辅助生殖技术对子代Pax6基因甲基化的影响及其在胰岛发育中的作用

It has been shown that the assisted reproductive technology (ART) children may be at an increased risk of developing diabetes mellitus. The ART process, which happens at the time of major epigenetic modulation for a fetus, may affect the DNA methylation process that plays an important role on islet differentiation, development and maintenance. Our previous study showed Pax6 participated in islet differentiation, development and maintenance. Thus, we hypothesize that the ART may block the demethylation of Pax6 gene and have adverse effect on islet development and function. By observing the effects of different ART procedures on the methylation status of mice blast and fetus, we try to analyze their association with the morphological and functional changes of islets and the metabolic status in the mouse models. In addition, Pax6 gene methylation will also be evaluated in the human embryonic stem cells as an in vitro system to mimic the in vivo process of islet differentiation. The effect of Pax6 gene methylation status on islet differentiation was observed by changing its methylation status through adding 5-Azacytidine (5AZD), or introducing DNA methyltransferase knockdown or overexpression in the system. Our study will be helpful to elucidate the mechanism of the adverse metabolic effects of ART, and may provide high quality data and evidences for further optimizing the ART process.

研究发现，辅助生殖技术（ART）后代糖尿病的发病风险增加。ART操作过程是胚胎表观遗传重建的关键期，可影响DNA甲基化，后者是胰岛发育分化和功能维持的重要调控机制之一。本课题组的前期工作显示，Pax6基因可调控胰岛分化、发育及成年后的功能维持。因此，我们推测ART过程可能导致Pax6基因去甲基化受阻，影响子代胰岛功能，增加糖尿病的发生风险。本研究拟观察ART不同操作步骤对小鼠胚胎期及成年后胰腺和外周组织Pax6基因甲基化修饰过程的影响，分析其变化对胰岛形态和功能的影响及其与ART子代代谢状态的相关性；利用人胚胎干细胞向胰岛细胞定向分化体系模拟体内胰岛发育过程，分析不同分化阶段Pax6基因甲基化的变化，应用5氮杂核苷、DNA甲基转移酶敲减或过表达体系改变Pax6甲基化状态，观察其对胰岛分化的影响。本研究为阐明ART过程对子代代谢影响提供理论基础，并为ART过程中各环节的条件优化提供实验依据。

本研究结果显示，体外受精-胚胎移植（IVF-ET）受孕小鼠的子代存在宫内生长受限，其出生体重低于自然受孕小鼠,体重差异出生后持续存在,高脂喂养也未改变。IVF-ET后代的血糖和胰岛素水平均高于对照组。高脂饮食诱导后，高血糖更明显，胰岛功能明显受损。提示IVF-ET后代存在胰岛功能紊乱和宫内生长受限，影响出生后的糖代谢。对15例IVF-ET受孕的成年妇女和年龄匹配的自然受孕妇女进行临床观察，发现IVF-ET的后代体重高于对照组，并观察到胰岛素抵抗、负荷后胰岛素分泌缺陷的存在。OGTT结果均正常，但IVF-ET后代的血糖水平高于对照组。体外实验表明，Pax6基因启动子区被甲基化修饰。甲基化程度越高，Pax6基因表达越低。甲基化是早期胚胎发育过程中Pax6基因表达的重要调控机制。体外实验采用人胚胎干细胞定向分化系统（hESCs）模拟胰岛发育。结果表明，不同分化阶段Pax6基因启动子的甲基化变化与Pax6的表达丰度成反比，从而影响胰岛的分化效率。本研究为阐明辅助生育过程对后代代谢影响的机制提供了理论依据，为优化辅助生育操作流程提供证据。