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| 重编程肝癌细胞系Huh7为多潜能干细胞样细胞 |
| 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* |
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(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
△共同第一作者
*通信作者) |
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| 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。 |
| 关键词: 肝细胞癌 诱导多潜能干细胞 慢病毒 重编程 |
| DOI：10.3724/SP.J.1008.2012.00603 |
| 投稿时间：2012-01-22修订日期：2012-04-01 |
| 基金项目:国家杰出青年科学基金(30925037). |
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| Reprogramming hepatocellular carcinoma cell Huh7 into pluripotent stem-like cells |
| ZHANG Jian1△,GAO Hai-xia2△,FAN Li3,JIN Hua-jun2,ZHANG Jing-lei4,LI Lin-fang2,LIU Tao2,QIAN Qi-jun2,3* |
(1. Department of Cell Biology, School of Biology and Basic Medical Sciences, Medical College, Soochow University, Suzhou 215123, Jiangsu, China
2. Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
3. Xinyuan Institute of Medicine and Biotechnology, School of life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, Zhejiang, China
4. Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai 200438, China
△Co-first authors.
*Corresponding author.) |
| Abstract: |
| 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. ConclusionHuh7 cells can be induced into pluripotent stem-like cells mediated by lentiviruses individually carrying Oct4, Sox2, Nanog, and Lin28. |
| Key words: hepatocellular carcinoma induced pluripotent stem cells lentivirus reprogramming |
