腺病毒介导的VEGF启动子—胸苷激酶系统对骨肉瘤细胞的杀伤试验

Selective killing of osteosarcoma cells by adenovirus-mediated transfer of vascular endothelial growth factor promoter-thymidine kinase gene in vitro

目的 探讨腺病毒介导的VEGF启动子调控的单纯疱疹病毒胸苷激酶基因 (HSV tk)系统 (AdVEGF tk)对体外培养的骨肉瘤细胞的选择性杀伤作用。方法 采用 2种重组腺病毒载体AdVEGF tk和AdCMV tk(CMV启动子调控tk基因表达 )体外感染低表达VEGF的成骨细胞系MC3T3 E1和高表达VEGF的骨肉瘤细胞系LM 8,并给予不同浓度的丙氧鸟苷 (GCV)处理 ,用MTT法检测感染细胞的增殖情况。结果 未感染病毒或感染空病毒的MC3T3 E1细胞和LM8细胞对GCV不敏感 ,感染AdCMV tk的MC3T3 E1细胞和LM 8细胞对GCV敏感。与感染空病毒的MC3T3 E1细胞相比 ,感染AdVEGF tk的MC3T3 E1细胞对GCV的敏感性轻度增加 ,用 5 0 μg/mlGCV处理时 ,30 %MC3T3 E1细胞被杀死 ;感染AdVEGF tk的LM8细胞对GCV的敏感性明显增加 ,用 5 0 μg/mlGCV处理时 ,95 %LM 8细胞被杀死 ,产生的细胞毒活性表现为剂量依赖性。结论 腺病毒介导的VEGF启动子 tk/GCV系统对高表达VEGF的骨肉瘤细胞具有杀伤作用。

Objective The purpose of this study was to investigate whether herpes simplex virus thymidine kinase (HSV-tk) gene expression driven by VEGF promoter followed by ganciclovir (GCV) administration would be effective in killing osteosarcoma cells in vitro. Methods Recombinant adenoviral vectors were constructed with HSV-tk under the control of VEGF promoter(AdVEGF-tk) or cytomegalovirus (CMV) promoter (AdCMV-tk). AdVEGF-tk and AdCMV-tk were transduced into osteoblastic cell line MC3T3-E1 with lower expression of VEGF and osteosarcoma cell line LM8 with higher expression of VEGF, followed by GCV treatment for 5 days. The sensitivity to GCV of infected cells was measured with MTT method. Results GCV did not inhibit the growth of both cell lines tested, but significantly inhibit the growth of both cell lines infected with AdCMV-tk. Lower cytotoxicity of GCV was observed in AdVEGF-tk infected MC3T3-E1 cell, 30% were killed at GCV concentration of 50 μg/ml. On the contrary, higher cytotoxicity of GCV was observed in AdVEGF-tk infected LM8 cell, 95% were killed at GCV concentration of 50 μg/ml. Conclusion Adenoviral vector containing VEGF promoter for the HSV-tk plus GCV treatment appears to be highly selective in killing osteosarcoma cell line in vitro.