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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 m RNA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-polym erase chain reaction,RT-PCR)、W estern 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,M TT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 m RNA,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(0.4vs.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 express FasL and induce apoptosis of T cells that express Fas, that is, “Fas counterattack” of tumor cells. In this study, by detecting the inhibition of anti-Fas ribozyme on mouse T cell apoptosis and exploring new ways to enhance T cell anti-tumor ability. Methods: The hammerhead ribozyme targeting Fas gene was designed and synthesized to detect the in vitro cleavage ability of Fas m RNA. The cells were transfected into the spleen T cells by electroporation. At the same time, blank control group and empty vector transfected group were established. Reverse transcriptase-polymerase chain reaction (RT-PCR) blot detection of Fas expression on the cells. The cells were treated with anti-Fas antibody (JO2), and the apoptosis was detected by flow cytometry. The proliferation of cells was detected by MTT assay before and after transfection. The killing activity of cytotoxic T cells was tested by lactate dehydrogenase assay. Results: The anti-Fas ribozyme can cleave Fas m RNA significantly and its cutting efficiency is 60%. The level of Fas on the cell surface of the ribozyme group was significantly lower than that of the control group and the empty vector group (0.4 vs 1.1, 1.0, P <0.01). After anti-Fas antibody treatment, the rate of apoptosis in the nuclease group was significantly lower than that in the control and empty vector groups (35% vs.86%, 87%, P <0.01) Control group and empty vector group (208% vs.100%, 98%, P <0.01). The in vitro cytotoxic activity of T cells in ribozyme group was significantly stronger than that in other two groups (67% vs.32%, 31%, P <0.01). CONCLUSION: Anti-Fas ribozyme can significantly reduce the level of Fas in splenic T lymphocytes of mice and prevent the apoptosis of the cells from Fas pathway, thus enhancing the antitumor activity of T cells.