白血病细胞表面可高表达FasL ,诱导表达Fas的T细胞凋亡 ,从而降低其抗白血病的作用 .以高表达Fas的小鼠淋巴瘤细胞系Yac 1作为模型 ,设计并合成了针对FasmRNA 5 96位点的锤头状核酶基因 ,用T7/SP6体外转录系统检测了该核酶对FasmRNA的体外切割效率 ,通过电穿孔转染法将其导入Yac 1细胞 ,通过RT PCR、Western印迹法检测细胞上Fas的表达 .细胞经抗Fas的抗体作用后 ,通过MTT法测细胞的增殖 ,annexin Ⅴ凋亡检测试剂盒测细胞凋亡 .该核酶体外切割活性达6 0 % ,且能有效降低细胞表面Fas的表达 ;细胞经抗Fas的抗体作用后 ,转染核酶的细胞增殖活性较对照增高 ,而凋亡率明显降低 .结果表明 ,构建的切割FasmRNA核酶在体内外均具备良好的切割FasmRNA的活性 ,使细胞免于Fas途径的凋亡 ,为研究抑制T细胞的凋亡从而增强其抗白血病效应提供实验基础 .
Abstract
Based on the fact that tumor cells expressing FasL can induce apoptosis of T cells expressing Fas and reduce their anti-tumor activity,mouse lymphoma cell line-Yac-1 highly expressing Fas was used as a model.A hammerhead ribozyme targeting position 596 of the Fas mRNA was synthesized and its in vitro transcription was constructed,which was transfected into Yac-1 cells by electroporation.Fas expression on Yac-1 cells was detected by RT-PCR and Western blotting.After being treated with anti-Fas antibody(JO2),Yac-1 cells viability was measured by MTT assay.Cells apoptosis was measured according to FITC labeled annexin-V apoptosis detecting kit.In vitro cleavage activity fo anti-Fas ribozyme was 60% and could decrease Fas expression on Yac-1 cells efficiently.Treated with anti-Fas antibody(JO2),apoptosis rates of cells transfected with ribozyme decreased remarkably and cells viability was enhanced compared with the control.The ribozyme cleaved Fas excellently in vitro and in vivo and made cells get rid of Fas-mediated apoptosis,which is very helpful for inhibiting apoptosis of T cells and enhancing its anti-tumor effects.
关键词
核酶 /
Fas /
Yac-1 /
凋亡 /
肿瘤免疫
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Key words
ribozyme /
Fas /
Yac-1 /
apoptosis /
tumor immunity
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参考文献
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