增强型肿瘤特异性启动子介导CDTK治疗肝癌的体内外研究

doi:10.13523/j.cb.20150601

中国生物工程杂志

2015, Vol. 35

Issue (6): 1-7 DOI: 10.13523/j.cb.20150601

研究报告

增强型肿瘤特异性启动子介导CDTK治疗肝癌的体内外研究

薛金锋, 薛志刚, 陈毅瑶, 李卓, 尹彪, 邬玲仟, 梁德生

中南大学医学遗传学国家重点实验室长沙 410078

In vitro and in vivo Gene Therapy Research of CDTK Genes Drove by Enhanced Tumor-specific Promoter in Liver Cancer

XUE Jin-feng, XUE Zhi-gang, CHEN Yi-yao, LI Zhuo, YIN Biao, WU Ling-qian, LIANG De-sheng

State Key Laboratory of Medical Genetics, Central South University, Changsha 410078, China

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摘要：

目的:探讨运用增强子上调肿瘤特异性启动子hTERT转录活性后,调控自杀融合基因CDTK对人肝癌细胞系Bel-7402的体内外杀伤作用。方法:将CMV增强子、hTERT启动子及CDTK自杀融合基因克隆入核糖体基因区打靶载体pHrn,构建pHr-CeTpCDTK-GFP 治疗载体并转染Bel-7402细胞,经RT-PCR、HPLC、MTT检测CDTK基因的表达和对肝癌细胞的体外杀伤效果。再将Bel-7402细胞接种到裸鼠皮下致瘤,以肿瘤杀伤载体pHr-CeTpCDTK-GFP进行瘤内转染并检测其抑瘤效果,此外将转染后的细胞接种致瘤,检测其成瘤时间及肿瘤生长曲线等,从两方面检测其体内杀伤效果。结果:成功构建了肿瘤特异性治疗载体,体外转染肝癌细胞后,经RT-PCR、HPLC证明CDTK基因能在肝癌中表达,且治疗载体在体外对肝癌细胞有明显毒性。动物实验结果发现裸鼠致瘤后治疗组血清中5-Fu浓度为7.694μg/ml,治疗组肿瘤体积与对照组相比减小6.5倍。而细胞转染治疗载体后致瘤,治疗组成瘤时间比对照组晚8天,且治疗组裸鼠的平均生存期较对照组延长16天。结论:pHr-CeTpCDTK-GFP载体能在体内外高效靶向杀伤肝癌细胞,为肝癌基因治疗提供了一种新的途径。

关键词： CDTK基因; 核糖体基因区载体; 肝癌; 基因治疗

Abstract:

Objective: To investigate the in vivo and in vitro killing effects of CDTK gene drove by enhanced hTERT promoter in liver cancer cell line Bel-7402. Methods: Cloned CMV enhancer, hTERT promoter and CDTK gene into hrDNA targeting vector pHrn and constructed pHr-CeTpCDTK-GFP vector. After transferred into Bel-7402 cells, CDTK gene expression and in vitro killing effects were detected by RT-PCR, HPLC and MTT. Finally, the pHr-CeTpCDTK-GFP vector mixed with liposome were injected directly into the tumor of nude mouse, which was produced by the injection of those liver cancer cells, meanwhile, transfected cells were injected into mouse, the tumor size and killing effects were closely observed. Results: The RT-PCR and HPLC results showed that the liver cancer cells transfected with the constructed pHr-CeTpCDTK-GFP vector could express CDTK successfully. The MTT showed that the vector is toxic to the liver cancer cell. Through the animal test, it was found that the concentration of 5-Fu was 7.694μg/ml in treatment group, the tumor volume of treatment group decreased 6.5 times compared with the control group. Using vector-transfected cells induce tumor, the tumor formation time of treatment group was 8 days later than control group, while the mean survival period of treatment group was 16 days longer than control group. Conclusions: The pHr-CeTpCDTK-GFP vector could kill the liver cancer efficiently and resulted in a new approach for the liver cancer gene therapy.

Key words: CDTK suicide gene rDNA targeting vector Liver cancer Gene therapy

收稿日期: 2015-03-03 出版日期: 2015-06-25

ZTFLH:

Q782

基金资助:

国家自然科学基金资助项目(30500302)

通讯作者: 梁德生 E-mail: liangdesheng@sklmg.edu.cn

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引用本文:

薛金锋, 薛志刚, 陈毅瑶, 李卓, 尹彪, 邬玲仟, 梁德生. 增强型肿瘤特异性启动子介导CDTK治疗肝癌的体内外研究[J]. 中国生物工程杂志, 2015, 35(6): 1-7.

XUE Jin-feng, XUE Zhi-gang, CHEN Yi-yao, LI Zhuo, YIN Biao, WU Ling-qian, LIANG De-sheng. In vitro and in vivo Gene Therapy Research of CDTK Genes Drove by Enhanced Tumor-specific Promoter in Liver Cancer. China Biotechnology, 2015, 35(6): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150601 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I6/1

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