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目的探讨在T系急性白血病的Jurkat细胞中,GPI类锚固蛋白CD59通过LAT介导的细胞增殖、活化、凋亡等相关的生物学效应,研究GPI锚固类膜蛋白分子对细胞调控的作用方式。方法分别构建LAT-EGFP、neg-EGFP融合蛋白慢病毒载体,转染Jurkat细胞,建立稳定表达株(LAT-EGFP作为实验组,neg-EGFP转染空病毒组作为对照组)。利用CD59单克隆抗体刺激实验组及对照组细胞后,通过CCK-8检测细胞的增殖活性,利用流式细胞术检测细胞凋亡状况,并通过激光共聚焦显微镜观察LAT与CD59分子在细胞中的定位表达。最后利用免疫印迹技术检测细胞内相关信号转导蛋白的表达情况。结果 CD59单抗交联刺激后,细胞增殖活性增加。实验组细胞出现明显晚期凋亡甚至坏死现象。而免疫荧光显示LAT-EGFP组细胞中LAT分子高亮聚集于脂筏区,相较于对照组细胞呈现明显的戒指环状。Western blot显示,CD59抗体交联后,ZAP70、Fyn、LCK的表达均下降且LATEGFP组蛋白表达量低于对照组。结论 GPI锚固蛋白CD59通过T细胞活化连接蛋白LAT对T细胞信号转导发挥正向调控作用,转染LAT-EGFP的Jurkat细胞处于活化状态,LAT呈戒指环状定位于脂筏。
Objective To investigate the biological effects of GPI anchored protein CD59 on cell proliferation, activation and apoptosis in T-cell acute leukemia Jurkat cells. Methods The lentiviral vector containing LAT-EGFP and neg-EGFP fusion protein was constructed and transfected into Jurkat cells. The stable expression strain (LAT-EGFP as experimental group, neg-EGFP transfected empty virus group as control group) was established. The cells were stimulated with CD59 monoclonal antibody to detect the proliferation of cells in the experimental group and the control group by CCK-8. The apoptosis of cells was detected by flow cytometry. The expression of LAT and CD59 in the cells was observed by laser scanning confocal microscopy Positioning expression. Finally, immunoblotting was used to detect the expression of intracellular related signal transducers. Results CD59 monoclonal antibody cross-linked stimulation, cell proliferation activity increased. Experimental group of cells showed significant late apoptosis or necrosis. Immunofluorescence showed LAT-EGFP cells in the LAT molecules highlight the lipid raft area, compared with the control group cells showed a ring ring. Western blot showed that the expression of ZAP70, Fyn and LCK were decreased and the expression of LATEGFP protein was lower than the control group after the cross-linking of CD59 antibody. Conclusion The GPI anchoring protein CD59 plays a positive role in T-cell signal transduction via T cell activation of LAT. Jurkat cells transfected with LAT-EGFP are activated, and LAT is located in the ram of the ring.