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目的探讨磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/AKT)信号通路对负性共刺激分子B7-H4细胞定位的调控作用。方法体外培养稳定转染B7-H4/HEK293细胞,采用PI3K/AKT特异性抑制剂LY294002和/或出核转运抑制剂来普霉素B(LMB)处理细胞之后,免疫荧光技术结合激光共聚焦显微镜检测B7-H4蛋白在B7-H4/HEK293细胞中亚细胞定位的变化;Western blot法检测B7-H4蛋白在细胞膜、细胞质和细胞核表达水平的变化。结果激光共聚焦显微镜观察结果显示,PI3K抑制剂LY294002作用于B7-H4稳定转染的B7-H4/HEK293细胞后,与空白对照组相比,B7-H4发生核转移,加入出核转运抑制剂LMB后,核转移的程度显著增加;Western blot法结果显示,LY294002抑制PI3K/AKT信号通路活性24 h后,B7-H4在细胞膜和细胞质中的定位均显著降低(P<0.05),而细胞核中B7-H4的定位显著增加(P<0.05)。结论 PI3K/AKT信号通路可抑制B7-H4的核转移。
Objective To investigate the regulatory effect of phosphatidylinositol 3-kinase / protein kinase B (PI3K / AKT) signaling on the location of B7-H4 negative costimulatory molecule. Methods After transfection of B7-H4 / HEK293 cells in vitro, the cells were treated with PI3K / AKT specific inhibitor LY294002 and / or nucleotransduction inhibitor leptomycin B (LMB). The immunofluorescence combined with laser confocal microscopy The changes of subcellular localization of B7-H4 protein in B7-H4 / HEK293 cells were detected by Western blot. The changes of B7-H4 protein expression in the cell membrane, cytoplasm and nucleus were detected by Western blot. Results Confocal laser scanning microscopy showed that B7-H4 nuclear translocation was induced by PI3K inhibitor LY294002 in B7-H4 / HEK293 cells stably transfected with B7-H4, and nuclear transport inhibitors LMB, the degree of nuclear metastasis was significantly increased. The results of Western blot showed that the localization of B7-H4 in the cell membrane and cytoplasm was significantly decreased after LY294002 inhibited PI3K / AKT signaling pathway for 24 h (P <0.05) The localization of B7-H4 was significantly increased (P <0.05). Conclusion The PI3K / AKT signaling pathway can inhibit the nuclear translocation of B7-H4.