更昔洛韦对转染HSV1-TK基因的人肺腺癌细胞A549的体内外杀伤效应

摘要

AIM: To observe the killing effects of ganciclovir (GCV) on the human pulmonary adenocarcinoma cell A549 transduced with Herpes simplex virus I type thymine kinase (HSV1-TK) gene in vitro and in vivo.METHODS: A retroviral vector containing the TK gene was constructed and transduced into a pulmonary carcinoma cell A549 by electroporation, to observe the sensitivity of the transfected cell to GCV in vitro and the bystander effect (MTT assay). Tumor cell apoptosis caused by the TK/GCV system was observed with a flow cell meter (FCM) and a scan electronic microscope (SEM). Recombination and expression of the TK gene were examined with DNA PCR and in situ hybridization,respectively. The therapeutic effect of GCV on subcutaneous tumor growth between transfected and parental cells was also compared. RESULTS: The sensitivity of the transfected cell to GCV was 46 times higher than that of the parental cell, and the bystander effect was stronger in high cell density than in low cell density. The subG0G1 peak was shown on the DNA histogram after A549-Tk cell was treated with 50 μmol/L GCV for 3days by FCM, but not in the A549 cell. A cell cycle analysis showed that the apoptotic cell in the A549-TK and A549 cells were (12.2± 1.7) % and (1.3±0.3) %, respectively ( P ＜0.01). The cell apoptosis features of nuclear condensation, apoptotic vesicle, and nuclear showing semimoon feature were found in the A549-TK cell by SEM, but not in the A549 cell.Recombination and expression of the TK gene were positive in the transfected cell. In vivo, the growth of tumors formed by the transfected cell was apparently inhibited by GCV, but not in the control group.CONCLUSION: The transfected cell obtained sensitivity to GCV and the bystander effect was closely related to intercellular touch. The TK/GCV system killing tumor cell was related to cell apoptosis. GCV inhibited the growth of tumors which were inoculated by A549-TK cell in vivo.%目的：活体内外观察更昔洛韦(ganciclovir，GCV)对转染HSV1-TK基因的人肺腺癌细胞A549杀伤效应.方法：构建一个包含TK基因的逆转录病毒表达载体，用电穿孔法转化人肺腺癌细胞A549，MTT法测转基因细胞对GCV敏感性和观察旁观者效应；流式细胞仪、扫描电镜检测GCV诱导转染细胞凋亡，PCR和原位杂交分别检测转染细胞TK基因整合和表达，活体比较GCV对转染细胞、亲代细胞接种裸鼠皮下肿瘤治疗效果. 结果：转染细胞对GCV IC50比亲代细胞提高46倍，旁观者效应在高细胞密度较低细胞密度明显.FCM发现转染细胞经GCV作用3天后DNA直方图呈现亚G0G1峰，亲代细胞无该峰形成.细胞周期分析表明亚G0G1细胞在A549-TK和A549分别为(12.2±1.7)％和(1.3±0.3)％(P＜0.01).扫描电镜发现转染细胞有明显的凋亡特征如核浓缩、凋亡小体和核呈半月征等，对照细胞无这些变化.PCR、原位杂交表明转染细胞有TK基因整合和表达，体内实验表明转染细胞接种肿瘤受GCV治疗抑制生长，亲代细胞接种肿瘤不受抑制.结论：转TK基因细胞获得GCV的敏感性，旁观者效应杀灭肿瘤细胞与细胞间密切接触有关，TK/GCV杀灭肿瘤与诱导凋亡有关，GCV活体内抑制转TK基因细胞接种肿瘤生长.