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目的观察Rab4突变体在树突状细胞(DC)内的定位,研究其对DC内源抗原递呈的影响。方法构建Rab4突变体的慢病毒表达质粒,以DNA-磷酸钙共沉淀法制备慢病毒,病毒感染DC-OVA细胞并使用流式细胞术检测感染效率,激光共聚焦显微镜观察DC-OVA细胞中Rab4突变体的表达与定位,最后用识别MHC I类分子-OVA肽复合物的T细胞杂交瘤B3Z细胞检测DC-OVA细胞内源抗原递呈的变化。结果慢病毒可高效感染DC-OVA细胞,感染后Rab4突变体可在DC-OVA中稳定表达,主要分布在细胞膜周围的囊泡结构中。抗原递呈检测结果显示Rab4对DC内源性抗原递呈具有正相关性影响。结论成功观察到不同Rab4突变体在DC内的表达与分布,初步证实Rab4蛋白可通过其活性的改变参与调节DC的内源性抗原递呈。
Objective To observe the localization of Rab4 mutant in dendritic cells (DCs) and study its effect on the endogenous antigen presentation of DCs. Methods The lentivirus expression plasmid of Rab4 mutant was constructed and the lentivirus was prepared by DNA-calcium phosphate co-precipitation method. The virus was infected into DC-OVA cells and the infection efficiency was detected by flow cytometry. The expression of Rab4 in DC-OVA cells was observed by laser scanning confocal microscopy Mutant expression and localization. Finally, the changes of endogenous antigen presentation in DC-OVA cells were detected by T cell hybridoma B3Z cells that recognize MHC class I-OVA peptide complexes. Results The lentivirus could efficiently infect DC-OVA cells. After infection, Rab4 mutant was stably expressed in DC-OVA and mainly distributed in the vesicle structure around the cell membrane. Antigen presentation showed that Rab4 had a positive correlation with the endogenous antigen presentation of DC. Conclusions The expression and distribution of different Rab4 mutants in DCs were successfully observed. It is preliminarily confirmed that Rab4 protein may be involved in the regulation of the endogenous antigen presentation of DC through the change of its activity.