ART小鼠肝脏INSIG-SREBP-SCAP基因表达改变机制研究

Since Louise Brown was born by in vitro fertilization and embryo-transfer (IVF-ET) in 1978, more than 4 million babies have been born using assisted reproductive technology (ART). Besides its successful application in solving many problems of human infertility, some negative effects on the developing conceptus have been found. Decreased birth weight and imprinting disorders are the two main potential risks for the health of ART children. The evidences already demonstrated that embryo/fetal changes in growth and epigenetic modification may predispose offspring to increased risk of metabolic and cardiovascular diseases in the adult life, or the concept of the embryo/fetal origins of adult diseases. Therefore, ART mouse models have been established in our research team. The abnormal adipose metabolism and increased level of serum cholesterol and altered hepatic expression of the key transcription factors for cholesterol homeostasis, insulin-induced gene (INSIG), sterol regulatory element binding proteins (SREBPs) and SREBP cleavage-activating protein (SCAP) were observed in the aged ART mice. For the further investing the underlying molecular mechanisms, we plan to measure the methylation rates of the differentiate methylation regions of INSIG-SREBP-SCAP in the liver of aged mice conceived from IVF, intracytoplasmic injection (ICSI), in vitro oocyte maturation (IVM) and in vivo fertilization (control). Followed by analyses of the expression and/or methylation situations of INSIG-SREBP-SCAP in ART mice at middle-age (7 months) and sexual maturity (8 weeks), new-birth, or preimplantation embryos and oocytes, the effects of the main factors and key time point of ART procedures on the metabolism of serum cholesterol and the hepatic expression of INSIG-SREBP-SCAP will be studied.

低出生体重和表观遗传异常是ART子代的二大健康风险，这与"胚胎/胎儿源性的成年心血管疾病"的出生儿病理改变相同。课题组因此建立小鼠模型，在观察到老年ART小鼠血脂指标改变同时，发现肝组织胆固醇和脂肪酸合成代谢调节通路：胰岛素诱导基因-类固醇调节元件结合蛋白-及其裂解激活蛋白（INSIG- SREBP-SCAP）基因表达显著改变。故此，拟在上述研究基础上，渐次进行ART老年小鼠肝组织INSIG -SREBP-SCAP通路基因调节区甲基化修饰，新生、性成熟和中年小鼠相关基因表达修饰动态变化，以及小鼠卵母细胞、胚胎相关基因表达研究，探索ART小鼠INSIG-SREBP-SCAP通路基因表达改变发生发展的机理，分析ART过程可能的作用环节和时间点，为ART子代成年血脂代谢异常风险评估和ART技术可能的改进途径提供科学依据。

通过建立ART C57BL/6J 小鼠模型，获得IVM/ICSI 成熟卵母细胞，新生、性成熟老年IVF、ICSI、IVM 和体内受精小鼠，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， 进行ART 小鼠模型的INSIG-SREBP-SCAP 基因表达和修饰变化研究，结果表明：ICSI 和/或IVM老年小鼠血压、心率、糖耐量水平、血胆固醇、血脂蛋白、血AST/ALT等指标的显著性改变；ART（IVF、ICSI、IVM）可影响小鼠肝脏和小鼠卵母细胞中INSIG -SREBP-SCAP 的部分基因表达和修饰水平。结合人ART 早孕胎儿组织和足月胎盘组织的INSIG -SREBP-SCAP基因表达和修饰变化等分析提示：1.ART老年小鼠肝脏组织INSIG -SREBP-SCAP表达改变起因于有关基因甲基化修饰和miRNA调节改变；2.ART小鼠肝脏组织INSIG -SREBP-SCAP表达、修饰改变贯穿小鼠从新生、性成熟、到老年的过程；3.ART过程对INSIG -SREBP-SCAP表达影响在IVM卵母细胞中已经呈现，表明IVM和体外培养在该通路基因表达改变中作用显著，减低或改变IVM操作步骤和时间，有助提高ART安全性。