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目的探讨tk基因系统在体外培养条件下对T细胞克隆的转导情况以及转导后的tk杀伤效应。方法将tk基因在体外培养条件下导入结肠抗原特异性T细胞克隆,观察基因转染、克隆凋亡情况,比较tk~+和tk~-T细胞克隆之白细胞介素(IL)-2分泌水平、细胞存活率、“旁观者效应”间差异。结果荧光显微镜下观察可见转染成功,tk基因导入淋巴细胞发生凋亡；当复合感染度(MOI)为0、1．0、5．0、20．0、50．0、100．0、200．0、500．0时,细胞存活率分别为(94．43±5．02)%、(85．33±4．12)%、(81．07±2．46)%、(73．51±3．89)%、(68．68±5．26)%、(54．16±3．72)%、(45．51±2．29)%、(22．16±1．77)%。随着MOI和丙氧鸟苷(GCV)浓度增加,细胞存活率呈现明显下降趋势；tk~+T细胞克隆实验组IL-2分泌水平明显下降,与对照组差异有统计学意义(P＜0．01)；各种tk~+T细胞比例条件下的存活率变化曲线与ADV/Empty组接近,未能产生类似肿瘤细胞的旁观者效应。结论tk基因对T细胞克隆具备杀伤效应,但tk~+T细胞克隆杀伤实验中未能见到与肿瘤细胞类似的“旁观者效应”。
Objective To investigate the transduction of T cell clones and the tk killing effect after transduction of tk gene system in vitro. Methods The tk gene was introduced into colonic antigen-specific T cell clones in vitro and the gene transfection and cloning and apoptosis were observed. The levels of interleukin (IL) -2 secretion in tk ~ + and tk ~ -T cell clones were compared , Cell survival, “bystander effect” between the differences. Results The results of fluorescence microscopy showed that transfection was successful and tk gene was induced to apoptosis in lymphocytes. When the MOI was 0, 1.0, 5.0, 20.0, 50.0, 100.0, 200 . The cell survival rates were (94.43 ± 5.02)%, (85.33 ± 4.12)%, (81.07 ± 2.46)%, (73.51 ± 3.89%, (68.68 ± 5.26)%, (54.16 ± 3.72)%, (45.51 ± 2.29)%, (22.16 ± 1.77)%, . With the increase of the concentration of MOI and ganciclovir (GCV), the cell survival rate showed a significant decrease; IL-2 secretion in tk ~ + T cell clones decreased significantly compared with the control group (P <0 .01). The curves of survival rate under various ratios of tk ~ + T cells were similar to those of ADV / Empty group, and no bystander effect similar to that of tumor cells was found. Conclusion The tk gene has the killing effect on T cell clones, but the “bystander effect” similar to tumor cells can not be seen in tk ~ + T cell cloning and killing experiment.