抗Fas核酶抑制小鼠T细胞凋亡

Inhibition of Apoptosis of Mouse T Cells by Anti-Fas Ribozyme

目的 研究抗Fas核酶对小鼠原代T细胞凋亡的抑制作用, 探索增强T细胞抗凋亡能力的新途径。方法设计并合成针对小鼠Fas基因的锤头状核酶, 通过电穿孔转染法将其导入小鼠脾脏T细胞, 通过RT -PCR、Westernblot检测细胞上Fas的表达, 细胞经抗Fas的抗体(JO2 ) 作用后, 通过Caspase -3 活性检测试剂盒测转染前后细胞Caspase 3活性的变化, MTT法测细胞的增殖, Annexin Ⅴ凋亡检测试剂盒测细胞凋亡。结果 ①与对照组、空载体和突变核酶转染组相比, 细胞表面的Fas水平明显降低; ②与抗Fas的抗体孵育后, 与转染空载体和突变核酶的细胞相比, 转染核酶的细胞Caspase- 3活性明显降低; ③经抗Fas抗体处理后, 细胞的增殖活性明显增高; ④与空载体和突变核酶转染组相比, 转染核酶的细胞凋亡率明显降低。结论 小鼠活化T细胞上高表达Fas, 抗Fas核酶能显著降低其Fas水平, 使细胞免于Fas途径的凋亡, 从而增强其抗凋亡能力。

Objective To investigate the inhibitive effects of anti-Fas ribozyme against apoptosis of mouse primary T cells,and explore a new way to enhance the ability of T cells against apoptosis.Methods A hammerhead ribozyme targeting the Fas mRNA was synthesized and transduced into mouse spleen T cells by using electroporation. The Fas expression in T cells was detected by using RT-PCR and Western blot. After the cells were treated with anti-Fas antibody (JO_ 2),T cells viability was measured by MTT assay,caspase-3 proteolytic activity before and after transfection was detected with caspase-3 kit,and cells apoptosis was measured by flow cytometry.Results As compared with control,blank-transfected and mutated ribozyme-transfected groups,the Fas expression in the ribozyme-transfected cells was obviously decreased. After the cells were treated with anti-Fas antibody (JO_ 2),caspase-3 activity and apoptotic rate of ribozyme-transfeced cells was significantly decreased as compared with blank-transfected and mutated ribozyme-transfected cells. Cells viability was increased evidently. Apoptotic rate of T cells transfected with anti-Fas ribozyme was reduced obviously as compared with blank-transfected and mutated ribozyme-transfected cells.Conclusion Fas is highly expressed in mouse activated spleen T cells,anti-Fas ribozyme can remarkably decrease the Fas expression and make the cells avoid Fas-mediated apoptosis,which enhanced their ability against apoptosis.