Zhang Min, Chen Zhi-Chao, Tian Lei, Liu Fang, Liu Ling-Bo, You Yong, Zou Ping
Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, P.R.China.
Ai Zheng. 2005 May;24(5):520-4.
BACKGROUND & OBJECTIVE: Tumor cells can express FasL, and induce apoptosis of activated T cells expressing Fas, which is so called "Fas counterattack". This study was to investigate inhibitory effect of anti-Fas ribozyme on apoptosis of mouse T cells, and explore a new way to enhance antitumor ability of T cells.
A hammerhead ribozyme targeting Fas mRNA was synthesized, its in vitro cleavage reaction was performed. Anti-Fas ribozyme was transfected into mouse spleen T cells by electroperation (ribozyme-transfected group), empty control and mock-transfected groups were set up. Fas expression on T cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. After treatment of anti-Fas antibody (JO(2)), cell apoptosis was measured by flow cytometry, proliferation of T cells was measured by MTT assay. In vitro killing activity of cytotoxic T lymphocytes (CTLs) was detected by lactate dehydrogenase (LDH) releasing assay.
Anti-Fas ribozyme cleaved Fas mRNA efficiently with a rate of 60%. mRNA level of Fas was significantly lower on ribozyme-transfected cells than on control and mock-transfected cells (0.4 vs. 1.1, and 1.0, P < 0.01)u its protein level was according to this result. After treatment of JO(2), cell apoptosis rate was significantly lower in ribozyme-transfected group than in the rest 2 groups (35% vs. 86%, and 87%, P < 0.01), but cell proliferation rate was significantly higher in ribozyme-transfected group than in the rest 2 groups (208% vs. 100%, and 98%, P < 0.01). In vitro killing activity of CTLs was significantly stronger in ribozyme-transfected group than in the rest 2 groups (67% vs. 32%, and 31%, P < 0.01).
Anti-Fas ribozyme can remarkably suppress Fas expression on mouse activated spleen T cells, and protect T cells from Fas-mediated apoptosis, which may enhance antitumor ability of T cells.
肿瘤细胞可表达FasL，并诱导表达Fas的活化T细胞凋亡，即所谓的“Fas反击”。本研究旨在探讨抗Fas核酶对小鼠T细胞凋亡的抑制作用，探索增强T细胞抗肿瘤能力的新途径。
合成靶向Fas mRNA的锤头状核酶，进行体外切割反应。采用电穿孔法将抗Fas核酶转染至小鼠脾T细胞（核酶转染组），设立空载体对照组和模拟转染组。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法检测T细胞上Fas的表达。用抗Fas抗体（JO(2)）处理后，采用流式细胞术检测细胞凋亡，采用MTT法检测T细胞增殖。采用乳酸脱氢酶（LDH）释放法检测细胞毒性T淋巴细胞（CTL）的体外杀伤活性。
抗Fas核酶能有效切割Fas mRNA，切割率为60%。核酶转染细胞的Fas mRNA水平显著低于对照组和模拟转染组（0.4比1.1和1.0，P<0.01），其蛋白质水平也符合此结果。用JO(2)处理后，核酶转染组的细胞凋亡率显著低于其余两组（35%比86%和87%，P<0.01），但核酶转染组的细胞增殖率显著高于其余两组（208%比100%和98%，P<0.01）。核酶转染组CTL的体外杀伤活性显著强于其余两组（67%比32%和31%，P<0.01）。
抗Fas核酶可显著抑制小鼠活化脾T细胞上Fas的表达，保护T细胞免受Fas介导的凋亡，可能增强T细胞的抗肿瘤能力。
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