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《China Biotechnology》 2015-05
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Construction of A Cell Line EGFP-Luc-Hepa1-6 and Its Application in the Mouse Model of the Hepatoma

YE Yu-chen;ZHAO Jun-long;WANG Lin;DUAN Juan-li;GAO Chun-chen;QIN Hong-yan;DOU Ke-feng;Department of Hepatobiliary Surgery,Xijing Hospital;State Key Laboratory of Cancer Biology,Department of Medical Genetics and Developmental Biology,Fourth Military Medical University;  
Objectives: We aimed to construct an eukaryotic expressive vector p CMV-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: p GL3-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 p IRES2-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 Lumina Ⅱimaging system. Confirming the expression of the vector in the cell line,we inoculated this cell line orthotopically in the liver of C57 BL /6 mouse to establish a hepatoma model. The growth of the hepatoma was consecutively monitored in vivo using Lumina Ⅱ 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 p CMVLuciferase-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.
【Fund】: 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
【CateGory Index】: R735.7;R-332
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【Secondary Citations】
Chinese Journal Full-text Database 1 Hits
1 JIA Feng Qi, WANG Wei Feng, JIANG Xiao Ling, YANG Qing, WEI Li Xin, YANG Guang Shun, WU Meng Chao (Tumor Immunology and Biotherapy Center, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China);Construction of adenoviral vector carrying murine MIP1-α and its expression in hepatocellular carcinoma[J];Academic Journal of Second Military Medical University;2002-05
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