玻璃化冷冻卵巢出生小鼠生长发育和糖代谢紊乱的风险和机制研究

Cryopreservation gamete and embryo by vitrification has been widely used in assistant reproductive technology, which makes its influence on the gamete and embryo follow-up development and the progeny genetic safety becomes a major concern. Our previous study has found that descendant birth from orthotropic transplanted vitrified-warmed ovaries showing bad health of low birth weight and growth retardation. We presume that it is concerned with vitrified-warmed process interfereing the descendant DNA methylation might induce overexpression of Grb10 to suppress PI3k/Akt/Mapk signal pathway via the mutual control mechanism between DNA methyltransferase 1(DNMT1) and microRNAs. This project intends to construct descendant derived from orthotopic transplanted vitrified-warmed ovaries, to obtain pancreas beta-cell and animal model. Then, we will use methylated DNA immunoprecipitation (MeDIP) assay, pyrophosphate sequencing, reverse transcriptase quantitative real-time PCR and Western blot to measure global DNA methylation level and DNMT1, Grb10 expression in the pancreas of descendant. Microarray technology will be used to screen specific miRNAs and to be verified. The specificity miRNAs precursor, the inhibitor and the target vector carrying DNMT1 will be constructed. Overexpression of DNMT1， silence DNMT1 and silence Grb10 and transfect oocytes to clarify the mechanism of specificity miRNAs regulating DNA methylation and DNMT1 negative feedback regulated specificity miRNAs. The purpose of the reasearch is to reveal the mutual control mechanism between DNMT1 and specificity miRNAs to participate in the vitrification freezing interference descendant DNA methylation. To determine the early warning markers and provide a theoretical basis for comprehensive assessment and source head prevention of descendant epigenetic security hidden danger induced by vitrification freezing technology.

玻璃化冷冻在辅助生殖中的广泛应用使其对配子、胚胎后续发育以及子代遗传安全性的影响备受关注。我们前期发现玻璃化冷冻卵巢出生小鼠具有出生低体重和生长发育迟缓的不良健康状态，推测与玻璃化冻融干扰子代DNA甲基化诱导Grb10过表达抑制PI3k/Akt/Mapk信号通路的机制有关。本项目拟建立玻璃化冷冻卵巢出生小鼠获得胰岛Beta细胞/动物模型，采用甲基化芯片、RT-qPCR和蛋白印迹等检测子代整体甲基化水平，DNMT1、 Grb10等变化；微阵列技术筛选特异性miRNAs并予以验证；构建特异性miRNAs前体、抑制物和携载DNMT1靶向载体；过表达DNMT1、DNMT1-siRNA和Grb10-siRNA转染beta细胞，旨在阐明miRNAs与DNMT1相互调控参与玻璃化冷冻干扰子代DNA甲基化影响生长发育和糖代谢的机制，确定预警标记物，为全面评估和防控玻璃化冷冻的表观遗传安全性隐患提供理论依据

卵巢冻融后再移植技术为罹患恶性肿瘤的患者提供了理想的生育力保存措施，但是研究表明，该技术在玻璃化冻存卵巢皮质时使其内配子不可避免地遭受氧化应激，又在移植过程中使其内配子经历缺血再灌注损伤，所以需要对后代远期的健康状况进行评估。 本项目观察了冷冻和移植过程对后代小鼠生长和葡萄糖代谢的影响，综合利用原代胎鼠肝细胞培养、分子生物学、RNA干扰、携载miR-145b-mimic及miR-145b-inhibit等技术手段，从体内外研究了miR-145b调控Grb10，负性调控IGF-1信号通路下游PI3K-Akt和ERK-MAPK途径影响葡萄糖代谢和细胞增殖的作用机制。. 结果表明，与正常小鼠相比，新鲜移植组及冷冻移植组仔鼠的体重均显著降低，而空腹血糖、空腹胰岛素水平和糖耐量均显著升高，首次发现：卵巢经历冷冻或移植出生的仔鼠出现生长迟缓和糖代谢紊乱的高风险。与正常小鼠相比，新鲜移植组和冷冻移植组子代肝脏内Grb10及DNA甲基转移酶（包括Dnmt1、Dnmt3a、Dnmt3b、Uhrf1）表达均显著增加，此外，胰岛素/IGF-1信号通路下游磷酸化因子P-IGF-1R、p-IRS1、 p-IRS2、 p-AKT、与p-MAPK的表达水平均显著下调，提示，冷冻卵巢移植出生子代DNA甲基转移酶表达变化，代谢相关性印记基因Grb10过表达，Grb10过表达抑制了IGF-1信号通路下游磷酸化因子的表达。 胎鼠肝细胞转染Grb10-siRNA显示IGF-1信号通路下游的因子磷酸化水平显著上调，证实：Grb10负性调控 IGF-1信号通路。另外，冷冻卵巢移植组子代肝脏内Grb10启动子区11个位点中的4个位点的DNA甲基化状态显著降低，可能是Grb10过表达的原因之一。通过荧光素酶报告实验以及携载miR-145b mimic和miR-145b inhibitor证实：miR-145b调控Grb10的表达，并且miR-145b表达在冷冻卵巢移植组和新鲜卵巢移植组子代肝脏内均较正常组降低。综合这些研究结果，本项目最终证实： 下调的miR.-145b通过调控Grb10过表达，抑制IGF-1信号通路PI3K-Akt和ERK-MAPK途径下游的磷酸化因子表达，从而影响葡萄糖代谢和细胞增殖可能导致冷冻卵巢出生的子代远期糖代谢紊乱的风险升高，为这项技术在临床应用前的安全性评估提供了实验依据。