论文部分内容阅读
为了研究抗Fas锤头状核酶对T细胞Fas表达及其凋亡的影响和探讨增强供者淋巴细胞输注时移植物抗白血病(GVL)效应的新策略,构建可有效切割FasmRNA的锤头状核酶真核质粒,用电穿孔法将其导入小鼠CTL细胞株CTLL-2之后,借助RT-PCR和Westernblot检测其Fas的表达,同时检测转染前后其胱冬酶-3(Caspase-3)活性和凋亡(AnnexinV-FITC法)的改变,并用MTT法检测空白对照组、空载体转染组及pU6-RZ596转染组CTLL-2细胞的增殖情况和体外杀伤小鼠急性粒-单核白血病细胞(WEHI-3)的活性。结果表明:构建的U6嵌合型锤头状核酶RZ596在细胞内能有效切割Fas,明显降低小鼠活化CTLL-2的Fas水平,与高表达Fas配体的WEHI-3孵育后,其存活率和体外杀伤WEHI-3活性明显高于对照组。结论:抗Fas核酶能显著降低小鼠活化CTLL-2的Fas表达,使其免于WEHI-3的膜Fas配体经Fas途径所致的凋亡,并提高CTL对小鼠急性粒-单核白血病细胞的杀伤力,从而阻抑小鼠急性粒-单核白血病细胞的免疫逃逸。
In order to study the effect of anti-Fas hammerhead ribozyme on Fas expression and apoptosis of T cells and to explore a new strategy to enhance graft versus leukemia (GVL) effects in donor lymphocyte transfusion, a hammerhead that effectively cleaves Fas mRNA Like eukaryotic plasmids were transfected into mouse CTL cell line CTLL-2 by electroporation. The expression of Fas was detected by RT-PCR and Western blotting. Caspase- 3) activity and apoptosis (AnnexinV-FITC method) changes, and MTT assay was used to detect the proliferation of CTLL-2 cells in blank control group, empty vector transfected group and pU6-RZ596 transfected group and in vitro killed mice acute granulocyte- Monocytic leukemia cells (WEHI-3) activity. The results showed that the constructed U6 chimeric hammerhead ribozyme RZ596 effectively cleaves Fas in the cells and significantly reduces the Fas level in mouse CTLL-2-activated cells. After incubation with WEHI-3, which is highly expressed Fas ligand, its survival WEHI-3 activity was significantly higher than that of control group in vitro and in vitro. CONCLUSION: Anti-Fas ribozyme can significantly decrease the expression of Fas in CTLL-2 cells in mice and prevent its apoptosis induced by Fas pathway of Fas ligand in WEHI-3 cells. Nuclear leukemia cell lethality, thereby inhibiting the mouse acute granulocyte-monocytic leukemia cells immune escape.