HSV1-TK/GCV自杀基因系统治疗卵巢癌的体内杀伤作用

HSV1-TK/GCV suicide gene therapy in nude mice with human ovarian cancer

目的:探讨Ⅰ型单纯疱疹病毒胸苷激酶(herpessimplexvirustypeⅠthymidinekinase,HSV1TK)/丙氧鸟苷(ganciclovir,GCV)自杀基因系统对卵巢癌的体内治疗效果。方法:用携带TK基因的重组逆转录病毒转染人卵巢癌细胞系SKOV3,用G418筛选抗性克隆(命名为SKOV3/TK)。PCR方法检测TK基因整合情况。将SKOV3和SKOV3/TK细胞按不同比例混合培养,给与10mg/LGCV治疗,观察体外旁观者效应。再分别用含有不同SKOV3/TK浓度的细胞建立荷瘤鼠模型作为3个TK基因转染组,并以接种单纯SKOV3细胞的动物模型作为对照组。TK基因转染组均行GCV20mg/(kg·d)腹腔注射共9d,对照组给等体积生理盐水腹腔注射。绘制肿瘤生长曲线,计算抑瘤率,并取瘤细胞做病理学检查。结果:体外混合培养细胞显示明显BSE,当SKOV3/TK细胞数占混合细胞数20%时,10mg/LGCV就可将约50%的混合细胞杀死。体内实验显示,20%、40%和100%TK基因转染组肿瘤体积与对照组比较,抑瘤率分别为38%、61%和99%,各组间两两比较差异有统计学意义,P<0.01(除20%TK基因转染组和40%TK基因转染组P值为0.001外,其余各组间P值均<0.001)。病理结果显示,TK基因转染组出现不同程度点、片状坏死,以100%TK基因转染组最重,残存的肿瘤均为坏死组织。结论:HSV1TK/GCV自杀基因系统对荷人卵巢癌皮下瘤裸鼠模型的肿瘤具有杀伤作用,同时存在BSE。这种杀瘤作用随着TK基因转染率的提高而增强。

OBJECTIVE： To study the killing effect and the bystander effect of a suicide gene therapy system of HSV1- TK/GCV （herpes simplex virus type I thymidine kinase/ganciclovir） for ovarian cancer in vivo. METHODS： Human ovarian cancer cell line SKOV-3 was transfected with the recombinant ret rovirus which contained HSV1-TK gene. Following selection by G418 medium, and resistant clones （named SKOV-3/TK） were identified by PCR. SKOV-3 and SKOV 3/TK cells were mixed in different proportions. Ten mg/L GCV was added into the mixed cells. The bystander effect was studied in vitro. Subcutaneous tumor models were induced in nude mice by subcutaneous injection with three different rations of SKOV-3 to SKOV-3/TK cells （8 ： 2, 6 ： 4,0 ： 10）. Nude mice subcutaneous injected with SKOV-3 cells were used as blank control. The former three groups were administered with GCV 20 mg/（kg · d） intraperitoneally for 9 days. The average tumor size were examined. At the end of the experiment, tumor specimens were processed for histopathological analysis. RESULTS： Significant bystander effect was observed in vitro. When the percentage of SKOV-3/TK cells reached 20%, the killing-rate of tumor cells was almost 50%. In vivo study, tumor growth inhibition rate of the former three groups were 38 %, 61% and 99% respectively, which was significantly different anong all groups, P〈0. 01. The extensive necrosis was observed in the treated mice with SKOV-3/TK tumor models, especially in the tumors with 100% SKOV-3/TK cells. CONCLUSIONS：There are great killing effect and bystander effect of HSV1-TK/GCV suicide gene therapy system for subcutaneous ovarian cancer nude mice models. The killing effect is enhanced by high gene transfer rate.