中文摘要：

卵母细胞特异性连接组蛋白（oocyte-specific linker histone,H1foo）是组蛋白家族的成员之一,特异性地定位于哺乳动物卵母细胞和早期胚胎中.在小鼠（Mus musculus）、牛（Bos taurus）和猪（Sus scrofa）等体外受精和体细胞核移植过程中均存在H1foo与其它成分的置换,并且这种置换有利于供核体细胞染色质构象的打开,实现基因组的完全重编程,恢复全能性.为了探索H1foo在诱导多能性干细胞（induced pluripotent stem cells,iPSCs）的过程中对iPSCs诱导效率的影响,本研究基于对猪H1foo的已有研究积累,选取猪胎儿成纤维细胞（porcine fetal fibroblast cell,PFF）,构建能同时表达八聚体转录因子-4（octamer-binding transcription factor-4,OCT4）、性别决定基因相关转录因子-2（SRY-related high-mobility-group（HMG）-box protein-2,SOX2）、Kruppel样转录因子（kruppel-like factor-4,KLF4）和禽髓细胞瘤病病毒原癌基因（cellular homologue of avian myelocytomatosis virus oncogene,c-MYC）四因子（OSKM）的强力毒素（doxycycline,DOX）调控载体,利用电转的方法在OSKM-PFF中瞬时表达猪pVenus-H1foo后添加DOX开启重编程,瞬时表达猪H1foo,转染得到稳定的OSKM-PFF.通过对所获得克隆进行鉴定,并对所获iPSCs克隆数进行统计分析,结果发现,转染有猪pVenus-H1foo和空载体pVenus的OSKM-PFF细胞中的碱性磷酸酶阳性克隆数目无显著差异.本研究首次证明H1foo对猪iPSCs的诱导效率无显著影响,这为进一步研究体细胞核移植与iPSCs诱导这两种重编程手段的内在机制提供了基础资料.

英文摘要：

Oocyte-specific linker histone（Hlfoo）, the member of histone families, is located specificly in mammalian oocytes and primary embryos. There exists fast replacement between Hlfoo and its counterpart during the process of fertilization and nuclear transfer of mouse （Mus musculus） , cattle（Bos taurus） and porcine（Sus scrofa）, which do help to open the chromosome in donor nuclear and achieve complete reprogramming. Induced pluripotent stem cell（iPSC） technology is another reprogramming strategy different from somatic nuclear transfer. Many reports showed that an open chromosomal structure facilitates somatic reprogramming and increases the induction efficiency of iPSCs. Therefore, in order to investigate whether Hlfoo has an effect on the induction efficiency of iPSCs, a transient expression of Hlfoo was conducted in the induction of porcine iPSCs. Two consecutive infections were conducted to the porcine fetal fibroblasts （PFF） by adding doxycycline（DOX）-inducible Lentiviral vectors expressing octamer-binding transcription factor- 4（0CT4）, SRY- related high- mobility- group（HMG）- box protein- 2（SOX2）, Kruppel- like factor- 4（Klf4） and cellular homologue of avian myelocytomatosis virus oncogene（c-MYC）. Vector pVenus-Hlfoo was electrotransferred into the OSKM-PFF and the cells transferred pVenus were acted as negative control. After several days, the obtained colonies were identified by IF and AP staining as iPSCs. By counting the number of iPSCs colonies, the results showed that the number of AP-positive colonies obtained in PFF with pVenus-Hlfoo or pVenus was almost the same. It was the first exploration about the effect of Hlfoo on induction of iPSCs which turned out there was no evident effect. The results above make basis data for the researches on mechnisms of reprogramming including somatic cell nuclear transfer and iPSCs.