田 慧，张智红，林居强，刘曼西，骆清铭
(华中科技大学生物医学光子学教育部重点实验室，湖北 武汉 430074 )
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构人肿瘤坏死因子(recombinant human tumor necrosis factor, rmhTNF)的联合使用，对人涎腺腺样囊性癌细胞(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 (thymidine kinase, 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，能更为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，为深入研究“旁观者效应”提供参考。
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Fluorescent Imaging Cytotoxic Effect of HSV-tk/GCV System and Recombinant TNF on the Tumor Cells
TIAN Hui, ZHANG Zhi-hong, LIN Ju-qiang , LIU Man-x i, LUO Qing-ming
( The Key Laboratory of Biomedical Photonics of Ministry of Education,
Huazhong University of Science and Technology, Wuhan 430074 , Hubei , China )
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 ACC-M 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. Result: 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 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".
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