抗Fas锤头状核酶增强小鼠T细胞杀伤粒-单核白血病细胞的研究

Enhancement of Killing Effects of Murine T Lymphocytes against Myelomonocytic Leukemia Cells by an Anti-Fas Ribozyme

目的研究抗Fas锤头状核酶对T细胞Fas表达及其凋亡的影响,探讨增强供者淋巴细胞输注时移植物抗白血病(graft versus leukemia,GVL)效应的新策略。方法构建可有效切割Fas mRNA的锤头状核酶真核质粒,电穿孔法将其导入小鼠细胞毒T淋巴细胞(cytotoxic T lymphocyte,CTL)细胞株CTLL-2中,借助RT-PCR和Western blot检测细胞Fas的表达,同时检测转染前后细胞Caspase-3活性的改变和细胞凋亡(AnnexinⅤ-FITC法)。MTT法检测空白对照组、转染空载体组及转染pU6-RZ596组CTLL-2细胞的增殖情况和体外杀伤粒-单核白血病细胞(WEHI-3)的活性。结果构建的U6嵌合型锤头状核酶RZ596在细胞内能有效切割Fas,RT-PCR电泳检测其与β-actin条带的灰度比值:CTLL-2细胞空白对照组为(1.06±0.12),转染空载体组为(0.98±0.15),转染pU6-RZ596组为(0.43±0.11)(n=3);Fas蛋白的Western blot检测显示:以空白对照组灰度值为1,转染空载体组和转染pU6-RZ596组电泳条带的灰度比值分别为(0.98±0.13)和(0.45±0.08)(n=3),提示抗Fas锤头状核酶可明显降低小鼠活化CTLL-2的Fas水平。与高表达Fas配体(FasL)的WEHI-3细胞共孵育,CTLL-2细胞的存活率和体外杀伤活性明显增加,空白对照组、转染空载体组和转染pU6-RZ596组的细胞凋亡率分别为88%、84%和37%,对WEHI-3细胞杀伤活性分别为32%、31%和67%。结论抗Fas锤头状核酶能显著降低小鼠活化CTLL-2细胞的Fas表达,使其免于Fas途径的凋亡。

Objective To investigate the inhibitory effects of anti-Fas hammerhead ribozyme on Fas expression and Fas-meidated apoptosis of CTLL-2 cells,and explore a new strategy to enhance the ability of T cells against leukemia in donor lymphocytes infusion.Methods CTLL-2 cells were transfected with pEGFP-RZ596 and pEGFPC1 via electroporation.Blank control group（group A）,vector transfection group（group B） and anti-Fas hammerhead ribozyme transfection group（group C） were set up.The Fas expression in T cells was detected by RT-PCR and Western blot.After being co-cultured with WEHI-3 highly expressing FasL,the viability of T cells,caspase-3 proteolytic activity,apoptosis and killing role of CTL in vitro were detected.Results Ribozymes could be successfully introduced into murine T cells and effectively incised Fas in them.Compared with β-actin,gray scale of Fas mRNA in group A,group B and group C was（1.06±0.12）,（0.98±0.15） and（0.43±0.11） respectively（n=3）.Western blot revealed that the gray scale of Fas protein in T cells of groups A,B and C was 1,（0.98±0.13） and（0.45±0.08） respectively（n=3）,suggesting that anti-Fas hammerhead ribozyme could significantly reduce the Fas levels in the murine activated CTLL-2.After co-culture with WEHI-3 cells,the viability of CTLL-2 cells in group C was significantly increased as compared with groups A and B with the apoptosis index being 88%,84% and 37%,and the killing activity of activated CTLL-2 cells to WEHI-3 being 32%,31% and 67% respectively in groups A,B and C.Conclusion Anti-Fas ribozyme can remarkably decrease the Fas expression of the murine activated CTLL-2 cells and make them escape the Fas-mediated apoptosis.