HSV1-TK自杀基因系统对小鼠肝癌细胞移植瘤的抑制效应  

Inhibitory effect of HSV1-TK suicide gene system induced by retrovirus on murine transplanted hepatocarcinoma

作　　者：吴映雅[1] 杜标炎[2] 谭宇蕙[1] 赵鹏[2] 王慧峰[2] 

机构地区：[1]广州中医药大学生化教研室,广东省广州市510405 [2]广州中医药大学病理教研室,广东省广州市510405

出　　处：《世界华人消化杂志》2005年第16期1959-1963,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30171201广州市科技计划项目;No.2002J1-C7041~~

摘　　要：目的:构建表达单纯疱疹病毒胸苷激酶基因(HSV1-tk)的重组逆转录病毒,建立HSV1-tk/GCV肿瘤自杀基因治疗系统并检测其对小鼠肝癌细胞H22移植瘤的抑制效应.方法:构建携带HSV1-tk基因的逆转录病毒载体pLXSN-tk,用PolyFectTransfection试剂介导重组逆转录病毒转染入包装细胞系PT67,通过G418筛选建立稳定产病毒的细胞株,将该重组逆转录病毒感染小鼠肝癌细胞H22,以G418筛选的抗性克隆(命名为H22/tk);将H22/tk细胞与未经基因修饰的H22细胞按1:4混合接种小鼠皮下,联合应用GCV,观察其抑瘤作用(n=19).结果:经酶切鉴定和DNA序列测定,证明HSV1-tk基因成功定向插入到pLXSN逆转录病毒载体中;重组病毒DNA转染包装细胞PT67,获取病毒滴度为4×107cfu/L的重组逆转录病毒培养液;感染小鼠肝癌细胞H22后再筛选出G418抗性克隆株H22/tk.H22/tk细胞与H22细胞混合所致荷瘤鼠在GCV治疗前,各组间肿瘤体积无显著性差异;GCV治疗后,与对照组相比,GCV治疗组肿瘤的生长明显受到抑制,到第8,10,12,14d,致瘤模型对照组的肿瘤体积分别为356±205,635±382,963±580,1509±1105mm3,而GCV治疗组分别为231±155(VS对照组,t=-2.25,P=0.03),413±252(vs对照组,t=-2.14,P=0.04),592±420(vs对照组,t=-2.38,P=0.02),939±847mm3(vs对照组,t=-1.92,P=0.06),GCV治疗组的肿瘤生长抑制率分别为35.0%,35.0%,38.5%,37.8%.结论:成功获取表达HSV1-tk基因的逆转录病毒,已建立显示出体内抑瘤活性的自杀基因治疗系统,为进一步研究中医药对自杀基因抗肿瘤的增效作用奠定基础.AIM： To establish the HSVI-tk/GCV tumor suicide system by constructing a recombinant retroviral vector, and to determine the inhibitory effect of this system on murine transplanted hepetocarcinoma in vivo. METHODS： The HSV-tk cDNA was orientationally cloned into the retroviral vector plasmid （pLXSN） using DNA recombinant technique. The recombinant plasmid （pLXSN-tk） was mixed with PolyFect Transfection reagent and was transfected into the packaging cell line PT67. Then, a stable virus-producing packaging cell lines was obtained by G418 screening, and was transfected into murine hepatocarcinomatous H22 cells. An anti-G418 clone named H22/tk was obtained by G418 screening. H22/tk and H22 wild type cells were mixed in a proportion of 1 ： 4. Then the mixed cells were inoculated subcutaneously into Kunming mice. The tumor-bearing mice were randomly divided into model control group and GCV treated group,and the tumor inhibitory effect was observed by measuring tumor sizes. RESULTS： HSV1-tkwas inserted into recombinant plas- mid pl_XSN-tk successfully. The anti-G418 positive cell strain PT67/tk, which could excrete retrovirus recombinant, was obtained. The titer of the virus was 4 × 10^7cfu/L. After the infection of H22, the anti-G418 positive cell strain H22/tk was also obtained. The sensibility to GCV showed in H22/tk cells was much higher than that in H22. Nearly all the cells lysed and became granule-like after treated with GCV （1 000 mg/L） for 72 h. The tumor growth in the GCV treated group was significantly inhibited as compared with that in the control group. There was no significant difference in tumor size between the two groups before the mice were treated with GCV. But at the 8^th, 10^th, 12^th, 14^th day after treatment with GCV, the tumor sizes in the GCV treated group were obviously decreased as compared with those in the control group （231±155 mm^3 vs 356 ± 205 mm^3, t = -2.25, P = 0.03; 413 ± 252 mm^3 vs 635 ± 382 mm^3, t= -2.14, P= 0.04; 592 ± 420 mm^3 vs 963 ± 580 mm^3, t =

关 键 词：单纯疱疹病毒胸苷激酶基因 自杀基因系统 肝癌 小鼠肝癌细胞H22 抑制效应 HSV1-TK HSV1-TK/GCV 重组逆转录病毒感染 细胞移植瘤 HSV1-tk基因 

分 类 号：R735.7[医药卫生—肿瘤]