Zerrouqi A, Rixe O, Ghoumari A M, Yarovoi S V, Mouawad R, Khayat D, Soubrane C
Laboratory of Medical Oncology Department, Pitié-Salpêtrière Hospital, Paris, France.
Cancer Gene Ther. 1996 Nov-Dec;3(6):385-92.
In this study, we investigated whether the regulation and the copy number of the herpes simplex virus thymidine kinase (HSVtk) gene increased the sensitization to ganciclovir (GCV) of glioma cell lines (Rat C6 and human U118-MG) using liposome-mediated gene transfer. Three recombinant plasmids carrying the HSVtk gene driven by the thymidine kinase promoter in single (pAGo) and double copy (pYED) or by the human cytomegalovirus promoter (pCMVtk) were used for the transfection. The DNA delivery was optimized by screening a panel of cationic liposomes using Lac-Z and luciferase as reporter genes. The efficiency of transfection reached 33% to 36% in vitro but only 18.6% in vivo after an intratumoral injection of DNA-liposome complexes. Moreover, after transfection of the three plasmids, the cell-killing effect of GCV was evaluated. A significant enhancement (four- to fivefold) of the cell sensitivity to GCV was shown in pCMVtk and pYED as compared with pAGo-transfected cells in both cell lines. According to the plasmid, the effect of the HSVtk/GCV system was confirmed by in vivo experiments and was objectified by a higher tumor weight reduction with pCMVtk (49%) than pAGo (27%). From these results, we conclude that (1) the gene transfer can be achieved by cationic liposomes both in vitro and in vivo and that (2) using this type of vector, the antitumor effect of the HSVtk/GCV system could be potentiated by the up-regulation of HSVtk gene duplication.
在本研究中，我们利用脂质体介导的基因转移，研究单纯疱疹病毒胸苷激酶（HSVtk）基因的调控和拷贝数是否会增加胶质瘤细胞系（大鼠C6和人U118 - MG）对更昔洛韦（GCV）的敏感性。使用了三种重组质粒进行转染，其中一种携带由胸苷激酶启动子驱动的单拷贝（pAGo）和双拷贝（pYED）HSVtk基因，另一种携带由人巨细胞病毒启动子驱动的HSVtk基因（pCMVtk）。通过使用Lac - Z和荧光素酶作为报告基因筛选一系列阳离子脂质体，优化了DNA递送。DNA - 脂质体复合物瘤内注射后，体外转染效率达到33%至36%，但体内仅为18.6%。此外，转染三种质粒后，评估了GCV的细胞杀伤作用。与两种细胞系中pAGo转染的细胞相比，pCMVtk和pYED转染的细胞对GCV的敏感性显著增强（四至五倍）。根据质粒，体内实验证实了HSVtk/GCV系统的作用，并且通过pCMVtk（49%）比pAGo（27%）更高的肿瘤重量减轻得以体现。从这些结果中，我们得出结论：（1）阳离子脂质体在体外和体内均可实现基因转移；（2）使用这种类型的载体，HSVtk基因复制的上调可增强HSVtk/GCV系统的抗肿瘤作用。
