重编程肝癌细胞系Huh7为多潜能干细胞样细胞  

Reprogramming hepatocellular carcinoma cell Huh7 into pluripotent stem-like cells

作　　者：张建[1] 高海霞[2] 范丽[3] 金华君[2] 张敬磊[4] 李林芳[2] 刘韬[2] 钱其军[2,3] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院细胞生物学系,苏州215123 [2]第二军医大学东方肝胆外科医院基因-病毒治疗实验室,上海200438 [3]浙江理工大学生命科学学院新元医学与生物技术研究所,杭州310018 [4]第二军医大学东方肝胆外科医院肝外三科,上海200438

出　　处：《第二军医大学学报》2012年第6期603-607,共5页Academic Journal of Second Military Medical University

基　　金：国家杰出青年科学基金(30925037)~~

摘　　要：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 human hepatocellular carcinoma cell Huh7 into pluripotent stem cells.Methods Four recombinant lentiviruses individually carrying Oct4,Sox2,Nanog,and Lin28 were constructed and used to co-infect Huh7 cells in vitro.Post infection,the obtained pluripotent stem-like cells were identified by Alkaline phosphatase staining,immunofluorescence assay,quantitative-PCR and immunohistochemistry.The differentiation capability of the cells was examined by teratogencity test.Results Pluripotent stem-like cell colonies（iHuh7） were observed in cultured Huh7 cells after lentivirus-based induction.These colonies were positive for alkaline phosphatase staining and immunofluorescence assay showed expression of pluripotent factors： Oct4 and TRA-1-60.Quantitative-PCR indicated that several endogenous pluripotency-associated genes and stem cell-specific microRNAs were highly expressed in these pluripotent stem-like cells.In vivo differentiation test showed that iHuh7 cells could lead to teratogencity.Conclusion Huh7 cells can be induced into pluripotent stem-like cells mediated by lentiviruses individually carrying Oct4,Sox2,Nanog,and Lin28.

关 键 词：肝细胞癌 诱导多潜能干细胞 慢病毒 重编程 

分 类 号：R329.29[医药卫生—人体解剖和组织胚胎学]