基础培养液和小分子对猪类胚胎干细胞建系效率的影响  

The Effects of Basal Medium and Small Molecules on the Efficiency of Porcine Embryonic Stem Cell-like Cell Lines Establishment

作　　者：李妍[1] 薛冰华[1] 张雪[1] 赫义龙 伟人悦 刘忠华[1] 

机构地区：[1]东北农业大学胚胎工程实验室,哈尔滨150030

出　　处：《中国细胞生物学学报》2015年第5期621-629,共9页Chinese Journal of Cell Biology

基　　金：国家重点基础研究发展计划(973计划)(批准号:2011CBA01000);国家自然科学基金(批准号:J1210069)资助的课题~~

摘　　要：猪是人类疾病模型的重要候选动物之一,建立真正的猪胚胎干细胞系将极大地推动该领域的进展。培养液和细胞信号通路是目前限制猪胚胎干细胞系建立的两个主要影响因素。该研究探索了基础培养液(KO-DMEM和DMEM/F12)和小分子(PD0325901和CHIR99021,简称2i)对猪类胚胎干细胞建系效率的影响。实验结果显示,发育至6 d的体外受精胚胎在KO-DMEM培养液中贴壁率和原代克隆形成率显著高于DMEM/F12培养液;添加2i后,两种培养液中的胚胎贴壁率和原代克隆形成率均下降。在KO-DMEM培养液中可获得稳定传代的细胞系,获得的细胞系呈碱性磷酸酶阳性,核型正常,表达Oct4、Sox2和Nanog,不表达Cdx2。细胞系可成功进行转基因操作。结果表明,以KO-DMEM为基础培养液的培养体系可以获得猪类胚胎干细胞,2i不利于胚胎贴壁和原代克隆形成。该研究为猪胚胎干细胞建系培养体系的选择及推断猪胚胎干细胞多能性细胞信号调控通路提供了重要的实验依据。Swine is considered as one of the most important candidates for human disease models, and establishment of bona fide porcine embryonic stem cells （pESC） will facilitate making disesase models. Culture medium and signalling pathway are two main factors that affect the establishment of porcine embryonic stem cells. In this study, we investigated the effects of basal medium （KO-DMEM and DMEM/F12） and small molecular inhibitors （PD0325901 and CHIR99021, 2i） on the efficiency of pESC establishment. The results showed that the rates of attached embryos and primary colonies in KO-DMEM basal medium were significantly higher than those in DMEM/F12 basal medium when IVF embryos of 6 days were seeded, and the rates were significantly decreased when embryos seeded in both of the two medium with addition of 2i. pESC-like cell lines were derived from KO- DMEM basal medium. These cells were alkaline phosphatase （AP） positive and had normal karyotypes, expressedpluripotent markers of Oct4, Sox2 and Nanog, but did not express Cdx2. The cell lines could be genetically modi- fied without losing of the pluripotency. These results indicated that KO-DMEM basal medium could support pESC- like cells derivation and 2i had negative effects on embryos attachment and primary colonies formation. This study provided important evidence for selection of basal medium and predicting of pluripotency related cell signalling in pESC establishment.

关 键 词：猪 类胚胎干细胞 基础培养液 小分子抑制剂 

分 类 号：Q813[生物学—生物工程]