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背景与目的:肿瘤细胞可表达FasL,从而诱导表达Fas的T细胞凋亡,即肿瘤细胞的“Fas反击作用”.本研究通过检测抗Fas核酶对小鼠T细胞凋亡的抑制作用,探索增强T细胞抗肿瘤能力的新途径.方法:设计并合成针对小鼠Fas基因的锤头状核酶,检测其对Fas mRNA的体外切割能力.通过电穿孔转染法将其导入小鼠脾脏T细胞,同时设立空白对照组和空载体转染组,通过逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)、Western blot检测细胞上Fas的表达.细胞经抗Fas的抗体(JO2)作用后,流式细胞仪检测细胞凋亡,MTF法测转染前后细胞的增殖,以乳酸脱氢酶试验检测细胞毒性T细胞的体外杀伤活性.结果:抗Fas核酶能显著切割Fas mRNA,切割效率为60%.核酶组细胞表面的Fas水平显著低于对照组及空载体组(0.4 vs.1.1,1.0,P＜0.01).经抗Fas抗体处理后,核酶组细胞的凋亡率明显低于对照组及空载体组(35%vs.86%,87%,P＜0.01);核酶组的细胞增殖活性显著高于对照组及空载体组(208%vs.100%,98%,P＜0.01);核酶组T细胞的体外杀伤活性明显强于其它2组细胞(67%vs.32%,31%,P＜0.01).结论:抗Fas核酶能显著降低小鼠脾脏T细胞的Fas水平,使细胞免于Fas途径的凋亡,从而增强T细胞的抗瘤能力.“,”BACKGROUND & OBJECTIVE: Tumor cells can express FasL, and induce apoptosis of activated T cells expressing Fas, which is so called “Fas counterattack”. This study was to investigate inhibitory effect of anti-Fas ribozyme on apoptosis of mouse T cells, and explore a new way to enhance antitumor ability of T cells. METHODS: A hammerhead ribozyme targeting Fas mRNA was synthesized, its in vitro cleavage reaction was performed. Anti-Fas ribozyme was transfected into mouse spleen T cells by electroperation (ribozyme-transfected group), empty control and mocktransfected groups were set up. Fas expression on T cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.After treatment of anti-Fas antibody (JO2), cell apoptosis was measured by flow cytometry, proliferation of T cells was measured by MTT assay. In vitro killing activity of cytotoxic T lymphocytes (CTLs) was detected by lactate dehydrogenase (LDH) releasing assay. RESULTS: Anti-Fas ribozyme cleaved Fas mRNA efficiently with a rate of 60%. mRNA level of Fas was significantly lower on ribozyme-transfected cells than on control and mocktransfected cells (0.4 vs. 1.1, and 1.0, P ＜ 0.01); its protein level was according to this result. After treatment of JO2, cell apoptosis rate was significantly lower in ribozyme-transfected group than in the rest 2 groups (35% vs. 86%, and 87%, P ＜ 0.01), but cell proliferation rate was significantly higher in ribozyme-transfected group than in the rest 2 groups (208% vs. 100%, and 98%, P ＜ 0.01). In vitro killing activity of CTLs was significantly stronger in ribozyme-transfected group than in the rest 2 groups (67% vs. 32%, and 31%, P ＜ 0.01). CONCLUSION: Anti-Fas ribozyme can remarkably suppress Fas expression on mouse activated spleen T cells, and protect T cells from Fas-mediated apoptosis, which may enhance antitumor ability of T cells.