EGFP-Luc-Hepa1-6细胞系的构建及其在小鼠肝癌模型中的应用

doi:10.13523/j.cb.20150501

中国生物工程杂志

2015, Vol. 35

Issue (5): 1-7 DOI: 10.13523/j.cb.20150501

研究报告

EGFP-Luc-Hepa1-6细胞系的构建及其在小鼠肝癌模型中的应用

叶雨辰¹, 赵俊龙², 王琳¹, 段娟丽¹, 高春辰², 秦鸿雁², 窦科峰¹

1. 第四军医大学西京医院西安 710032;
2. 第四军医大学基础部医学遗传学与发育生物学教研室肿瘤生物学国家重点实验室西安 710032

Construction of A Cell Line EGFP-Luc-Hepa1-6 and Its Application in the Mouse Model of the Hepatoma

YE Yu-chen¹, ZHAO Jun-long², WANG Lin¹, DUAN Juan-li¹, GAO Chun-chen², QIN Hong-yan², DOU Ke-feng¹

1. Department of Hepatobiliary Surgery, Xijing Hospital, Xi'an 710032, China;
2. State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, Fourth Military Medical University, Xi'an 710032, China

全文: PDF(1505 KB) HTML

摘要：

目的: 构建带有增强型绿色荧光蛋白EGFP及荧光素酶luciferase的真核表达载体pCMV-Luciferase-IRES2-EGFP,对肝癌细胞系Hepa1-6转染后进行稳定筛选,并将表达该载体的细胞系应用于小鼠原位肝癌模型构建,以对小鼠肝癌细胞进行稳定标记与活体示踪。方法: 对pGL3-Basic质粒进行Xba I酶切、Klenow片段补平黏端、Xho I酶切获得luciferase小片段,与Xho I/Sma I 酶切pIRES2-EGFP质粒获得的载体大片段连接,获得重组质粒pCMV-Luciferase-IRES2-EGFP。酶切鉴定、测序比对确认序列完全正确后,以重组载体转染Hepa1-6细胞进行稳定筛选,以荧光显微镜观察EGFP表达,报告基因实验与LuminaⅡ成像系统检测荧光素酶活性。确认该细胞系中的质粒得到表达后,以该细胞系进行C57BL/6小鼠肝脏原位接种构建肝癌模型,以LuminaⅡ成像系统连续活体监测肝癌的生长,取离体的肝癌组织制备石蜡切片(HE染色)和冰冻切片(免疫荧光染色)分别观察离体肿瘤组织病理学特征及其中肝癌细胞绿色荧光蛋白表达情况。结果: 成功构建表达pCMV-Luciferase-IRES2-EGFP载体的Hepa1-6细胞系(EGFP-Luc-Hepa1-6),并以该细胞系成功构建C57BL/6小鼠原位肝癌模型,实现小鼠肝癌细胞的活体示踪与体外标记。结论:成功构建EGFP-Luc-Hepa1-6细胞系,且以此细胞系构建的小鼠原位肝癌模型可以同时实现小鼠肝癌生长的连续活体监测与离体组织检测。

关键词： 肝癌; Hepa1-6; EGFP; Luciferase; 小鼠; 载体

Abstract:

Objectives: We aimed to construct an eukaryotic expressive vector pCMV-Luciferase-IRES2-EGFP which contains luciferase and enhanced green fluorescent protein(EGFP) and steadily screen Hepa1-6 cell line transfected with this recombinant vector,with the purpose of stably labeling and in vivo tracking the hepatoma cells in orthotopic hepatoma model established using this cell line. Methods: pGL3-Basic vector was digested by Xba I,end-repaired by Klenow Fragment and then redigested by Xho I to acquire the luciferase gene,which was ligated with the larger fragment of pIRES2-EGFP vector through Xho I/Sma I double digestion to construct the recombinant vector. Authenticated by endonuclease digestion and sequencing,the recombinant product presented the correct sequence. The expression of EGFP and activity of luciferase of Hepa1-6 cells transfected with this vector were,respectively,dectected by fluorescent microscope and dual-luciferase reporter assay and LuminaⅡ imaging system.Confirming the expression of the vector in the cell line, we inoculated this cell line orthotopically in the liver of C57BL/6 mouse to establish a hepatoma model.The growth of the hepatoma was consecutively monitored in vivo using LuminaⅡ imaging system and both paraffin sections(HE staining) and frozen sections (immunofluorescent staining) were prepared from the ex vivo tumor tissue to observe the pathological features and EGFP expression of the hepatoma cells. Results: Hepa1-6 cell line expressing the recombinant vector pCMV-Luciferase-IRES2-EGFP(EGFP-Luc-Hepa1-6) was successfully constructed.Using this cell line,an orthotopic hepatoma model in mice was established with the capability of in vivo tracking and ex vivo labeling the hepatoma cells. Conclusions: The EGFP-Luc-Hepa1-6 cell line was constructed successfully and the orthotopic hepatoma model established using this cell line can satisfy the needs of detecting the progression of the hepatoma both in vivo and ex vivo.

Key words: Hepatoma Hepa1-6 EGFP Luciferase Mouse Vector

收稿日期: 2015-02-13 出版日期: 2015-05-25

ZTFLH:

R735.7

基金资助:

国家自然科学基金重大研究计划培育项目(91029731),国家自然科学基金青年科学基金(81100307),国家自然科学基金青年科学基金(81401940),肿瘤生物学国家重点实验室(第四军医大学)开放基金(Cbskl201408)资助项目

通讯作者: 窦科峰 E-mail: gdwkdkf@163.com

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引用本文:

叶雨辰, 赵俊龙, 王琳, 段娟丽, 高春辰, 秦鸿雁, 窦科峰. EGFP-Luc-Hepa1-6细胞系的构建及其在小鼠肝癌模型中的应用[J]. 中国生物工程杂志, 2015, 35(5): 1-7.

YE Yu-chen, ZHAO Jun-long, WANG Lin, DUAN Juan-li, GAO Chun-chen, QIN Hong-yan, DOU Ke-feng. Construction of A Cell Line EGFP-Luc-Hepa1-6 and Its Application in the Mouse Model of the Hepatoma. China Biotechnology, 2015, 35(5): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150501 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I5/1

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