抗Fas核酶抑制Fas介导的小鼠肝脏细胞凋亡的研究

Inhibition of Fas-mediated apoptosis in mouse hepatic cells via an anti-Fas ribozyme

目的 研究抗Fas锤头状核酶对原代培养的小鼠肝脏细胞Fas表达及其凋亡的影响。方法 采用胶原酶灌流法分离小鼠肝脏细胞 ,同时构建了针对FasmRNA的锤头状核酶真核质粒 ,经纳米载体Effectene将其导入肝细胞 ,通过RT PCR、Westernblot检测细胞Fas的表达 ,以Caspase 3活性检测试剂盒测转染前后细胞Caspase 3活性的变化 ,AnnexinⅤ FITC凋亡检测试剂盒测转染前后细胞凋亡 ,MTT法测细胞的增殖。结果 小鼠原代肝细胞上表达Fas ,抗Fas核酶能显著降低肝细胞上Fas水平 ,经抗Fas抗体作用后 ,转染核酶的细胞Caspase 3活性明显降低 ,该细胞增殖活性较对照组增强 ,而凋亡率明显降低。结论 抗Fas核酶能显著降低小鼠原代肝细胞上Fas的表达 ,使其免于Fas途径的凋亡。

Objective To investigate the effects of anti-Fas hammerhead ribozyme on Fas expression and apoptosis in primary cultured mouse hepatic cells. Methods Mouse hepatic cells were isolated by using collagenase irrigation,and a hammerhead ribozyme targeting the Fas mRNA was constructed,which was transfected into mouse hepatic cells via Effectene. Then Fas expression in mouse hepatic cells was detected by using RT-PCR and western blot. After being treated with anti-Fas antibody (JO 2),hepatic cell viability was measured by MTT assay,Caspase-3 proteolytic activity was detected,and apoptosis was measured according to AnnexinⅤ-FITC apoptosis detecting kit.Results The expression of Fas was detected in primary mouse hepatic cells. The Fas expression in hepatic cells transfected with anti-Fas ribozyme was decreased remarkably and correlated with resistance to Fas-mediated apoptosis as determined by flow cytometry and Caspase-3 proteolytic activity.Conclusion Anti-Fas ribozyme can remarkably decrease the Fas expression in mouse hepatic cells,which make hepatic cells get rid of Fas-mediated apoptosis.