本文選題：妊娠期高血糖 + DNA甲基化　； 參考：《華中科技大學》2016年博士論文

【摘要】：目的：宮內暴露于高糖環(huán)境可增加子代代謝性疾病的發(fā)生風險。胎兒可能通過DNA甲基化編程介導這一發(fā)病機制,本部分利用焦磷酸測序的方法測定GDM胎盤及臍血中PPARGC1A (PGC-1α)啟動子DNA的甲基化水平。方法：本部分研究共納入58例在我院行常規(guī)剖宮產術的孕母,記錄孕婦的年齡、血糖值、體重指數、產次等。剖宮產后立即收集胎盤組織以及臍血組織,提取胎盤以及臍血組織中的基因組DNA,并經過亞硫酸氫鹽轉化,通過PCR技術擴增PGC-1α啟動子的目的序列,最后用焦磷酸測序(pyrosequencing)的方法對PGC-1α啟動子的CpG位點-841,-816,-783,-617,-652,-260的甲基化水平進行測定；測定臍血血糖及胰島素。并利用SPSS軟件對PGC-1α甲基化水平進行分析。結果：根據IADPSG標準納入GDM組24例,正常血糖組34例。GDM組妊娠24-28周血糖水平(空腹血糖、餐后1h血糖、餐后2h血糖)以及臍血血糖水平高于正常對照組(p0.01)。GDM組臍血HOMA-IR略高于正常組(p0.05)。兩組的胎盤與臍血中PGC-1α啟動子特異性CpG位點的平均甲基化水平無明顯差異。同一位點臍血的PGC-1α啟動子特異性CpG位點的甲基化水平高于胎盤組織。結論：PGC-1α啟動子特異性CpG位點的DNA甲基化水平具有組織特異性,可能不存在具體血糖閾值以評估高糖風險。目的：宮內暴露于高糖環(huán)境可增加后代代謝性疾病的發(fā)病風險。孕母高血糖所致胎兒編程的潛在機制尚未完全明確,近來的研究報道表明表觀遺傳修飾可能在這一過程中起重要的作用。過氧化物酶體增殖物激活受體γ的共激活因子-1α (PGC-1α)在多種生物過程中發(fā)揮著重要的作用,可介導機體能量平衡和代謝過程,是聯(lián)系外界環(huán)境與能量代謝的樞紐。本部分旨在分析PGC-1α啟動子的DNA甲基化水平與孕婦妊娠期血糖水平的相關性。同時測定胎盤組織的線粒體含量和PGC-1α蛋白的表達情況,并分析其與PGC-1α啟動子的DNA甲基化水平之間的相關性。為健康和發(fā)育起源學說提供實驗依據。方法：本研究納入同濟醫(yī)院58例實行常規(guī)剖宮產術的母親,母孕期血糖濃度在孕24-28周時需經75g OGTT實驗測定,并根據孕期血糖的水平將58例孕婦分為GDM組以及正常對照組。并記錄孕婦的年齡、血糖值、體重指數、產次等參數。此外,在臨床實驗室測定相關代謝參數。通過western blot技術對胎盤PGC-1α蛋白水平進行測定；通過Real time PCR的方法對胎盤線粒體含量進行測定。并利用SPSS軟件分析PGC-1α啟動子特異位點的甲基化水平與代謝參數的相關性。結果：在本部分共分為GDM組和正常對照組兩個組。孕婦血糖水平與胎盤PGC-1α啟動子的甲基化水平呈正相關,與臍血PGC-1α啟動子特異位點的甲基化水平呈負相關。在GDM組,胎盤PGC-1α啟動子特異位點的甲基化水平與OGTT餐后2h血糖呈顯著正相關。胎盤PGC-1α啟動子特異性的CpG位點與胎盤PGC-1α蛋白之間無相關性。PGC-1α啟動子特異位點DNA甲基化水平與mtDNA/nDNA之間存在負相關。胎盤CpG位點甲基化水平與臍血HOMA-IR之間呈正相關。其他代謝參數與PGC-1α甲基化水平尚未發(fā)現顯著相關性。結論：宮內暴露于高糖環(huán)境可能介導了PGC-1α啟動子DNA甲基化水平的改變,這可能是參與其子代代謝編程的重要機制之一。目的：宮內暴露于高糖環(huán)境可增加子代代謝性疾病的發(fā)生風險。Betatrophin是促進胰島p細胞增值和調節(jié)脂類代謝的一個關鍵因子。Betatrophin對營養(yǎng)信號高度敏感,多種病理生理條件下可誘導其改變。我們假設宮內暴露于高糖環(huán)境的新生兒臍血中betatrophin的濃度升高,并且臍血betatrophin與妊娠期血糖水平及胎盤PGC-1α啟動子甲基化水平相關。方法：本部分研究納入了同濟醫(yī)院54例實行常規(guī)剖宮產術的孕母。母孕期血糖濃度通過孕24-28周的75g OGTT實驗來測定,并據此數據分為妊娠糖尿病(GDM)及正常組。分娩后立即收集臍血和胎盤組織,在臨床實驗室測定相關代謝參數。用熒光定量PCR (real time PCR)方法檢測胎盤的線粒體含量。臍血betatrophin的濃度通過市售的Elisa試劑盒測定。結果：GDM組臍血betatrophin濃度水平高于正常對照組(P0.05)。臍血betatrophin的濃度與胎盤PGC-1α啟動子甲基化水平無明顯相關性。臍血betatrophin的濃度與母孕期2-h血糖濃度(P0.05)、臍血胰島素(P0.01)、以及HOMA-IR(P0.01)水平呈正相關。GDM組胎盤線粒體含量高于正常對照組,且與臍血betatrophin水平呈負相關(p0.01)。結論：臍血betatrophin可能是宮內暴露于高血糖及胎兒胰島素抵抗的潛在生物標志物,并可能預測GDM對子代遠期代謝的不良影響。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R714

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