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目的重编程肝癌细胞系Huh7细胞为多潜能干细胞。方法包装携带有Oct4、Sox2、Nanog、Lin28基因的慢病毒,将包装好的病毒共感染Huh7细胞,诱导多潜能干细胞样克隆细胞。采用碱性磷酸酶染色、免疫荧光、实时定量RT-PCR等方法对诱导出的细胞进行鉴定,并采用畸胎瘤实验鉴定细胞的分化潜能。结果 Huh7细胞被重编程为多潜能干细胞样细胞(命名为iHuh7),碱性磷酸酶染色呈阳性,免疫荧光实验证明其表达多潜能因子Oct4和TRA-1-60,实时定量RT-PCR实验证明其高水平表达内源性的多潜能相关基因及干细胞特异的microRNAs,体内分化实验结果表明iHuh7可以形成畸胎瘤。结论通过携带4种多潜能基因Oct4、Sox2、Nanog、Lin28的慢病毒介导的重编程,Huh7细胞可以被诱导为多潜能干细胞样细胞。
Objective To reprogram the hepatoma cell line Huh7 cells into pluripotent stem cells. Methods Lentivirus carrying Oct4, Sox2, Nanog and Lin28 genes was packaged and the packaged virus was co-infected with Huh7 cells to induce pluripotent stem cell-like clonal cells. The induced cells were identified by alkaline phosphatase staining, immunofluorescence and real-time quantitative RT-PCR. Teratoma experiment was used to identify the differentiation potential of the cells. Results The Huh7 cells were reprogrammed into pluripotent stem cell-like cells (named iHuh7) with positive staining for alkaline phosphatase. The immunofluorescence assay showed that the expressed Huh7 cells were pluripotency factor Oct4 and TRA-1-60, and real-time quantitative RT-PCR The high-level expression of endogenous pluripotency-related genes and stem cell-specific microRNAs, in vivo differentiation experiments show that iHuh7 can form teratoma. Conclusion Huh7 cells can be induced to pluripotent stem cell-like cells by lentivirus-mediated reprogramming of the four pluripotency genes Oct4, Sox2, Nanog, Lin28.