【摘要】 目的:应用光学成像方法检测HSV-tk/GCV自杀基因系统与重组改造的人肿瘤坏死因子(recombinanthuman tumor necrosis factor,rmhTNF)的联合使用,对人涎腺腺样囊性癌细胞(adenoid cystic carcinoma,ACC-M)的杀伤效率。方法:实验分为四组,细胞模型对照组、单纯HSV-tk/GCV自杀基因系统治疗组、rmhTNF处理组、自杀基因联合rmhTNF治疗组。将稳定表达胸苷激酶-绿色荧光蛋白(thymidine kinase-green fluorescen protenin,tk-GFP)的ACC-M细胞(ACC-M-tk-GFP)按5 000个/孔接种到96孔板中,24 h后给药治疗;分别于加药前、给药后6 h、给药后24 h进行荧光成像。检测绿色荧光蛋白(green fluorescent protein,GFP)的荧光用于准确显示胸苷激酶(tk)基因表达的肿瘤细胞,检测碘化丙啶(propidium iodide,PI)的荧光用于显示死亡的肿瘤细胞。通过细胞的透射图及其PI和GFP荧光图像,分别计算tk+细胞和tk-细胞的死亡率。结果:HSV-tk/GCV自杀基因系统与rmhTNF联合治疗组的细胞在加药后6 h就已经出现细胞死亡迹象,而单纯HSV-tk/GCV自杀基因系统治疗组的细胞在加更昔洛韦(ganciclovir,GCV)后24 h才出现细胞死亡,单纯rmhTNF治疗组在加药后24 h还没有细胞死亡发生。对于给药后24 h的tk+细胞死亡率,联合治疗组(85.88%)明显高于GCV(38.13%)和rmhTNF单独治疗组(2.97%),并且联合治疗组给药24 h后会引发部分tk-细胞死亡(9.83%)。结论:HSV-tk/GCV自杀基因系统与rmhTNF联合治疗具有显著的协同抗肿瘤效应,能够明显提高对肿瘤细胞的杀伤率;结合光学成像方法,能为更直观方便地检测各组药物分别对tk+细胞和tk-细胞的杀伤率,可为深入研究"旁观者效应"提供参考。更多还原

【Abstract】 Objective: To investigate the synergistic effect of HSV-tk/GCV system combining with Recombinant Human Tumor Necrosis Factor(rmhTNF) on adenoid cystic carcinoma(ACC-M) cells using fluorescent imaging.Method: HSV-tk-GFP gene transfected into ACCM cells for obtaining tk-GFP stable expressing ACC-M cells(ACC-M-tk-GFP cells).The object was divided into four groups: control group,Ganciclovir(GCV) treated group,rmhTNF treated group and GCV/rmhTNF combination treatment group.24 hours after ACC-M-tk-GFP cells inoculated,we treated the last 3 groups with GCV and(or) rmhTNF.The effect was monitored using fluorescent imaging just before treatment and 6 hours and 24 hours after treatment,respectively.GFP fluorescence showed the amount of tk+ cells and PI fluorescence reflected the amount of the death cells.The cytotoxic effect of drugs on tk+ cells and tk-cells could be calculated through the transmission images and the fluorescent images of PI and GFP.Results: The apoptosis first appeared in GCV/rmhTNF combination group at 6 hours after treatment and became greater and greater with time;then appeared in GCV treated group 24 hours after treatment;while nearly no apoptosis appeared in rmhTNF treated group and control group and the cells of which kept growing during 24 hours after treatment.The apoptosis rate of tk+ cells in GCV/ rmhTNF combination group(85.88 %) was much higher than GCV treated group(38.13 %) and rmhTNF treated group(2.97 %) at 24 hours after drug treatment.Furthermore,some tk-cells(9.83 %) in GCV/ rmhTNF combination group could be induced apoptosis at 24 hours after treatment.Conclusion:The effect of the therapy combining HSV-tk/GCV system with rmhTNF on ACC-M-tk-GFP cells is better than the therapy of HSV-tk/GCV system alone.GCV/rmhTNF combination has significant synergistic anti-cancer effect.Fluorescent imaging provided the anti-cancer research with more effective detecting technique and references for further research on "By-stander effect".更多还原