LAT介导GPI锚固蛋白CD59对Jurkat细胞的生物学效应研究

发布时间：2019-06-10 20:20

【摘要】：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。
[Abstract]:Objective to investigate the biological effects of GPI anchoring protein CD59 on cell proliferation, activation and apoptosis mediated by LAT in Jurkat cells of T line acute leukemia, and to study the regulation of GPI anchoring membrane protein molecules on cells. Methods LAT-EGFP,neg-EGFP fusion protein lentivirus vector was constructed and transformed into Jurkat cells, and a stable expression strain was established (LAT-EGFP as experimental group and neg-EGFP transgenic empty virus group as control group). After stimulating the cells of the experimental group and the control group with CD59 monoclonal antibody, the proliferation activity of the cells was detected by CCK-8, and the apoptosis was detected by flow cytometry. The localization and expression of LAT and CD59 molecules in cells were observed by laser confocal microscope. Finally, the expression of intracellular signal transduction proteins was detected by immunoblotting. Results after cross-linking stimulation of CD59 monoclonal antibody, the cell proliferation activity increased. In the experimental group, the cells in the experimental group showed obvious apoptosis and even necrosis in the late stage. Immunofluorescence showed that the LAT molecules in the LAT-EGFP group were highlighted in the raft area, which was significantly higher than that in the control group. Western blot showed that after the CD59 antibody was crosslinked, ZAP70,Fyn, was found to be the most prominent ring in the cells of the control group. Western blot showed that the ZAP70,Fyn, antibody was crosslinked. The expression of LCK decreased and the expression of LATEGFP histone was lower than that of the control group. Conclusion GPI anchoring protein CD59 plays a positive role in T cell signal transduction through T cell activated junction protein LAT. Jurkat cells transfected with LAT-EGFP are activated and LAT is located in lipid raft with ring.
【作者单位】：青岛大学医学院免疫学教研室;
【基金】：国家自然科学基金(81273206)
【分类号】：R392.12

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