Zhang Min, Liu Fang, You Yong, He Wei, Zou Ping, Chen Zhi-chao, Liu Zhong-ping, Liu Ling-bo
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430022, China.
Zhonghua Xue Ye Xue Za Zhi. 2004 Dec;25(12):705-8.
To investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in mouse cytotoxic T lymphocyte (CTL) cell line--CTLL-2 cells, and explore a novel approach to enhance the ability of T cells against leukemia in donor lymphocytes infusion (DLI).
A hammerhead ribozyme targeting the Fas mRNA was synthesized and transfected into CTLL-2 cells by electroporation. Fas expression in CTLL-2 cells was detected by using RT-PCR, Western blot and flow cytometry, CTLL-2 cells viability was measured by MTT assay, caspase-3 proteolytic activity by caspase-3 detection kit, and cell apoptosis by flow cytometry. Killing activity of CTLL-2 was detected by LDH releasing assay in vitro.
Expression of Fas mRNA and protein in CTLL-2 cells was reduced to 50% after transfection with anti-Fas ribozyme. Being treated with anti-Fas antibody (JO(2)), compared with control and mock-transfected cells, viability of CTLL-2 cells transfected with anti-Fas ribozyme increased by 1-fold, caspase-3 activity and apoptosis rate of ribozyme-transfected cells decreased to 50% and 37%, respectively, and cell killing activity was enhanced by 2-fold.
Anti-Fas ribozyme can cleave Fas efficiently and inhibit Fas-mediated apoptosis of CTLL-2 cells, resulting in improvement of their viability.
研究抗Fas锤头状核酶对小鼠细胞毒性T淋巴细胞（CTL）细胞系——CTLL-2细胞中Fas表达及Fas介导的凋亡的抑制作用，探索一种在供者淋巴细胞输注（DLI）中增强T细胞抗白血病能力的新方法。
合成靶向Fas mRNA的锤头状核酶，通过电穿孔法转染至CTLL-2细胞。采用RT-PCR、蛋白质印迹法及流式细胞术检测CTLL-2细胞中Fas的表达；采用MTT法检测CTLL-2细胞活力；采用caspase-3检测试剂盒检测caspase-3蛋白水解活性；采用流式细胞术检测细胞凋亡。通过乳酸脱氢酶（LDH）释放法体外检测CTLL-2细胞的杀伤活性。
用抗Fas核酶转染后，CTLL-2细胞中Fas mRNA和蛋白的表达降低至50%。用抗Fas抗体（JO(2)）处理后，与对照细胞和空载体转染细胞相比，抗Fas核酶转染的CTLL-2细胞活力增加1倍，核酶转染细胞的caspase-3活性和凋亡率分别降至50%和37%，细胞杀伤活性增强2倍。
抗Fas核酶可有效切割Fas，抑制CTLL-2细胞Fas介导的凋亡，从而提高其活力。
