HBx蛋白对人精子表观遗传修饰及胚胎发育影响的研究

The epigenetic information of sperm plays an important role in the male infertility and early development of embryos. Hepatitis B virus X protein (HBx) can be widely involved in the epigenetic modification of the host cell. But the effect mechanism of HBx on hunman sperm function and the development of embryos is still remains unclear.In our pre-experiments, we found that the histone-protamine alternation of HBV infected patients’s sperm is abnomal and the expression of histone methyltransferase Suv39h1 were reduced.Futhermore, the methylation level of promoter area in imprinted gene IGF2、KCNQ1OT1 is abnomal. So we speculate that HBx may be involved in the epigenetic modification of hunman sperm and lead to the abnormality of imprinted gene in the histone retained district. The sperm which carried the abnormal epigenetic information might lead to abnormal embryonic development. The purpose of this project is to analyze the correlation between the semen parameters and the sperm histone-protamine abnormal alternation rate of HBV infected patients, developing the histone-protamine abnormal alternation standard. We will study the mechanism of formation of abnormality of imprinted gene in the histone retained district through co-immunoprecipitation and mass spectrum and plasmid transfection of spermatogonial stem cells arrays.In animal level,we will discuss aberrant imprinted genes on the development of embryos by overexpression or silencing of imprinted gene IGF2、 KCNQ1OT1. This study can elucidate the molecular mechanisms of the effect of HBV on hunman sperm function and embryo development. Aslo,it can provide a new theoretical basis for the diagnosis and develop treatment measures for HBV patients’s fertility.

精子表观遗传信息对男性不育及胚胎发育具有重要作用。乙肝病毒x蛋白（HBx）广泛参与宿主细胞表观遗传修饰，但其对受精及胚胎发育影响的机制不够明确。申请人前期研究发现，HBV感染者精子组蛋白转换异常且组蛋白甲基转移酶Suv39h1等基因表达下调，印迹基因IGF2、KCNQ1OT1甲基化异常，故我们推测：HBx可参与人精子表观遗传修饰，引起组蛋白异常保留区印迹基因甲基化异常，携带异常印迹基因的精子进而影响胚胎发育。本项目拟探究HBV感染者精液参数与组蛋白异常转换率的相关性，制定HBV感染者组蛋白异常转换率标准；采用免疫共沉淀及鼠精原干细胞质粒转染等技术明确组蛋白异常保留区异常印迹基因形成的机制；在动物水平，过表达/沉默IGF2、KCNQ1OT1探究其对胚胎发育的影响。本研究能从表观遗传角度阐明HBV导致人精子功能及胚胎发育异常的分子机制，为HBV感染者生育力的诊断及治疗措施的研发提供新的理论依据。

HBV感染影响男性生育及胚胎发育的机制尚不明确。在本项目的资助下，我们对以下内容展开初步探讨：（1）HBV感染导致精子Suv39h1基因差异表达的机制；（2）父系印记基因异常开启对胚胎发育的影响。（3）采用CRISPR/Cas9技术对人胚胎及干细胞进行基因精确修饰。发现：（1）HBV感染者精子中KCNQ1OT1、Suv39H1、IGF2基因的表达量较正常组显著降低。（2）HBV感染者男性精子中Suv39h1基因启动子区甲基化水平与正常男性精子相比无显著改变；（3）在人精子中未发现HBx可与组蛋白甲基转移酶及DNA甲基转移酶发生直接相互作用；（4）干扰HBx基因转录因子表达后，HBx表达水平降低、Suv39h1基因表达显著升高。（5）KCNQ1OT1、IGF2基因过表达实验， KCNQ1OT1、IGF2基因对早期胚胎发育影响不显著。（6）应用CRISPER/Cas9系统对26个多PN胚胎进行基因编辑，4个胚胎被成功编辑，证明了基因编辑技术对于HIV免疫的可能性。(7)应用CRISPER/Cas9系统对β-地中海贫血病致病突变进行基因修复；(8)建立无精症患者非基因整合诱导多能干细胞系。本研究初步从表观遗传角度阐明HBV导致人精子功能及胚胎发育异常的分子机制；证实人类可以通过基因编辑技术，高效安全的对胚胎、干细胞DNA进行编辑，预防或治疗遗传性疾病及传染性疾病。