RAS-COLI/COLIII表达异常对IVF小鼠心血管发育和功能影响及其机制研究

Since Louise Brown was born using the technology of in vitro fertilization and embryo-transfer (IVF-ET) in 1978, millions of babies have been born using assisted reproductive technology (ART). Besides its successful application in solving many problems of human infertility, some negative effects have been found. Epidemiological investigations of ART safety showed a significant increased incidence of cardiovascular malformations and more predisposing risk factors of adult cardiovascular diseases in ART offspring. Our preliminary work found some up-regulated expression of the genes of RAS system in the myocardial tissue of newborn, adult and aged IVF mouse model. The altered expression levels of COLI/COLIII and RAS-COLI/COLIII related epigenetic regulators: miR-100、miR-297 and miR-758 were also observed, which indicates that IVF process may cause abnormal expression of RAS-COLI/COLIII in the offspring through microRNA-mediated pathway. To further understand the cause and effect of abnormal expression of RAS-COLI/COLIII in IVF mouse, we plan to measure the expressions and related regulating mechanisms of RAS-COLI/COLIII pathway using RT-PCR and western blot. Then fluorescent histochemistry will be applied to measure the changes of expression of related genes in mouse IVF oocytes and preimplantational embryos. Finally, in vitro tests of mouse embryonic stem cell and myocardial cell cultures treated with high concentration of oxygen, overexpression and inhibition of microRNAs will be carried out. Thus, the main effects of IVF process on the RAS-COLI/COLIII pathway will be studied and the mechanisms involved in cardiovascular development and function will be illuminated.

ART安全性流调报告显示ART儿心血管畸形发生率上升，成年心血管疾病易感风险因素增多。本课题前期研究发现，各年龄段IVF小鼠心肌组织RAS系统基因表达水平升高，伴COLI/COLIII和RAS-COLI/COLIII通路调节因子miR-100、miR-297和miR-758的表达改变，提示IVF过程可激活RAS，经microRNA介导，引发可传递给出生子代的RAS-COLI/COLIII表达异常。旨在了解IVF小鼠RAS-COLI/COLIII表达改变的原因和效应，拟在完成各年龄段小鼠心肌RAS-COLI/COLIII通路基因表达和调节分析后，在开展IVF卵母细胞和胚胎的基因表达分析同时，通过mES和心肌细胞体外培养，探索IVF小鼠RAS-COLI/COLIII表达改变发生时间和机制，了解该通路基因对心血管发育和功能的影响，为ART子代心血管畸形增高和成年心血管疾病风险评估提供科学依据。

通过构建完成的体内受精、IVF近交系小鼠模型及其材料，首先进行该三阶段IVF小鼠心肌RAS组分基因、CTGF、COLI以及COLIII等基因的表达改变检测，开展幼年和成年IVF小鼠心肌组织miR-100、miR-297和miR-758的表达水平分析，完成IVF出生小鼠从幼年到老年RAS-AT1-CTGF-COLI/COLIII通路的表达变化和microRNA表观遗传学修饰改变的研究。利用心肌细胞系的体外培养和microRNA基因表达诱导和干扰实验等,分析IVF出生子代RAS-AT1-CTGF-COLI/COLIII表达异常的调控机制及其对心肌细胞结构功能以及心血管系统疾病发生风险的影响。建立小鼠胚胎干细胞(mES)体外实验，通过高雌激素环境等体外培养、拟胚体形成以及类心肌细胞分化分析，挖掘RAS-COLI/COLIII表达异常对IVF小鼠心血管发育可能的影响和分子机制。