抗Fas锤头状核酶阻抑小鼠急性粒-单核白血病细胞免疫逃逸的研究

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Fas mRNA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Western blot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（Annexin V—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。

Depressing the Immune Escape of Acute Myelomonocytic Leukemia via an Anti-Fas Ribozyme

In order to investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in CTLL-2 cells (mouse CTL cell line), and to explore a new way for enhancing the ability of T cells against Leukemia in donor lymphocytes infusion, CTLL-2 cells were transfected with pEGFP-RZ596 and pEGFPC1 (mock-transfected) via electroporation. Fas expression on CTLL-2 cells was detected by RT-PCR and Western blot. The killing effect of CTL against WEHI-3 (mouse acute myelomonocytic leukemia cell line) highly expressing FasL in vitro was detected by MTT assay. The caspase-3 proteolytic activity and the apoptosis rate of CTLL-2 cells were detected by means of BD AproAlert Caspase-3 Colorimetric kit and FITC labeled Annexin-V apoptosis detecting kit respectively. The results showed that the anti-Fas ribozyme could be successfully introduced into mouse CTLL-2 cells; Fas expression on the surface of cells transfected with the ribozyme was obviously decreased, in comparison with control and mock-transfected cells; after cocultured with WEHI-3 cells, the viability of CTLL-2 cells transfeced with the ribozyme was significantly increased, as compared with other two groups; caspase-3 activity and apoptosis rate of the ribozyme-transfeced cells were significantly decreased, the killing effect of CTLL-2 transfected with the ribozyme was stronger than that of other groups. It is concluded that anti-Fas ribozyme can remarkably decrease Fas expression on CTLL-2 cells, so as to avoid Fas-mediated apoptosis by Fas ligand on WEHI-3 cells, and to enhance their killing activity against WEHI-3 cells, as a result, the immune escape of acute myelomonocytic leukemia was depressed.