锤头状核酶抑制CTLL-2细胞凋亡而增强其对Yac-1细胞杀伤活性的实验研究

目的　研究抗fas的锤头状核酶对小鼠细胞毒性T淋巴细胞 (CTL)系CTLL 2细胞fas基因的表达及其介导的细胞凋亡抑制作用 ,探索供者淋巴细胞输注 (DLI)时增强T细胞抗白血病的新途径。方法设计并合成抗fas的锤头状核酶基因 ,将其导入CTLL 2细胞 ,通过RT PCR、Westernblot和流式细胞仪分别检测核酶对CTLL 2细胞fasmRNA和Fas蛋白表达的抑制 ,以MTT法检测CTLL 2细胞与Fas抗体结合后的增殖活性 ,以半胱天冬酶 3(caspase 3)活性检测试剂盒检测细胞caspase 3蛋白酶活性 ,以流式细胞仪检测细胞凋亡 ,以乳酸脱氢酶法检测CTLL 2细胞的体外杀伤活性。结果锤头状核酶基因导入CTLL 2细胞后 ,可使其表面的Fas表达降低达 5 0 % ,细胞经Fas抗体 (JO2 )处理后 ,与空白对照、转染空载体的细胞相比 ,转染核酶的细胞增殖活性增加了 1倍 ,caspase 3活性降低近 5 0 % ,而细胞的凋亡率显著降低 ,只有 37% ,且CTLL 2细胞的体外杀伤活性为对照组的 2倍。结论该核酶具有切割fas基因的良好活性 ,并可抑制Fas介导的CTLL 2细胞凋亡 ,增加该细胞存活率 ,从而增强对Yac 1细胞的杀伤活性。

Experimental study of inhibiting CTLL-2 cell apoptosis and enhancing cytotoxicity to Yac-1 cell by hammerhead ribozyme

OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: Anti-Fas ribozyme can cleave Fas efficiently and inhibit Fas-mediated apoptosis of CTLL-2 cells, resulting in improvement of their viability.