逆转录病毒载体LNSHyTK的构建及与丙氧鸟苷联合杀伤B16细胞的研究

Construction of a retroviral vector containing HyTK gene and its cyto-toxic effect on B16 cells when combined with ganciclovir treatment

构建了一个能表达ＨｙＴＫ的重组逆转录病毒载体ＬＮＳＨｙＴＫ，经包装细胞ＰＡ３１７包装成假型逆转录病毒，并利用此病毒感染了小鼠黑色素瘤细胞株Ｂ１６．聚合酶链反应结果表明假病毒中不含复制活性病毒、ｔｋ基因已成功地被导入了Ｂ１６细胞。总ＲＮＡ斑点杂交显示转染细胞中有稳定的ｔｋ基因转录产物。细胞毒性实验显示，０．０５μｍｏｌ／Ｌ的丙氧鸟苷即可明显降低转染细胞的存活率，表明逆转录病毒介导的ＨyＴＫ基因转移使Ｂ１６细胞获得了对丙氧鸟苷的敏感性。因此，ＨyＴＫ基因／ＧＣＶ可能成为实体瘤的基因治疗途径。

A recombinant retroviral vector expressing HyTK fusion protein was constructed. The pseudo- type virus was packaged and rescued by transfection in PA317 cells and was used to infect murinemelanoma cell line B16. Using polymerase chain reaction,no replication- competent retrovirus in the pseudo- type virus stock was detected and the presence of tk gene in the infected cells was confirmed.Transcription-al product of the tk gene was also detected by RNA dot blot hybridization. The survival rate of HyTK gen- etransfected B16 cells was significantly decreased when cocultured vvith ganciclovir at a concentration of0. 05μmol/L,which suggested that B16 cells aquired sensitivity to ganciclovir by retrovirus- mediated HyTKgene transfer.The result implies the potential value of HyTK/GCV in gene therapy for solid tumors.