【作者】 时小艳；

【导师】 刘红林；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 原始生殖细胞(Prmordial germ cells,PGCs)是生殖细胞的祖细胞,存于8.5～13.5dpc(days post coitum)的鼠胚,具有迁移、增殖和性别分化等特性。近年研究证明PGCs发育过程中发生着大量的DNA及组蛋白的修饰,说明PGCs的特性可能与表观遗传修饰相关,从组蛋白变体角度分析是表观遗传学研究新的热点。本实验通过间接免疫荧光检测了不同发育时期含有PGCs的组织石蜡切片、单细胞及培养两天的PGCs中组蛋白H2A三种变体—macroH2A、H2A.X和H2A.Z的分布。研究结果如下:1免疫荧光检测小鼠PGCs中组蛋白H2A变体的分布分别采集8.5～13.5dpc五个时间段的含有PGCs的胚胎组织和PGCs单细胞,对组织进行石蜡切片间接免疫荧光,对单细胞进行间接免疫荧光检测。macroH2A在PGCs进入生殖嵴(10.5dpc)和性别分化以后(13.5dpc),分布状态发生变化;H2A.X在性别分化后(13.5dpc)分布发生变化;H2A.Z在进入生殖嵴和性别分化过程中(12.5dpc)及以后(13.5dpc)分布发生变动。说明组蛋白H2A的这三种变体确实参与了PGCs的发育。2免疫荧光检测培养后PGCs中组蛋白H2A变体的分布采集主要时间段的小鼠PGCs体外培养两天,分别将体外培养1d和2d的PGCs通过间接免疫荧光检测组蛋白H2A三种变体的分布,以体内同期的PGCs做标准对照,结果发现:macroH2A的分布在培养的细胞中较对照细胞迟缓;H2A.Z分布基本与对照基本一致;H2A.X在培养的进入生殖嵴前(8.5dpc)的PGCs中很快消失,之后各阶段培养的PGCs中H2A.X分布基本正常。另外,实验还发现,11.5dpc时PGCs的性别发育方向可能已确定。更多还原

【Abstract】 Specification of prmordial germ cells(PGCs) fate is an essential event during the life cycle of the organism,which segregates PGCs that will eventually generate the totipotent state,from somatic lineages that progress toward terminally differentiated states.Studies on the early mouse PGCs have demonstrated the importance of epigenetic reprogramming including erasure and re-establishment of parental imprints.There has been an increased focus on the role of histone H2A variants in epigenesis.In this study,immunofluorescent staining were used for studying the distritution of histone variants in tissue、single PGCs and cultured PGCs.1 Distritution of histone variants in fresh PGCsDissect the tissues contain PGCs of 8.5～13.5dpc mouse embryo and single PGCs using for immunofluorescent staining.The distritution of macroH2A changed while PGCs enter into Genital ridge(10.5dpc) and after sex distinct(13.5dpc);The change of H2A.X distritution occurred after the sex of PGCs is distinct(13.5dpc);The distritution of macroH2A changed while PGCs enter into Genital ridge(10.5dpc) and fore-and-aft sex distinct(12.5dpc and 13.5dpc).The result showed that the three variants of histone H2A must be concerned with the development of PGCs.2 Distritution of histone variants in cultured PGCsSeparate PGCs of 8.5～13.5dpc mouse embryo and culture them for lday and two day. Immunofluorescent staining were used for studying the distritution of histone variants in cultured PGCs.It reveal that the distritution of macroH2A in cultured PGCs slower than the corresponding period PGCs developed in vivo,H2A.Z in cultured PGCS showed in gear; H2A.X disappear in 8.5dpc cultured PGCs,however it is normal in 10.5dpc～13.5dpc cultured PGCs.Furthermore,the H2A.X distritution in cultured 11.5dpc PGCs reveal that PGCs distinct may had destined before 11.5dpc.更多还原