过表达重编程转录因子藏羚羊成纤维细胞异种克隆研究  

Inter-species Cloned Embryo Development with the Transcription Factor Induced Tibetan Antelope(Pantholops hodgsoni) Fibro Blast Cells

作　　者：苏广华[1] 李雪[1] 刘雪菲[1] 刘坤[1] 白春玲[1] 魏著英[1] 李光鹏[1] 旭日干[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特010070

出　　处：《农业生物技术学报》2016年第7期957-967,共11页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2014ZX08007-002)

摘　　要：藏羚羊(Pantholops hodgsoni)为濒危珍稀物种。本研究利用小鼠(Mus musculus)来源的POU5f1转录因子(octamer-binding transcription factor 4,OCT-4)、性别决定基因相关转录因子2(sex determining region Y-Box 2,SOX-2)、Kruppel样因子4(Kruppel-like factor 4,KLF-4)和鸟类骨髓细胞瘤元癌基因同源基因(avian myelocytomatosis viral oncogene homolog,C-MYC)等4种重编程因子,对藏羚羊体细胞进行诱导重编程,并将诱导后的细胞移植入牛(Bos taurus)去核的卵母细胞中,进行异种克隆操作,观察异种克隆胚胎(inter-species somatic cell nuclear transfer,i SCNT)的体外发育情况。结果表明,经过4因子诱导之后的藏羚羊细胞(简称i PS-ZLY)并没有形成胚胎干细胞(embryonic stem cell,ESc)样细胞克隆,细胞增殖能力和形态都发生了改变。细胞周期分析发现,i PS-ZLY进入G2-M期的比率高于藏羚羊成纤维细胞(简称ZLY)(21.5%vs 16.7%)。i PS-ZLY细胞在培养的第4天进入生长的平台期,而ZLY细胞则在第6天进入平台期。核型分析发现,i PS-ZLY的正常2N核型比例与ZLY细胞相似(68.3%vs 69.6%)。i PS-ZLY细胞表达OCT-4基因。当把i PS-ZLY细胞移植入去核的牛卵母细胞后,i PS-ZLY异种克隆桑葚胚和囊胚形成率分别为8.5%和2.4%,而ZLY异种克隆胚胎桑葚胚和囊胚形成率则分别为3.8%和0.95%。以上研究表明,经过4种因子诱导之后藏羚羊体细胞,其增殖能力发生显著改变,细胞生长周期加快,表达多能基因OCT-4;诱导细胞的i SCNT发育率显著高于普通细胞,诱导细胞对于i SCNT发育有促进作用。本研究首次利用重编程因子诱导藏羚羊体细胞,进而对诱导之后的细胞进行了包括细胞周期、染色体核型以及多能性基因的检测,虽然没有形成干细胞样细胞克隆形态,但是多能性基因OCT-4开始表达,是一个处于分化的体细胞和多能性细胞的中间状态,这样的中间状态有助于i SCNT的发育。本研究结果对重编程机理研究以�The Tibetan antelope（Pantholops hodgsoni） is endemic to the Tibetan Plateau and becomes endang ered species, which is national class Ⅰprotected animals in China. Inter-species somatic cell nuclear transfer（i SCNT） would be a possible rescue strategy for these species. i SCNT has been regarded as a potential alternative for rescuing highly endangered species and can be used as a model for studying nuclearcytoplasmic interactions. However, i SCNT embryos often fail to produce viable offspring. In this study, the Tibetan antelope fibroblasts were treated by the 4 classic mouse（Mus musculus） transcription factors of octamer-binding transcription factor 4（OCT-4）, sex determining region Y-Box 2（SOX2）, Kruppel-like factor4（KLF4） and（avian myelocytomatosis viral oncogene homolog（C- MYC） to induce the cell reprogramming.The pluripotent gene expression, cell growth curve and cell cycle of the induced cells were detected. The induced cells（i PS- ZLY） were then used as donors to transfer to the enucleated bovine（Bos taurus） oocytes.The results showed that the induced Tibetan antelope fibroblast cells（i PS- ZLY） had numerically higher cell mass in the G2- M stage than the Tibetan antelope fibroblasts（ZLY）（21.5% vs 16.7%）. Cell growth curve indicated that i PS-ZLY cells proliferated faster than the ZLY cells, i PS- ZLY cells gone into the growth of the plateau in 4 d, while the ZLY cells gone into a plateau in 6 d. The karyotypes of both i PS-ZLY and ZLY cells were similar（68.3%vs 69.6%）. The pluripotent gene OCT-4 expressed only in the i PS-ZLY. When transfered of the i PS-ZLY and ZLY cells, irrespectively to enucleated bovine oocytes, the i PS-ZLY cells resulted in 2.4%cloned blastocyst development, while the ZLY cells yielded 0.95% cloned blasatocysts. These results suggested that treatment of Tibetan antelope fibroblast cells with transcriptional factors enhanced cell proliferation, induced pluripotent gene expression and improved inter- species cloned embryo developm

关 键 词：诱导多能干细胞 异种克隆胚胎(iSCNT) 细胞周期 转录因子 

分 类 号：Q291[生物学—细胞生物学]