| ObjectiveExplore the mechanism about the autophagy of dendritic cells related to the occurrence and development of asthma and MSCs effects on the autophagy and the immune function of DCs.MethodsA.Detection the autophagy of lung-DCs in asthmatic.Balb/c mice were divided into asthma group and control group.Mice models of asthma group were induced with OVA.Hematoxylin-Eosin(H-E)staining was used to observe the pathological changes in lung tissues of mice;ELISA,Western Blot were respectively used to detect the serum level of OVA-specific Ig E,IL4/IL13 of BALF,the level of autophagy on different lung DCs.B.Explore the mechanism about the autophagy of dendritic cells related to the occurrence and development of asthma.1.Balb/c mice were divided into three groups using the random numbertable:group of asthma(asthma group),group of asthma treated with autophagy inhibitor(asthma+CQ group)and group of control(con group).Mice models of asthma group and asthma+CQ group were induced with OVA,and meanwhile mice of asthma+CQ group were treated with autophagy inhibitor CQ.Hematoxylin-Eosin(H-E)staining was used to observe the pathological changes in lung tissues of mice;ELISA,Western Blot and,flow cytometery were respectively used to detect the serum level of OVA-specific Ig E,the level of autophagy,the surface co-stimulatory molecules and the major histocompatibility complex class II(MHC class ?)on lung DCs.2.Bone marrow-derived DCs were treated with Autophagy inhibitor 3-MA in vitro and detected the surface expression of co-stimulatory molecules and MHC class ?.3.Sorting CD4+ T cells from spleens of OT-?mice,then co-cultured with DCs from mice of different groups(T cell:DCs=1:10),and detect the activation and proliferation of T cells with flow cytometery.C.Explore the mechanism about MSCs effects on the autophagy and the immune function of DCs in asthma.1.Balb/c mice were divided into three groups using the random numbertable:group of asthma(asthma group),group of asthma treated with MSCs(MSC group)and group of control(con group).Mice models of asthma group and asthma+NSC group were induced with OVA,and meanwhile mice of asthma+MSC group were treated with MSC by intravenous injection.Hematoxylin-Eosin(H-E)staining was used to observe the pathological changes in lung tissues of mice;ELISA,flow cytometery were respectively used to detect the serum level of OVA-specific Ig E,IL4/IL13 of BALF,the surface co-stimulatory molecules and the major histocompatibility complex class II(MHC class ?)on lung DCs.2.Bone marrow DCs were divided into control group(CON group),LPS pretreated DCs group(CON-DC group)and Co-cultrued DCs with MSCs(MSC-DC),Western Blot and,flow cytometery were respectively used to detect the the level of autophagy,the surface co-stimulatory molecules and the major histocompatibility complex class II(MHC class ?)on different grounp DCs.3.Sorting CD4+ T cells from spleens of OT-?mice,then co-cultured with different grounp DCs(T cell:DCs=1:10),and detect the activation and proliferation of T cells with flow cytometery.ResultsA.The autophagy in the lung DCs of Asthma was higher1.Asthma serum–Ig E,BALF IL-4 and IL-13 was higher than Normal Control(p<0,05);2.HE staining: Normal Control group normal,asthma worse than Control group.3.The ratio of LC3?/? and LC3?/ACTIN in the lung DCs of Asthma was higher than Normal Control;B.Inhibition of the autophagy of dendritic cells improve asthma condition1.Asthma Cntrol-Ig E was higher than Normal Control-Ig E(p<0.05),CQ group were lower than Aasthma Cntrol-Ig E(p<0,05);.2.HE staining: Normal Control group normal,Aasthma Cntrol group the worst,CQ group better than Aasthma Cntrol group3.The autophagy of lung DCs:the ratio of LC3?/? and LC3?/ACTIN in Asthma Cntrol-lung DCs was higher than Normal Control,CQ group were lower than Aasthma Cntrol;4.The surface co-stimulatory molecules and the major histocompatibility complex class II(MHC class ?)on different grounp lung-DCs: Asthma Control higher than Normal Control(p<0.05),CQ group were lower than Aasthma Cntrol(p<0,05);5.The surface co-stimulatory molecules and the major histocompatibility complex class II(MHC class ?)on different grounp DCs besides 3MA-DCs: OVA Control higher than Normal Control(p<0.05),3MA group were lower than Aasthma Cntrol(p<0,05);6.Number of T proliferation : Asthma Control higher than Normal Control(p<0.05),CQ group were lower than Aasthma Cntrol(p<0,05)7.T activation response : Asthma Control higher than Normal Control(p<0.05),CQ group were lower than Aasthma Cntrol(p<0,05)C.MSCs cut the autophagy of dendritic cells and improve asthma.1.Asthma Control-Ig E was higher than Normal Control-Ig E(p<0.05),MSC group were lower than Aasthma Control-Ig E(p<0,05);.2.HE staining: Normal Control group normal,Aasthma Cntrol group the worst,MSC group better than Aasthma Cntrol group.3.BALF IL-4 and IL-13: Aasthma Cntrol group was higher than Normal Control group(p<0.05),MSC group were lower than Aasthma Cntrol group(p<0,05);4.The autophagy of lung DCs:the ratio of LC3?/? and LC3?/ACTIN in LPS-DCs was higher than Con-DCs,MSC-DCs were lower than LPS-DCs;5.The surface co-stimulatory molecules and the major histocompatibility complex class II(MHC class ?)on different grounp DCs: LPS-DCs was higher than Con-DCs(p<0,05),MSC-DCs were lower than LPS-DCs(p<0,05);6.Number of T proliferation : LPS-DCs was higher than Con-DCs(p<0,05),MSC-DCs were lower than LPS-DCs(p<0,05).Conclusion1.Autophagy inhibitors improve the pathologic conditon of allergic asthma through obstructing autophagy in DCs,down-regulate the surface expression of co-stimulatory molecules and MHC ?on DCs,and further inhibit the proliferation of T cells.2.Bone marrow mesenchymal stem cells(MSCs)can inhibit bone marrow DCs autophagy,reduce the the surface expression of co-stimulatory molecules and MHC ?on DCs,and further inhibit the proliferation of T cells,and improve asthma condition. |
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