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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。
Objective To investigate the anti-fas hammerhead ribozyme on mouse cytotoxic T lymphocyte (CTL) line CTLL 2 cells fas gene expression and apoptosis-mediated inhibition, to explore donor lymphocyte infusion (DLI) When enhanced T cell anti-leukemia new way. Methods The anti-fas hammerhead ribozyme gene was designed and synthesized and introduced into CTLL 2 cells. The expression of fas mRNA and Fas protein in CTLL 2 cells was detected by RT-PCR, Western blot and flow cytometry respectively. MTT assay The proliferation activity of CTLL-2 cells after binding with Fas antibody was detected. The activity of caspase 3 protein was detected by caspase 3 activity assay kit. The apoptosis of CTLL-2 cells was detected by flow cytometry. The activity of lactate dehydrogenase Assay of CTLL 2 cells in vitro killing activity. Results The hammerhead ribozyme gene was transfected into CTLL 2 cells, and the expression of Fas on the surface of CTLL 2 cells was decreased by 50%. After the cells were treated with Fas antibody (JO 2) The activity of ribozyme increased by 1-fold and the activity of caspase-3 decreased by nearly 50%, while the apoptosis rate of the cells decreased significantly, only 37%. The cytotoxic activity of CTLL-2 cells in vitro was twice that of the control group. Conclusion The ribozymes have the good activity of cutting fas gene and inhibit the Fas-mediated apoptosis of CTLL 2 cells, increase the cell survival rate and enhance the killing activity of Yac 1 cells.