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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 a naive-like induced pluripotent stem (iPS) cell line and to label it with red fluorescence, which will lay the foundation for the related studies on the development and differentiation of porcine naive i PS cells. Methods: The murine OCT4, SOX2, KLF4 and c-MYC transcription factor Tet O-FUW-OSKM were transfected into porcine embryonic fibroblast (PEF) by nuclear transfection At the same time, it was transferred into the activation vector FUW-M2rt TA, cultured in a culture system of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (b FGF) After adding doxycycline hyclate (DOX) to induce, the pig i PS cell line was established, and the pluripotency of the cell line was identified. On this basis, iPS cell lines were transfected into the red fluorescent protein expression vector, which was labeled, and whether the cells labeled with red fluorescence still have pluripotency. Results: The cloned i PS cell line was three-dimensional and could be subcultured to more than 30 passages. The karyotype was normal, the alkaline phosphatase staining was positive, and many stem cell multipotent factors were expressed. Using LIF / STAT3 Signaling pathway to maintain its proliferation, differentiated in vitro to form embryoid-like bodies that express the genes associated with the three germ layers, is a pig naive-like i PS cell line. Successful establishment of the i PS cell line by red fluorescent labeling, alkaline phosphatase staining and immunofluorescence staining results showed that the pluripotency of the red fluorescence-labeled i PS cells still exist. Conclusion: Pig naive-like i PS cell line stably expressing red fluorescent protein was successfully established.