CAJ | 학술논문

目的探讨乏氧调节人成熟树突状细胞(mDCs)骨桥蛋白(OPN)表达的机制.方法免疫磁珠法分离人外周血CD14+单核细胞,分别在常氧和乏氧条件下经GM-CSF及IL-4体外诱导生成mDCs;ELISA检测培养上清中OPN水平,RT-PCR检测OPN基因水平表达;使用腺苷替代物(NECA)、特异性A2R激动剂(CGS21680)和特异性A2R拮抗剂(SCH58261)探讨A2R在调节人mDCs表达OPN中发挥的作用;利用重组人TGF-β1(rhTGF-β1)及抗TGF-β1单克隆抗体(anti-TGF-β1Mcab)检测TGF-β1在这一过程中发挥的作用.结果乏氧显著增加mDCs对OPN mRNA及OPN蛋白的表达,A2R拮抗剂抑制乏氧对OPN的诱导作用;常氧条件下,腺苷替代物(NECA)和A2R激动剂均能显著上调mDCs对OPN mRNA的表达及OPN蛋白的分泌,A2R拮抗剂可特异性抑制A2R激动剂对OPN的诱导;A2R参与调控免疫调节因子TGF-β1水平,通过rhTGF-β1和阻断抗体证实TGF-β1
목적 탐토핍양조절인성숙수돌상세포(mDCs)골교단백(OPN)표체적궤제.방법 면역자주법분리인외주혈CD14+단핵세포,분별재상양화핍양조건하경GM-CSF급IL-4체외유도생성mDCs;ELISA검측배양상청중OPN수평,RT-PCR검측OPN기인수평표체;사용선감체대물(NECA)、특이성A2R격동제(CGS21680)화특이성A2R길항제(SCH58261)탐토A2R재조절인mDCs표체OPN중발휘적작용;이용중조인TGF-β1(rhTGF-β1)급항TGF-β1단극륭항체(anti-TGF-β1Mcab)검측TGF-β1재저일과정중발휘적작용.결과 핍양현저증가mDCs대OPN mRNA급OPN단백적표체,A2R길항제억제핍양대OPN적유도작용;상양조건하,선감체대물(NECA)화A2R격동제균능현저상조mDCs대OPN mRNA적표체급OPN단백적분비,A2R길항제가특이성억제A2R격동제대OPN적유도;A2R삼여조공면역조절인자TGF-β1수평,통과rhTGF-β1화조단항체증실TGF-β1
Objective To investigate the mechanism of hypoxia regulate osteopontin (OPN) secreting by mature dendritic cells (mDCs). Methods CD14 + cells were enriched using anti-CD14 immunomagnetic beads, for inducing to mDCs, CD14 + cells were cultured with GM-CSF and IL-4 in hypoxia or normoxiain vitro. Concentration of OPN and TGF-β1 in supernatant were detected by sandwich ELISA, OPN mRNA detected by RT-PCR. Approach regulating function of A2 R in expressing of OPN by mDCs by using NECA (surrogate of adenosine), A2R agonist (CGS21680), A2R antagonist (SCH58261) and investigate role of TGF-β1 in this process by using rhTGF-β1 and anti-TGF-β1 Ab. Results Hypoxia inreased the level of OPN and OPN mRNA in mDCs, and this effect could be reversed by A2 R antagonist. Under normoxia,both NECA and A2R agonist (CGS21680) could upregulate the level of OPN and OPN mRNA in mDCs significantly, but this positive effect could be reversed by A2 R antagonist. A2 R played a role in regulating TGF-β1, and confirmed TGF-β1 involved in regulation of OPN by using rhTGF-β1 and anti-TGF-β1 Ab. Conclusion High adenosine induce the generation of TGF-β1 through the A2R on mDCs, and then TGF-β1 raise the OPN secreting by mDCs.