基于四环素诱导型Hnf1β和Foxa3表达载体的小鼠胚胎成纤维细胞转分化为肝干细胞的实验体系研究

doi:10.3969/j.issn.1004-616x.2021.03.002

癌变·畸变·突变 ›› 2021, Vol. 33 ›› Issue (3): 163-171.doi: 10.3969/j.issn.1004-616x.2021.03.002

基于四环素诱导型Hnf1β和Foxa3表达载体的小鼠胚胎成纤维细胞转分化为肝干细胞的实验体系研究

虞欣璐¹, 于兵¹, 王辰², 张红霞¹, 朱海英¹

1. 海军军医大学基础医学院细胞生物学教研室, 上海 200433;
2. 解放军960医院检验科, 山东济南 250031

收稿日期:2020-11-03 修回日期:2021-04-12 出版日期:2021-05-30 发布日期:2021-06-09
通讯作者: 朱海英，E-mail：zinnia69@163.com E-mail:zinnia69@163.com
作者简介:虞欣璐，E-mail：yuxinludy@sina.com;于兵，E-mail：smmucellyu@163.com。
基金资助:
国家自然科学基金（31471284）

Induction of hepatic stem-like cells from mouse embryonic fibroblasts using tetracycline-controlled Hnf1β and Foxa3 expression vectors

YU Xinlu¹, YU Bing¹, WANG Chen², ZHANG Hongxia¹, ZHU Haiying¹

1. Department of Cell Biology, Basic Medical School, Navy Medical Universtiy, Shanghai 200433;
2. Department of Laboratory Medicine, PLA 960 Hospital, Jinan 250031, Shandong, China

Received:2020-11-03 Revised:2021-04-12 Online:2021-05-30 Published:2021-06-09

摘要/Abstract

摘要： 目的：建立基于四环素诱导型Hnf1β和Foxa3表达载体的小鼠胚胎成纤维细胞（MEF）转分化为肝干细胞（iHepSCs-Dox）的诱导实验体系，为后续转分化过程所涉及的分子机制研究提供有效工具。方法：构建TetO-Hnf1β-EGFP和TetO-Foxa3-mCherry四环素诱导型慢病毒载体，经慢病毒介导将其转染入293FT细胞，利用实时荧光定量PCR （qPCR）检测不同浓度的多西环素（Dox）诱导外源基因表达水平的差异，确定最佳Dox诱导浓度；将两个诱导型表达载体转染到MEF细胞中，参照先前建立的诱导体系，在合适的Dox浓度下启动Hnf1β和Foxa3基因的表达，诱导MEF细胞的转分化。对经过20 d诱导出现的上皮样细胞集落进行扩增，获得iHepSCs-Dox细胞系。利用CCK-8法、克隆形成实验、碱性磷酸酶染色、体外诱导分化以及反转录PCR （RT-PCR）等实验，对所获得的iHepSCs-Dox细胞系的生物学特性作鉴定，同时与前期获得的诱导型肝干细胞系（iHepSCs）作对比，以此对基于四环素诱导型表达载体的转分化体系的诱导效果进行评价。结果：qPCR结果显示，在培养基中添加100 ng/mL的Dox作用24 h即可启动外源基因表达，撤去Dox 48 h后，外源基因的表达显著降低甚至关闭；RT-PCR结果显示，iHepSCs-Dox细胞表达胆管细胞的标志（CK19）、肝胆共同标志（CK18）以及肝脏干/前体细胞标志（Dlk1、Sox9、EpCAM），碱性磷酸酶染色结果显示阳性；具有形成克隆的能力；能够在体外诱导分化为肝干细胞，以上干性特征与前期构建的iHepSCs具有较大相似性。当从培养基中撤掉Dox之后，随着双因子表达的停止，iHepSCs-Dox细胞的增殖速率明显下降，CK18、EpCAM的表达下调；失去克隆形成能力，在体外无法诱导其分化为肝细胞。结论：成功建立了基于四环素诱导型双转录因子表达载体的MEF细胞转分化为肝干细胞的诱导体系，该诱导体系可以作为后续研究转分化过程中所涉及的分子机制的有效工具。另外，结果提示外源基因的持续表达是iHepSCs-Dox细胞干性维持的必要条件。

关键词: 四环素诱导型表达载体, 转分化, 小鼠胚胎成纤维细胞, 诱导性肝干细胞, Hnf1β, Foxa3

Abstract: OBJECTIVE： To induce trans-differentiation of mouse embryonic fibroblasts (MEF) into hepatic stem (iHepSCs)-like cells using a tetracycline-controlled Hnf1β and Foxa3 expression system，and to provide a model for investigations into molecular mechanisms involved in the reprogramming process. METHODS： TetO-Hnf1β-EGFP and TetO-Foxa3-mCherry tetracyclic-control vectors were constructed. 293FT cells were used to determine the optimal dosages of doxycycline (Dox) in the trans- differentiation system of MEF. After vectors were transfected into 293FT cells，expression of Hnf1β and Foxa3 genes under different dosages of Dox were detected by real-time fluorescence quantification PCR (qPCR). After 20 days of induction and then amplification of the treated cells，an iHepSCs-Dox cell line was obtained. Biological characteristics of the cell line were documented using the CCK-8 method，colon formation assay，alkaline phosphatase staining，in vitro induced differentiation and reverse transcription-PCR (RT-PCR)，etc. In addition，the induction effects of tetracyclic-controlled reprogramming system were evaluated. RESULTS： The qPCR results show that expressions of exogenous Hnf1β and Foxa3 genes were turned on at 24-h after treatment with 100 ng/mL of Dox and were turned off at 48-h after Dox was removed. After treatment with Dox，markers for bile duct cells (CK19)，common marker of liver and bile (CK18)，and liver stem/precursor cell markers (Dlk1，Sox9 and EpCAM) were detected. Positive alkaline phosphatase staining and cell clones were observed. Furthermore， iHepSCs-Dox cells could be induced into hepatic cells which were similar to iHepSCs. With cessation of Hnf1β and Foxa3 expressions after Dox was removed from the medium， proliferation rates of iHepSCs- Dox cells and expressions of CK18 and EpCAM decreased significantly. In addition， cell clones formation and inductive differentiation to hepatic cells failed as well. CONCLUSION： The trans-differentiation system of MEF which was controlled by tetracyclic has been successfully established. This model can be used for investigations into molecular mechanisms involved in cellular reprogramming processes. Additionally，our results indicate that continuous expressions of exogenous Hnf1β and Foxa3 were necessary for maintenance of iHepSCsDox cell stemness.

Key words: tetracycline-controlled expression vectors, transdifferentiation, mouse embryonic fibroblast cells, induced hepatic stem cells, hepatocyte nuclear factor 1-β, forkhead box A3

中图分类号:

R329.2+8

虞欣璐, 于兵, 王辰, 张红霞, 朱海英. 基于四环素诱导型Hnf1β和Foxa3表达载体的小鼠胚胎成纤维细胞转分化为肝干细胞的实验体系研究[J]. 癌变·畸变·突变, 2021, 33(3): 163-171.

YU Xinlu, YU Bing, WANG Chen, ZHANG Hongxia, ZHU Haiying. Induction of hepatic stem-like cells from mouse embryonic fibroblasts using tetracycline-controlled Hnf1β and Foxa3 expression vectors[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2021, 33(3): 163-171.

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基于四环素诱导型Hnf1β和Foxa3表达载体的小鼠胚胎成纤维细胞转分化为肝干细胞的实验体系研究

Induction of hepatic stem-like cells from mouse embryonic fibroblasts using tetracycline-controlled Hnf1β and Foxa3 expression vectors

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