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目的:探讨人类白细胞抗原B(HLA-B)在喉癌Hep2细胞凋亡调控中的作用机制。方法:免疫共沉淀结合蛋白质印迹技术鉴定HLA-B与S100结合蛋白A8(S100A8)、S100A8与S100A9及HLA-B与S100A9在体外发生相互作用的情况,免疫细胞化学对HLA-B、S100A8和S100A9在Hep2细胞中的表达进行定位,同时验证三者之间可能的关系;进一步应用RNA干涉技术,通过Real-ti mePCR及蛋白质印迹评估相互作用基因的表达情况。结果:Hep2细胞中HLA-B和S100A8蛋白在体外存在相互作用,而S100A8和S100A9并没有以异源二聚体的形式存在。免疫细胞化学结果显示,Hep2细胞中S100A8蛋白主要表达于细胞质中,HLA-B主要定位于细胞质和细胞膜上,而S100A9蛋白主要分布于细胞核中。另外,与转染PBS及无义小分子干扰(si RNA)组相比,RNA干涉HLA-B基因能明显下调S100A8 mRNA和蛋白的表达水平,F值分别为553.024、603.582,P值均为0.000;而RNA干涉S100A8基因对HLA-B mRNA和蛋白表达变化无显著影响,F值分别为1.266、1.087,P值分别为0.348、0.395。结论:HLA-B与S100A8相互作用,部分通过调节S100A8/Bcl-2的表达而激发喉癌Hep2细胞凋亡发生。
Objective: To explore the mechanism of human leukocyte antigen B (HLA-B) in regulating the apoptosis of Hep2 cells. Methods: The interaction of HLA-B with S100 binding protein A8 (S100A8), S100A8 and S100A9 and HLA-B and S100A9 in vitro were detected by co-immunoprecipitation and Western blotting. Immunocytochemistry was used to detect the interaction of HLA-B, S100A8 and S100A9 The expression of interacting genes in Hep2 cells was determined and the possible relationships among the three were verified. Real-ti mePCR and Western blot were used to evaluate the expression of interacting genes by RNA interference. Results: There was interaction of HLA-B and S100A8 proteins in Hep2 cells in vitro, while S100A8 and S100A9 did not exist as heterodimers. Immunocytochemistry showed that S100A8 protein mainly expressed in the cytoplasm of Hep2 cells, HLA-B mainly located in the cytoplasm and cell membrane, while S100A9 protein mainly distributed in the nucleus. In addition, RNA interference of HLA-B gene significantly down-regulated the expression of S100A8 mRNA and protein compared with transfection of PBS and nonsense siRNA (si RNA) group, with F values of 553.024 and 603.582, P values of 0.000 ; While S100A8 RNA interference had no significant effect on the expression of HLA-B mRNA and protein, with F values of 1.266 and 1.087, respectively, with P values of 0.348 and 0.355, respectively. Conclusion: HLA-B interacts with S100A8 and partly induces apoptosis of Hep2 laryngeal carcinoma by regulating the expression of S100A8 / Bcl-2.