CAJ | 학술논문

目的研究全反式视磺酸(atRA)对树突状细胞(DC)抗原提呈作用的影响是作用在骨髓细胞向DC的分化过程还是作用在已分化形成的DC,以及NF-κB信号通路是否参与到此调节过程.方法以MOG35-55免疫C57BL/6J小鼠,给予或不予atRA干预.分离小鼠脾脏DC及CD4细胞进行交互培养,同时给予IL-12或IL-23干预CD4细胞的分化.测定CD4细胞向Th1及Th17细胞的分化情况及相应细胞因子的产生能力.分离小鼠骨髓细胞(BMC),IL-4及GM-CSF诱导其向DC分化,并在不同时间点给予RA干预.比较DC表面分子CD11c、MHCⅡ的表达情况及其提呈抗原对效应细胞的激活作用；Western blot检测DC中NF-κB p65的磷酸化及p65向胞核转位的情况.最终比较RA对DC的影响是否可以被选择性RA受体(RAR)拮抗剂所拮抗.结果 RA体内干预显著降低脾脏DC的抗原提呈作用；在体外RA则可抑制BMC向DC分化过程,降低CD11c及MHCⅡ分子在细胞表面的含量,并抑制DC的抗原提呈功能；但是对已分化形成的DC却没有类似效应.RA可显著抑制DC中NF-κB p65的磷酸化及胞核中NF-κB p65的含量,并伴随着DC抗原提呈功能的减弱；其作用可被RARβγ拮抗剂所拮抗.结论 RA可抑制DC的提呈功能,其作用环节可能在于DC的分化过程并与NF-κB信号通路密切相关.
목적 연구전반식시광산(atRA)대수돌상세포(DC)항원제정작용적영향시작용재골수세포향DC적분화과정환시작용재이분화형성적DC,이급NF-κB신호통로시부삼여도차조절과정.방법 이MOG35-55면역C57BL/6J소서,급여혹불여atRA간예.분리소서비장DC급CD4세포진행교호배양,동시급여IL-12혹IL-23간예CD4세포적분화.측정CD4세포향Th1급Th17세포적분화정황급상응세포인자적산생능력.분리소서골수세포(BMC),IL-4급GM-CSF유도기향DC분화,병재불동시간점급여RA간예.비교DC표면분자CD11c、MHCⅡ적표체정황급기제정항원대효응세포적격활작용；Western blot검측DC중NF-κB p65적린산화급p65향포핵전위적정황.최종비교RA대DC적영향시부가이피선택성RA수체(RAR)길항제소길항.결과 RA체내간예현저강저비장DC적항원제정작용；재체외RA칙가억제BMC향DC분화과정,강저CD11c급MHCⅡ분자재세포표면적함량,병억제DC적항원제정공능；단시대이분화형성적DC각몰유유사효응.RA가현저억제DC중NF-κB p65적린산화급포핵중NF-κB p65적함량,병반수착DC항원제정공능적감약；기작용가피RARβγ길항제소길항.결론 RA가억제DC적제정공능,기작용배절가능재우DC적분화과정병여NF-κB신호통로밀절상관.
Objective To clarify whether the regulatory effect of all-trans-retinoic acid (atRA) on the antigen presentation function of dendritic cell(DC) is tightly associated with NF-κB signaling pathway.Also to clarify atRA mainly affected differentiated DC or influence the differentiation procedure from bone marrow cell to DC.Methods MOG35-55 immunized C57BL/6J mice received administration of atRA or not,splenic DC and CD4 cells isolated from immunized mice were subjected to in vitro cross-culture,treated with IL-12 and IL-23 respectively.Th1 and Th17 polarization of stimulated CD4 cells were determined by intracellular staining and FACS analysis,while the production of their corresponding cytokines,IFN-γ and IL-17,were measured by ELISA.Bone marrow cells were isolated from the femurs of the na(i)ve mice,treated with IL-4 and GM-CSF with or without synergistic RA treatment.MHC Ⅱ and CD11c molecules on the DC were assayed by immune staining and FACS analysis,their antigen presentation functions were decided by the proliferation and cytokine production of the Th1 effecter cells stimulated by antigen pulsed DC.To investigate the status of the NF-κB signaling pathway,the amount of phospho-Ser536 NF-κB p65 in the whole DC lysate and total NF-κB p65 in the nucleus were detected by Western blot.Finally,selective RA receptor antagonists were studied to figure out which receptor was majorly involved in the regulatory effect of RA on DC.Results Splenic DC from RA treated mice showed significantly decreased function of presenting antigen to stimulate CD4 cell polarization and cytokine production.Compared with untreated control,RA in vitro treated DC showed decreased expression of MHC Ⅱ and CD11c on its cell surface,which was accompanied with depressed function of stimulating Th1 cell proliferation and IFN-γ production.Further study revealed that RA mainly affect the differentiation procedure from BMC to DC,however,has no significant effect on differentiated DC as the aspects of MHC Ⅱ,CD11c expression,responder cell proliferation and cytokine production were evaluated.Decreased amount of phosphor-Ser536 NF-κB p65 in the whole cell lysate and total NF-κB p65 in the nucleus was investigated in RA treated DC,and decreased antigen presentation function of RA treated DC always came together with diminished activation of NF-κB signaling pathway.Finally it was demonstrated that RARβγ antagonist,but not RARα antagonist,could entirely block the RA effect on DC.Conclusion Retinoic acid inhibit the differentiation of DC as well as decrease its antigen presentation function,which might be resulted from the inactivation of NF-κB p65 signaling pathway and mediated by RARβγ.