猪naive-like诱导性多能干细胞系的建立及红色荧光蛋白标记

Establishment of porcine naive-like induced pluripotent stem cell line and labelling with red fluorescent protein

目的 :建立猪naive-like诱导性多能干(induced pluripotent stem,i PS)细胞系,并对其进行红色荧光标记,为通过示踪猪naive i PS细胞发育和分化的相关研究奠定基础。方法:首先利用核转染技术向巴马小型猪胎儿成纤维细胞(porcine embryonic fibroblast,PEF)中转入鼠源OCT4、SOX2、KLF4和c-MYC转录因子表达载体Tet O-FUW-OSKM,并同时转入激活表达载体FUW-M2rt TA,采用白血病抑制因子(leukemia inhibitory factor,LIF)结合碱性成纤维细胞生长因子(basic fibroblast growth factor,b FGF)的培养体系进行培养,通过在培养液中添加盐酸多西环素(doxycycline hyclate,DOX)进行诱导,建立起猪i PS细胞系,并对细胞系的多能性进行鉴定。在此基础上,向i PS细胞系转入红色荧光蛋白表达载体,对其进行标记,并鉴定被红色荧光标记后的细胞是否仍然具有多能性。结果:所建立的i PS细胞系克隆呈三维立体生长,可以进行单细胞传代培养至30代以上,核型正常,碱性磷酸酶染色呈阳性,表达多种干细胞多能因子,利用LIF/STAT3信号通路维持其增殖,体外可分化形成表达三胚层相关基因的类胚体,为猪naive-like i PS细胞系。成功对所建立的i PS细胞系进行红色荧光标记,碱性磷酸酶染色和免疫荧光染色结果显示被红色荧光标记的i PS细胞的多能性依然存在。结论:成功建立了稳定表达红色荧光蛋白的猪naive-like i PS细胞系。

Objective： To establish porcine naive-like induced pluripotent stem（iPS） cell line followed by labelling with the red fluorescent protein via nucleofection. The labelled porcine naive-like i PS cells could be used for tracing their development and differentiation. Methods： Firstly, bama miniature pig embryonic fibroblasts（PEF） were nucleofected with Tet O-FUW-OSKM（OSKM： mouse derived transcriptional factors OCT4, SOX2, KLF4 and c-MYC） and FUW-M2rt TA vectors. Then, these cells were incubated in the medium with both leukemia inhibitory factor（LIF） and basic fibroblast growth factor（b FGF）, supplemented with doxycycline hyclate.The established i PS cell lines were identified for their pluripotency. Secondly, the i PS cells were nucleofected with p EF1alpha-Ds Red-Express Vector which could express the red fluorescent protein, then the maintainence of pluripotent characters of these labeled cells were analyzed. Results： The established i PS showed domed morphology and could survive long-term single-cell passaging（30 passages）. They were alkaline-phosphatase positive, maintained a normal karyotype, expressed various pluripotency markers and required LIF/STAT3 signaling pathway for self renewal. The i PS cells could form embryonic bodies（EB）, which expressed markers of three germ layers after culture in LIF free medium. The i PS cells were transfected with red fluorescent protein vector successfullyand kept showing red fluorescence for 10 passages. The results of alkaline phosphatase staining and immunofluorescence staining suggested that the pluripotency of these cells had not been compromised after red fluorescent protein labelling. Conclusion： The red fluorescent protein labeled porcine naive-like i PS cell lines were established successfully.