摘要目的 探讨囊泡运输相关蛋白VAP-33在小鼠树突状细胞肉瘤细胞株DCS细胞中的表达及其功能.方法 用1%TritonX-114提取DCS细胞膜蛋白,用已制备的DCS细胞多克隆抗体(pAb)及蛋白A+G琼脂糖进行免疫沉淀,所得样品利用质谱技术进行分析,检测到VAP-33蛋白在DCS细胞中有表达,继而DCS细胞经不同量抗原(150、850、1500μl)刺激24、48、72 h后观察细胞形态及吞噬能力的变化,同时通过间接免疫荧光、共聚焦显微镜、Western blot等方法检测了VAP-33的分布及表达变化.0.5 mol/L胰岛素刺激DCS细胞20 min后,采用Western blot检测DCS细胞全蛋白、胞质蛋白、膜蛋白中VAP-33、葡萄糖转运蛋白4(GLUT-4)的表达变化,共聚焦方法观察胰岛素刺激前后VAP-33和GLUT-4在DCS细胞中的表达及定位变化.实验中均以常规培养的DCS细胞作为对照.结果 VAP-33蛋白主要表达在DCS细胞膜和胞质中;在外来抗原刺激下,随抗原量的增加及作用时间的延长DCS细胞趋向成熟树突状细胞,VAP-33表达量降低,对辣根过氧化物酶的吞噬能力增强.胰岛素刺激后,VAP-33与GLUT-4有共定位.结论 VAP-33在树突状细胞来源的肿瘤细胞中表达,与树突状细胞的抗原加工有关,在葡萄糖的转运中起一定作用.

abstractsObjective To elucidate the expression and function of VAP-33 gene in dendritic cell sarcoma (DCS) cell line. Methods The expression of VAP-33 in DCS ceils was investigated by mass spectrum with immunoprecipitation membrane protein. DCS cells were treated with antigens in different dosages (150, 850, and 1500 μl) for 24, 48 and 72 h respectively. Cell morphology and phngocytosis activity of DCS cells were measured. Indirect immunofluorescence, confocal microscopy and Western blotting were used to study the distribution and expression changes of VAP-33. Moreover, DCS cells were treated with 0.5 mol/L insulin for 20 min first and followed by Western blotting to detect changes of VAP-33 and glucose transfer protein 4 (GLUT-4) in the total cellular protein, cytoplasmic protein and membrane protein.Confocal microscopy was used to document the expression and distribution changes of VAP-33 and GLUT-4 in DCS cells. Results VAP-33 expression was obtained at the cell membrane and in the cytoplasm of DCS cells. Upon antigen stimulation, DCS cells showed more active phagocytosis and morphologically became more elongated with branched protrusions. The expression of VAP-33 was decreased by the antigen stimulation. Upon the insulin stimulation, the expression of VAP-33 and GLUT-4 were increased and co-localized. Conclusions VAP-33 expression in DCS originated from the dentritic cells (DC) seemed relating to the vesicle transportation during antigen processing in DC. Additionally, VAP-33 and GLUT-4 also take part in the glucose transportation in the cells.