The Study Of Endosomal Relocation Of Antigens Mediated By Clathrin And Phagocytosis Of Antigens Mediated By AKT

Dendritic cells(dendritic cells,DC)within the antigen of annexation combination in Major histocompatibility complex(Major histocompatibility complexes,MHC)molecules is the premise and foundation of T cell activation and immune response.Antigen uptake by DC includes phagocytosis,macropinocytosis or receptor-mediated endocytosis.Clathrin-mediated endocytosis involves the endocytosis of receptors,such as Fc receptor,transferrin receptor,DEC205(CD205),dc-sign and Mannose receptor(MR).MR is necessary for in vitro DC uptake of soluble antigens such as ovalbumin(OVA)mediated cross-presentation.Early endosome is the key organ chamber after MR ingestion of soluble antigens into cells,and Rab5 is the marker molecule on the early endosome.MR binds to the FcR chain,which is necessary for MR to locate on the cell membrane and for antigens to enter the cell.Laboratory studies have found that nicotine can up-regulate the expression of mannose receptor through the PI3K/AKT pathway,and enhance antigen endocytosis and endosomal translocation.To investigate the mechanism of mannose receptor-mediated cross presentation of antigens into the endosome.In this study,mouse mononuclear macrophage(Raw264.7)and mouse bone marrow derived dendritic cells were used as the cell model,OVA was used as the antigen model,and the activation of cell signaling pathway by antigen load was detected by Western Blot.Secondly,flow cytometry was used to detect the effect of PI3K/AKT inhibitor wortmannin treatment on the phagocytic antigen capacity and localization of Raw264.7 and dendritic cells.Then with clathrin co-immunoprecipitation and immunofluorescence technique to detect the clathrin active control after the clathrin with MR,signal kinase AKT,early the body mark molecular Rab5 and antigen interaction in the situation,found that AKT activity regulating antigen to consume and clathrin involved in mannose receptor mediated antigen to the inner body the role of dislocation.The results showed that:firstly,OVA antigen load can activate PI3K/AKT,Rac1 and Cbl;Secondly,the PI3K/AKT inhibitor wortmannin can affect the entry of antigens into cells and is co-immunoprecipitated with Rab5,an early endosomal marker molecule.Then,the co-immunoprecipitation of clathrin showed that Rab5,OVA and MR interact with clathrin,suggesting that clathrin may be involved in the translocation of MR receptor-mediated OVA into the early endosome.Finally,both AKT signaling pathway inhibitors and AKT siRNA transfection could inhibit OVA translocation into the endosome and regulate the co-localization of clathrin,MR and Rab5,the early endosomal marker protein.It suggests that MR,OVA and clathrin may form complexes and enter the endosome,which are regulated by clathrin.