肝癌抑制基因-1肿瘤靶向性表达及其特异性抗肿瘤作用  

Tumor-targeting expression of a new tumor suppressor gene HCCS1 and its tumor-selective inhibitory effects on hepatocellular carcinoma

作　　者：张剑[1] 甘愉[1] 胡晶莹[1] 赵新泰[1] 

机构地区：[1]上海交通大学医学院、上海市肿瘤研究所细胞与分子免疫实验室,200032

出　　处：《中华肝脏病杂志》2008年第5期355-359,共5页Chinese Journal of Hepatology

基　　金：国家自然科学基金（30330350）

摘　　要：目的建立肝癌抑制基因-1（HCCS1）肿瘤靶向性表达载体，提高肿瘤治疗的安全性。方法活细胞计数试剂盒测定HCCS1高表达对正常细胞和肿瘤细胞的影响，荧光素酶试验检测肿瘤特异性启动子PEG3p在正常肝细胞和肝癌细胞中的相对转录活性，AdEasyTM系统包装并利用PCR鉴定重组腺病毒Ad—PEG-3p—HCCS1，Westernblot检测重组腺病毒感染后HCCS1在正常细胞和肿瘤细胞中的表达情况，结晶紫试验和四甲基偶氮唑盐试验观察该重组腺病毒体外抗肿瘤的靶向性。结果HCCS1高表达对肿瘤细胞株BEL7404和SW-620的生长抑制作用明显超过正常细胞株L02和正常人肺成纤维细胞，96h抑制率达60％。荧光素酶试验显示，PEG-3p在BEL-7404、BEL7405、QGY-7703中的相对转录活性分别为L02的3．9、4．7、1．5倍。成功构建了新抑癌基因HCCS1肿瘤靶向性表达的重组腺病毒Ad—PEG3p—HCCS1，Westernblot检测结果显示，在BEL7404和QGY7703中，HCCS1表达高于在L02中的表达。结晶紫试验和四甲基偶氮唑盐试验显示，Ad—PEG-3p—HCCS1与Ad—CMVHCCS1相比，在不降低对肿瘤抑制效果的前提下，明显降低了对正常细胞的杀伤作用。结论肿瘤细胞对HCCS1的生长抑制作用更为敏感，PEG3p在肝癌细胞中也具有肿瘤特异性，AdPEG-3p—HCCS1可特异性地在肿瘤细胞中表达HCCS1，从而提高HCCS1基因治疗的安全性。Objective To construct a tumor-targeting recombinant adenovirus vector containing hepatocellular carcinoma suppressor gene HCCS 1 to enhance the safety of tumor treatment. Methods CCK- 8 assay was used to observe different inhibitory effects on normal and malignant liver cells with high expressions of HCCS 1 protein. The relative transcriptional activity of PEG-3p was quantified by luciferase assay. Recombinant adenovirus Ad-PEG-3p-HCCS 1 was packaged with AdEasyTM system and confirmed by PCR. The tumor-targeted expression of HCCS 1 protein in cells infected with Ad-PEG-3p-HCCS 1 was determined by Western blot. Crystal violet assay and MTT assay were applied to observe the selective anti-tumor effects of the newly constructed virus in vitro. Results A higher inhibitory rate of about 60% was found in BEL- 7404 and SW-620 than that in L02 and NHLF 96 h after the high expression of HCCS 1. Luciferase assay showed 3.9-, 4.7-, and 1.5-fold transcriptional activity in BEL-7404, BEL-7405 and QGY-7703 respectively, in comparison with that in L02. Ad-PEG-3p-HCCS 1 was constructed successfully and was verified by PCR. Western blot indicated that high expression of HCCS 1 could be induced in BEL-7404 and QGY-7703 but not in L02. Crystal violet assay and MTT assay showed that it remarkably reduced the toxicity to L02 but still had enough antitumoral effect on Ad-CMV-HCCS 1. Conclusions With high expression of HCCS 1 the tumor cells we used are being inhibited more. PEG-3p has the tumor-selective driving function in malignant liver cells. Our recombinant adenovirus Ad-PEG-3p-HCCS 1 can tumor-targetingly induce HCCS 1 expression intumor cells, which can improve the safety of gene therapy with HCCS1.

关 键 词：癌 肝细胞 基因 抑制 肿瘤 腺病毒载体 

分 类 号：R735.7[医药卫生—肿瘤]