等参数。此外,在临床实验室测定相关代谢参数。通过western blot技术对胎盘PGC-1α蛋白水平进行测定；通过Real time PCR的方法对胎盘线粒体含量进行测定。并利用SPSS软件分析PGC-1α启动子特异位点的甲基化水平与代谢参数的相关性。结果：在本部分共分为GDM组和正常对照组两个组。孕妇血糖水平与胎盘PGC-1α启动子的甲基化水平呈正相关,与脐血PGC-1α启动子特异位点的甲基化水平呈负相关。在GDM组,胎盘PGC-1α启动子特异位点的甲基化水平与OGTT餐后2h血糖呈显著正相关。胎盘PGC-1α启动子特异性的CpG位点与胎盘PGC-1α蛋白之间无相关性。PGC-1α启动子特异位点DNA甲基化水平与mtDNA/nDNA之间存在负相关。胎盘CpG位点甲基化水平与脐血HOMA-IR之间呈正相关。其他代谢参数与PGC-1α甲基化水平尚未发现显著相关性。结论：宫内暴露于高糖环境可能介导了PGC-1α启动子DNA甲基化水平的改变,这可能是参与其子代代谢编程的重要机制之一。目的：宫内暴露于高糖环境可增加子代代谢性疾病的发生风险。Betatrophin是促进胰岛p细胞增值和调节脂类代谢的一个关键因子。Betatrophin对营养信号高度敏感,多种病理生理条件下可诱导其改变。我们假设宫内暴露于高糖环境的新生儿脐血中betatrophin的浓度升高,并且脐血betatrophin与妊娠期血糖水平及胎盘PGC-1α启动子甲基化水平相关。方法：本部分研究纳入了同济医院54例实行常规剖宫产术的孕母。母孕期血糖浓度通过孕24-28周的75g OGTT实验来测定,并据此数据分为妊娠糖尿病(GDM)及正常组。分娩后立即收集脐血和胎盘组织,在临床实验室测定相关代谢参数。用荧光定量PCR (real time PCR)方法检测胎盘的线粒体含量。脐血betatrophin的浓度通过市售的Elisa试剂盒测定。结果：GDM组脐血betatrophin浓度水平高于正常对照组(P0.05)。脐血betatrophin的浓度与胎盘PGC-1α启动子甲基化水平无明显相关性。脐血betatrophin的浓度与母孕期2-h血糖浓度(P0.05)、脐血胰岛素(P0.01)、以及HOMA-IR(P0.01)水平呈正相关。GDM组胎盘线粒体含量高于正常对照组,且与脐血betatrophin水平呈负相关(p0.01)。结论：脐血betatrophin可能是宫内暴露于高血糖及胎儿胰岛素抵抗的潜在生物标志物,并可能预测GDM对子代远期代谢的不良影响。
[Abstract]:Objective: intrauterine exposure to high glucose can increase the risk of subgeneration metabolic diseases. Fetus may mediate this mechanism by DNA methylation. This part uses pyrosequencing method to determine the methylation level of PPARGC1A (PGC-1 alpha) promoter DNA in GDM placenta and umbilical cord blood. Methods: 58 cases were included in this study. The maternal age, blood sugar, body mass index (BMI) of pregnant women were recorded, the placental tissue and umbilical blood tissue were collected immediately after cesarean section, the placenta and umbilical cord blood tissues were collected, the genomic DNA of the placenta and umbilical cord blood were extracted, and the sequence of PGC-1 alpha promoter was amplified by PCR technique, and the pyrophosphoric acid was used at the end. The method of sequencing (pyrosequencing) was used to determine the level of CpG loci -841, -816, -783, -617, -652, and -260 of the PGC-1 alpha promoter; determine the blood glucose and insulin in the umbilical cord blood. And the PGC-1 alpha methylation level was analyzed by SPSS software. Results: 24 cases were included according to the IADPSG standard, and 34 cases of normal blood glucose group were 24-28 weeks of pregnancy. Blood glucose levels (fasting blood glucose, postprandial 1H blood sugar, postprandial 2H blood sugar) and umbilical blood glucose levels were higher than normal control group (P0.01).GDM group HOMA-IR slightly higher than normal group (P0.05). The average methylation level of the PGC-1 alpha promoter specific CpG site in the two group was not significantly different from that in the umbilical cord blood. The PGC-1 alpha promoter of the umbilical cord blood at the same site was specific. The methylation level of the sex CpG locus is higher than that of the placental tissue. Conclusion: the DNA methylation level of the specific CpG loci of the PGC-1 alpha promoter is tissue specific, and there may be no specific blood glucose threshold to assess the risk of high glucose. The potential mechanism of programming has not been fully defined. Recent studies have shown that epigenetic modification may play an important role in this process. The CO activation factor -1 alpha (PGC-1 a) of peroxisome proliferator activated receptor gamma plays an important role in a variety of biological processes, which can mediate the body's energy balance and metabolic processes. The purpose of this section is to analyze the correlation between the level of DNA methylation of PGC-1 alpha promoter and pregnancy glucose levels in pregnant women, and to determine the mitochondrial content of the placental tissue and the expression of PGC-1 alpha protein, and to analyze the correlation between the level of DNA methylation and the PGC-1 alpha promoter. The developmental origin theory provides experimental basis. Methods: This study included 58 mothers of Tongji Hospital who performed routine cesarean section. The blood glucose concentration during pregnancy was measured by 75g OGTT test, and 58 pregnant women were divided into GDM group and normal control group according to the level of blood glucose during pregnancy. The age, blood sugar, weight index of pregnant women were recorded. In addition, the related metabolic parameters were measured in the clinical laboratory. The level of PGC-1 alpha protein in placenta was measured by Western blot technique and the content of the placenta mitochondria was measured by Real time PCR. The correlation between the level of methylation of the PGC-1 alpha promoter specific site and the metabolic parameters was analyzed by the SPSS software. Results: in this part, there were two groups in the GDM group and the normal control group. The blood glucose level of pregnant women was positively correlated with the level of the methylation of the placental PGC-1 alpha promoter, and was negatively correlated with the level of methylation of the PGC-1 alpha promoter at the umbilical cord. In the group GDM, the level of methylation of the placental PGC-1 alpha promoter special point and the 2H blood glucose after the OGTT meal was shown to be significant. There is a positive correlation. There is no correlation between the CpG loci of the placental PGC-1 alpha promoter and the placental PGC-1 alpha protein. There is a negative correlation between the level of DNA methylation of the special site of the.PGC-1 alpha promoter and the mtDNA/nDNA. The level of the methylation of the placental CpG locus is positively correlated with the HOMA-IR of the umbilical cord blood. The other metabolic parameters and the level of PGC-1 alpha methylation have not been found yet. Significant correlation. Conclusion: intrauterine exposure to high glucose may mediate the changes in the level of DNA methylation of PGC-1 alpha promoter, which may be one of the important mechanisms involved in its progeny metabolic programming. Objective: intrauterine exposure to high glucose environment can increase the risk of subgeneration metabolic diseases,.Betatrophin is to promote the increment and modulation of islet P cells. A key factor in lipid metabolism,.Betatrophin, is highly sensitive to nutritional signals and can induce changes in a variety of pathophysiological conditions. We hypothesized that the concentration of betatrophin in umbilical blood exposed to high glucose environment and umbilical blood betatrophin and gestational blood glucose level and placental PGC-1 alpha promoter methylation level Methods: this part of the study included 54 pregnant women who performed routine cesarean section in Tongji Hospital. Blood glucose concentration during pregnancy was measured by 75g OGTT experiment of pregnant 24-28 weeks. The data were divided into gestational diabetes mellitus (GDM) and normal group. After delivery, the umbilical blood and placenta tissue were collected and the related metabolic parameters were measured in clinical laboratory. The mitochondrial content of placenta was detected by the light quantitative PCR (real time PCR) method. The concentration of betatrophin in umbilical blood was measured by the marketed Elisa kit. Results: the concentration of betatrophin in the umbilical cord blood of the GDM group was higher than that of the normal control group (P0.05). The concentration of betatrophin in the umbilical cord blood was not significantly correlated with the level of the methylation of the PGC-1 alpha promoter in the placenta. The concentrations of Hin, 2-h glucose concentration (P0.05), umbilical blood insulin (P0.01), and HOMA-IR (P0.01) level were positively correlated with the normal control group, and were negatively correlated with the level of betatrophin in umbilical cord blood (P0.01). Conclusion: umbilical cord blood betatrophin may be the potential of intrauterine exposure to hyperglycemia and fetal insulin resistance. Biomarkers may predict the adverse effects of GDM on the long-term metabolism of offspring.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714

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