ART子代DNA动态突变率上升与DNA损伤修复酶基因表达和甲基化修饰改变相互作用分子机理研究

Low birth weight and birth defect are two of the main potential risks for the health of ART offspring. Multiple pregnancy and altered DNA epigenetic modification are common explanations for the increased risks of the newborns conceived by ART. Whether DNA instability also contributes to the birth defects have remained uncertain until we recently demonstrated that ART increased the frequency of trinucleotide repeat dynamic mutations in offspring conceived through ART. Meanwhile, the up-regulated expression of the genes of some DNA damage repair enzymes has been observed and the decreased methylation in CpG islets of those genes has also been found. To understand the detailed molecular mechanisms among ART procedures, genes of DNA damage repair enzymes and dynamic mutation, placenta conceived by singleton pregnancy either from IVF or naturally will be selected. Alterations of the expression and methylation modification of the genes of DNA damage repair enzymes involved in dynamic mutation will be investigated by RT-PCR, western blot and pyrosequencing. Then fluorescent histochemistry will be applied to measure the changes of expression of DNA damage repair enzymes in mouse and human IVF oocytes and preimplantational embryos. Finally, in vitro tests of mouse embryo stem cell cultures treated with high concentration of estrogen and oxygen, over- or down-expression of the genes of DNA damage repair enzymes by transfection of recombined virus or RNA interference will be carried out. Thus, the relationship among IVF procedures, the expression and methylation modification of the genes of DNA damage repair enzymes and the increased ratio of DNA dynamic mutation in ART will be illuminated.

ART子代存在出生体重下降和出生缺陷率上升等风险，ART过程对DNA稳定性的影响也是可能原因，但一直未能定论。课题组通过子代动态突变基因三核苷酸重复序列拷贝数突变频率分析，在确认ART可增加动态突变发生风险的同时，发现其胎盘组织数个与动态突变发生相关DNA损伤修复酶基因表达和甲基化修饰的显著性改变，使我们有理由假设ART影响子代DNA动态突变发生涉及DNA损伤修复酶基因及其作用途径。故此，拟以人IVF胎盘组织为对象，以与动态突变发生相关DNA损伤修复酶基因为目标，分析各基因表达和甲基化修饰改变；通过小鼠IVF卵母细胞和着床前胚胎免疫组化，辅以人IVF卵和胚胎验证，分析ART过程影响DNA损伤修复酶基因表达作用时间点和环节；经mES培养高雌激素和/或高氧环境处理，DNA损伤修复酶基因过表达和干扰等体外实验，分析ART过程中DNA损伤修复酶基因与动态突变率改变相互作用的分子机制。

通过收集ART胎盘和早期胎儿组织和建立小鼠模型，研究ART子代DNA甲基化和DNA损伤修复基因的影响机制。同时应用 Real-Time PCR、 Western-blot、荧光免疫细胞化学、亚硫酸氢盐测序PCR（BSP）及 pyrosequencing 等技术，研究其基因动态突变相关的DNA损伤修复基因的表达和DNA甲基化修饰变化状态。利用小鼠胚胎，探索体外操作和培养环境条件对小鼠胚胎中DNA损伤修复基因的影响，并通过对不同培养条件下小鼠胚胎干细胞（mouse embryonic stem cell, mESC）中DNA损伤修复基因的表达和甲基化修饰状态进行研究，分析ART过程中DNA损伤修复酶基因与动态突变率改变相互作用的分子机制。结果表明：1. IVF和ICSI早孕期胎儿组织中部分DNA损伤修复基因呈现低表达，其发生可以用DNA损伤修复基因启动子区域CpG岛的高甲基化水平解释。2. IVF出生小鼠脑组织中部分DNA损伤修复基因的表达及其启动子区域CpG岛的甲基化修饰水平存在改变；虽然3周龄的IVF小鼠脑组织中部分DNA损伤修复基因的表达和甲基化修饰水平改变在小鼠生长至10周龄时可以被部分纠正，但1.5岁高龄的IVF小鼠脑组织中此类基因又出现更显著的基因表达、甲基化修饰及其蛋白水平的改变。3.小鼠囊胚中存在DNA损伤修复基因表达改变可能与高氧浓度暴露有关，而与雌激素暴露无明显联系。4. 胚胎培养环境中的高氧浓度暴露可能导致DNMT1基因表达上调，导致DNA损伤修复基因甲基化状态和基因表达差异，这可能是IVF-EC囊胚DNA损伤修复基因表达改变的可能机制。5. 胚胎培养环境中的高氧浓度暴露是ART出生子代DNA损伤修复基因表达改变的重要原因，推广使用体外低氧浓度的胚胎培养系统，有利于降低子代DNA损伤修复基因表达和甲基化修饰改变可能引发的安全性风险。