巴马小型猪胎儿成纤维细胞重编程为诱导多能性细胞的研究

Induction of reprogrammed embryonic fibroblasts into pluripotent stem cells in Bama miniature pig

目的探讨巴马小型猪胎儿成纤维细胞重编程为诱导多能性细胞的方法。方法应用经典的逆转录病毒介导方法,将Oct4,Sox2,c-Myc和Klf4四种人源性转录因子转导入巴马小型猪的胎儿成纤维细胞中,建立形态和特征类似猪上胚层细胞来源的猪多能性细胞(pig induced pluripotent cells,piPCs)。免疫荧光化学方法检测多能性细胞标记物,RT-PCR检测了该细胞体外三胚层相关基因表达水平。结果巴马小型猪胎儿成纤维细胞重编程的piPCs细胞呈AKP染色阳性,体外长期传代都保持正常的染色体核型,猪上胚层多潜能性细胞标记分子Oct4,Sox2,Nanog,Tra-1-60,Tra-1-81和SSEA-4呈阳性表达,并且表达小鼠胚胎干细胞(ES)多潜能性细胞标记分子SSEA-1,体外诱导分化后具有三胚层特异基因的表达,但是体内分化能力存在缺陷。结论巴马小型猪胎儿成纤维细胞重编程成功诱导成多能性细胞。

Objective To study the induction of reprogrammed embryonic fibroblasts into pluripotent stem cells in Bama miniature pig. Methods Using Yamanaka’ s classical method, four human originated transcription factors hOct4,hSox2, hc-Myc, and hKlf4 were transfected into Bama miniature pig fibroblast to establish pig induced pluripotent cells（piPCs） which were similar to pig epiblast originated cells in morphology and characterizations. The characteristics of the piPCs cells were proved via Alkaline Phophatase（AP） staining, immunofluorescence was used to detect the embryonic stem cells surface antigens, and RT-PCR was appllied to analyze the relative genes expression. Results The bama miniature pig fibroblasts were reprogrammed into pig induced pluripotent cells（piPCs） by Yamanaka’s classical method. The piPCs resembled pig epiblast cells in their morphology and showed Oct4, Sox2, Nanog, Tra-1-60, Tra-1-81, SSEA-4 and SSEA-1positive, with a normal karyotype and forming embryoid bodies and expressing 3 germ layers gene markers in vitro, but without teratoma formation in nude mice. Conclusion The Bama miniature pig fibroblasts were reprogrammed successfully into pig induced pluripotent cells（piPCs）.