摘要
目的 研究单纯疱疹病毒胸苷激酶 (HSV1 TK)基因转染并联合抗病毒药更昔洛韦 (Ganciclovir ,GCV)对人胶质母细胞瘤细胞的杀伤效应。方法 采用基因工程技术构建带HSV1-TK基因的逆转录病毒重组体pLX SN TK ,采用脂质体介导入PA317包装细胞 ,建立重组逆转录病毒载体分泌细胞株PA317 TK ;用该细胞上清液转导人胶质母细胞瘤细胞 ,用不同浓度GCV作用人胶质母细胞瘤细胞SW0 38 C2 TK和野生型SW0 38 C2 ,采用噻唑蓝(MTT)比色法检测 72h后细胞存活率 ,并求出半杀伤浓度。结果 重组逆转录病毒载体pLXSN TK能有效地将TK基因导入SW0 38-C2细胞内 ,并使其获得对GCV的敏感性 ;体外细胞毒实验 :在 2 0 μmol LGCV存在的情况下 ,野生型细胞无明显改变 ,而实验组细胞明显死亡 ,半杀伤浓度IC50 为 0 8μmol L ,与SW0 38 C2相比 ,对GCV的敏感性提高了 6 0 0倍 ,旁杀效果也较明显。结论 SW0 38 C2转染TK基因后 ,能有效地被GCV杀灭。
Objective To study the efficacy of gene therapy with the herpes simplex virus thymidine kinase(HSV1\|TK) gene transfection associated with ganciclovir(GCV) on human multiform glioblastoma cells SW038\|C2. Methods\ A recombinant retroviral vector, pLXSN\|TK, expressing the suicide gene herpes simplex virus thymidine kinase gene was constructed. Then the recombinant plasmid was transfected into PA317 packaged cell and productive cell line of recombinant of cell culture PA317/TK was established. The supernatant of cell culture was used to infect the human multiform glioblastoma cell line SW038\|C2. The sensitivity of SW038\|C2/TK cells and wild type SW038\|C2 cells to GCV was expressed as IC 50 which was measured with MTT assay after treating both cells for 72 hrs. Results\ The recombinant retroviral vector pLXSN\|TK could transfer HSV1\|TK gene into the genome of SW038\|C2 cell line and make it sensitive to GCV. In vitro study showed that the SW038\|C2/TK cells were apparently killed in a 20μmol/L GCV solution while the wild type SW038\|C2 cells grew normally under the same condition. The IC 50 of SW038 C2/TK cell in GCV solution was 0 8μmol/L and that of SW038 C2 cells 500μmol/L, showing a 600 fold difference. The “bystander effect” of HSV1 TK on SW038 C2 cells was significant. Conclusion\ GCV could specifically and effectively kill the TK transfected SW038 C2 cells \%in vitro.\% This result suggested that the HSV1 TK/GCV system might have a clinical potential for gene therapy.
出处
《中国实验动物学报》
CAS
CSCD
2003年第2期65-68,F003,共5页
Acta Laboratorium Animalis Scientia Sinica
基金
广东省科委重点项目
粤科计字 [199]号
