摘要
用逆转录病毒载体将单纯疱疹病毒胸苷激酶基因（ＨＳＶｔｋ）导入恶性肿瘤细胞，随后可应用药物９－（１，３－二羟基－丙氧基－甲基）鸟嘌呤（ｇａｎｃｉｃｌｏｖｉｒ，ＧＣＶ）选择性地杀死肿瘤细胞．将ＨｙＴＫ基因替换逆转录病毒载体ＧｌＮａ中的ｎｅｏ基因，构建成重组逆转录病毒载体ＧＴＫ，转染混合包装细胞（双噬性ＰＡ３１７细胞和单噬性ＧＰ＋Ｅ－８６细胞），通过“乒乓效应”获得高滴度重组病毒．用该重组病毒转染小鼠恶性黑色素瘤细胞系Ｂ１６细胞，用ｈｙｇｒｏｍｙｃｉｎＢ筛选出阳性细胞克隆（ＨｙＴＫ＋），经ＰＣＲ方法检测证明ＨｙＴＫ基因已成功地导入肿瘤细胞中，且不含可复制的辅助病毒．分别用不同浓度的ＧＣＶ作用于ＨｙＴＫ－及ＨｙＴＫ＋的Ｂ１６细胞，光镜下观察２４ｈ和４８ｈ后细胞形态及进行活细胞计数．结果表明，ＧＣＶ浓度大于０．
It is shown that transfer of the Herpes simplex virus thymidine kinase(HSVtk)gene into malignant tumor cells could confer the tumor cells susceptibility to the antiviral drug ganciclovir(GCV),and thus produce “killing”effect on the tumor cells exposed to GCV.A recombinant retroviral vector GTK was constructed by substituting HyTk gene for neo gene in the retroviral vector GlNa and was transduced into mixed packaging cells (PA317 cell and GP+E 86 cell).High titer retroviral supernatant (4 6×10 5 CFU/ml)was obtained by ping pong effect and the HyTK gene was then transferred into mouse melanoma cell line B16 cells.PCR results showed that the HyTK gene was transferred successfully into B16 cells and replication competent virus was absent.The cytotoxic effect on B16/HyTK + cells exposed to GCV(>0.1 μmol/L)was demonstrated by investigating under light microscope and by counting the living cells.
