CAJ | 학술논문

本研究旨在探讨人脐静脉内皮细胞(HUVEC)对树突状细胞(DC)发育的影响.首先,利用酶消化法从人脐带分离获得HUVEC,从细胞形态、表型和功能进行鉴定;进一步将获得的HUVEC结合细胞因子配伍与CD14+单核细胞共培养,检测HUVEC对CD14+细胞向DC分化的影响;流式细胞术检测分化细胞的表型,混合淋巴细胞反应检测分化细胞的免疫学功能;采用中和抗体实验和Western blot检测对细胞增殖和分化起重要调控作用的IL-6和VEGF以及ERK和p38MAPK通路的改变.结果表明,从脐静脉分离获得的细胞呈长梭形形态,细胞表型为vWF+ CD31+ CD73+CD45-HLA-DR-CD86-CD34low,Dil-Ac-LDL吸收实验为阳性,且细胞可诱导形成血管样结构,提示分离获得了HUVEC;进一步将HUVEC与CD14+单核细胞共培养,流式细胞仪检测结果表明HUVEC可抑制CD14+单核细胞向DC的分化,诱导获得的细胞CD1a表达显著降低,混合淋巴细胞检测结果显示,与HUVEC共培养获得的DC刺激T细胞增殖作用显著降低,且具有剂量依赖性;中和抗体实验分析其可能的作用机制表明,IL-6和VEGF在CD14+单核细胞向DC分化过程中具有重要调控作用,Western blot检测结果说明其主要通过ERK和p38通路完成调控作用.结论:人内皮细胞参与DC的发育,且起到抑制作用.
본연구지재탐토인제정맥내피세포(HUVEC)대수돌상세포(DC)발육적영향.수선,이용매소화법종인제대분리획득HUVEC,종세포형태、표형화공능진행감정;진일보장획득적HUVEC결합세포인자배오여CD14+단핵세포공배양,검측HUVEC대CD14+세포향DC분화적영향;류식세포술검측분화세포적표형,혼합림파세포반응검측분화세포적면역학공능;채용중화항체실험화Western blot검측대세포증식화분화기중요조공작용적IL-6화VEGF이급ERK화p38MAPK통로적개변.결과표명,종제정맥분리획득적세포정장사형형태,세포표형위vWF+ CD31+ CD73+CD45-HLA-DR-CD86-CD34low,Dil-Ac-LDL흡수실험위양성,차세포가유도형성혈관양결구,제시분리획득료HUVEC;진일보장HUVEC여CD14+단핵세포공배양,류식세포의검측결과표명HUVEC가억제CD14+단핵세포향DC적분화,유도획득적세포CD1a표체현저강저,혼합림파세포검측결과현시,여HUVEC공배양획득적DC자격T세포증식작용현저강저,차구유제량의뢰성;중화항체실험분석기가능적작용궤제표명,IL-6화VEGF재CD14+단핵세포향DC분화과정중구유중요조공작용,Western blot검측결과설명기주요통과ERK화p38통로완성조공작용.결론:인내피세포삼여DC적발육,차기도억제작용.
This study was aimed to investigate the effect of human umbilical vein endothelial cells(HUVEC)on dendritic cell(DC)development.First,HUVEC were isolated from human umbilical cord by collagenase digestion,and then the morphology,immunophenotypes and functions were identified.Furthermore,the HUVEC were cocultured with CD14+ monocytes under the cytokine condition for detecting the influence of HUVEC on differentiation of CD14+ cells to DC.The phenotype of dendritic cells derived from CD14+ cells was analyzed by flow cytometry,the immunoregulatory function of DC was tested by mixed lymphocyte reaction(MLR).The change of IL-6 and VEGF as well as EPK and p38 signal pathway were analyzed by neutral antibody experiment and Western blot.The results showed that HUVEC isolated from human umbilical cord were characterized by spindle-shaped morphology,homogenous immunophenotypes (vWF+ CD31+ CD73+ CD45-HLA-DR-CD86-CD34(low),Dil-Ac-LDL incorporation ability and forming capillary-like structures.Following stimulation with granulocyte-macrophage colony-stimulating factor(GM-CSF)plus interleukin-4 (IL-4),HUVEC cocultures could inhibit the initial differentiation of CD14+ monocyte to DC.Interestingly,IL-6 and VEGF enhanced the suppression effect of HUVEC on generation of DC via activation of the ERK or p38 mitogen activated protein kinase pathway.It is concluded that HUVEC are involved in DC development and can suppress the differentiation of monocyte to DC.