论文部分内容阅读
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 IC_(50)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G_0G_1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。
OBJECTIVE: To observe the killing effects of ganciclovir (GCV) on human lung adenocarcinoma cell line A549 transfected with HSV1-TK gene both in vitro and in vivo. METHODS: A retroviral expression vector containing TK gene was constructed. Human lung adenocarcinoma cell line A549 was transformed by electroporation. The sensitivity of GCV and the bystander effect of transgenic cells were measured by MTT assay; GCV was detected by flow cytometry and scanning electron microscope. Apoptosis was induced in transfected cells. PCR and in situ hybridization were used to detect the integration and expression of TK gene in the transfected cells. The effects of GCV on transfected cells and parental cells inoculated with subcutaneous tumors in nude mice were compared. RESULTS: Transfected cells had a 46-fold increase in GCV IC50 (50) compared to parental cells, and the bystander effect was significantly lower at high cell densities. FCM found that the DNA histogram showed a sub-G0G1 peak after transfection of cells with GCV for 3 days, and the parent cell did not have such a peak. Cell cycle analysis showed that subcellular G_0G_1 cells were (12.2±1.7)% and (1.3±0.3)% (P<0.01) in A549-TK and A549, respectively. Scanning electron microscopy revealed that the transfected cells had obvious apoptotic characteristics such as nuclear condensation, The apoptotic bodies and nucleus are half moon, etc., and the control cells do not have these changes. PCR and in situ hybridization showed that the transfected cells had TK gene integration and expression. In vivo experiments showed that transfected cells were inoculated with tumors inhibited by GCV treatment, and parental cells inoculated tumors were not inhibited. Conclusions: Transgenic TK gene cells are sensitive to GCV. Bystander effect kills tumor cells and it is closely related to the contact between cells. TK/GCV kills tumors and induces apoptosis. GCV inhibits the growth of tumor cells by transfecting TK gene cells in vivo.