hTERT启动子调控的TRAIL基因表达载体的构建及其对肝癌细胞增殖的抑制作用

J4 ›› 2010, Vol. 36 ›› Issue (5): 825-831.

hTERT启动子调控的TRAIL基因表达载体的构建及其对肝癌细胞增殖的抑制作用

李金华^1,2|刘扬¹|王宏芳^1,2|王志成¹|吴嘉慧¹|龚守良¹|李娟²

1. 吉林大学公共卫生学院卫生部放射生物学重点实验室|吉林长春 130021；2. 吉林大学公共卫生学院卫生检验教研室|吉林长春 130021

收稿日期:2010-05-12 出版日期:2010-09-28 发布日期:2010-09-28
通讯作者: 龚守良;李娟 E-mail:gongsl@163.com
基金资助:
国家自然科学基金资助课题(30471665)；吉林省科技发展计划项目资助课题(200905133)

Construction of hTERT promoter-driven TRAIL expression vector and its inhibitory effects on hepatoma cell proliferation

LI Jin-Hua^1,2, LIU Yang¹, WANG Hong-Fang^1,2, WANG Zhi-Cheng¹, TUN Jia-Hui¹, GONG Shou-Liang¹, LI Juan²

Key Laboratory of Radiobiology|Ministry of Health|School of Public Health|Jilin University,Changchun 130021,China;2. Department of Health Laboratory,School of Public Health,Jilin University,Changchun 130021,China

Received:2010-05-12 Online:2010-09-28 Published:2010-09-28

摘要/Abstract

摘要：

目的：构建hTERT启动子调控的肿瘤坏死因子相关的凋亡诱导配体(TRAIL)基因表达载体，探讨其对肝癌细胞SMMC7721和肝细胞HL7702增殖的抑制作用。方法：采用分子生物学方法构建重组质粒pshuttle-TRAIL和pshuttle-hTERT-TRAIL，经PCR和酶切鉴定正确后，通过脂质体转染SMMC7721和HL7702细胞，同时转染pcDNA3.1+-GFP并用荧光显微镜检测转染效率。MTT法检测重组质粒对细胞增殖变化的影响。结果：成功构建重组质粒pshuttle-TRAIL和pshuttle-hTERT-TRAIL，荧光显微镜检测结果显示：质粒与脂质体的比例为1∶3时细胞转染效率最高。pshuttle-hTERT-TRAIL组SMMC7721细胞增殖抑制率从16 h开始明显增加，与对照组比较差异有显著性（P<0.001），且从24 h开始与pshuttle-TRAIL组比较细胞增殖抑制率明显增加（P<0.05）；但重组质粒对HL7702细胞增殖抑制率无影响。结论： hTERT启动子调控的TRAIL可明显抑制肝癌细胞的增殖，而对人肝细胞的增殖无影响，其作用效果明显优于TRAIL单基因治疗。

Abstract:

Abstract:Objective
To construct the hTERT promoter-driven TRAIL expression vector and explore its inhibitory effects on the proliferation of hepatoma SMMC7721 cells and HL7702 hepatocytes. Methods The recombinant plasmids pshuttle-TRAIL and pshuttle-hTERT-TRAIL were constructed with molecular biological methods. They were confirmed correctly by endonucleases digestion and PCR identification,and transfected into SMMC7721 and HL7702 cells through liposome,meanwhile,the pcDNA3.1+-GFP was transfected to detect the transfection efficacy by fluorescence microscopy. MTT was used to detect the effects of the recombinant plasmids on cell proliferation in transfected cells. Results The recombinant plasmid pshuttle-TRAIL and pshuttle-hTERT-TRAIL were constructed successfully. And the transfection efficacy was highest by fluorescence microscopy when the ratio of plasmid to liposome was 1∶3. As compared with control group, the inhibitory rates of SMMC7721 cells from 16 h after transfection in pshuttle-hTERT-TRAIL group was increased significantly (P<0.001);and as compared with pshuttle-TRAIL group,the inhibitory rates of SMMC7721 cells from 24 h after transfection in pshuttle-hTERT-TRAIL group was increased significantly (P<0.05). However,as compared with control group,the inhibiory rates of HL7702 cells in recombinant plasmids groups did not change significantly. Conclusion hTERT promoter-driven TRAIL could significantly inhibit the hepatoma cell proliferation,especially superior to that in pshuttle-TRAIL,however,no effects on hepatocyte proliferation.

Key words: TNF-related apoptosis inducing ligand;hTERT promoter;tumor targeting therapy;cell proliferation

中图分类号:

李金华|刘扬|王宏芳|王志成|吴嘉慧|龚守良|李娟. hTERT启动子调控的TRAIL基因表达载体的构建及其对肝癌细胞增殖的抑制作用[J]. J4, 2010, 36(5): 825-831.

LI Jin-Hua, LIU Yang, WANG Hong-Fang, WANG Zhi-Cheng, TUN Jia-Hui, GONG Shou-Liang, LI Juan. Construction of hTERT promoter-driven TRAIL expression vector and its inhibitory effects on hepatoma cell proliferation[J]. J4, 2010, 36(5): 825-831.