CAJ | 학술논문

目的研究IL-4诱导THP-1细胞表达DC-SIGN的信号调节通路,探索DC -SIGN表达的信号调控网络.方法以佛波脂(PMA)刺激THP-1细胞24h后加入IL-4作用48 h诱导DC-SIGN的表达,并设ERK阻断剂、NF-κB阻断剂、JAK-STAT阻断剂和MAPK阻断剂处理组.用RT-PCR检测DC-SIGN的mRNA表达,Western blot检测胞质内DC-SIGN蛋白的表达,流式细胞术检测细胞表面DC-SIGN的表达.另外,提取IL-4诱导0、10、20、30、60和120 min的THP-1细胞胞质和胞核蛋白,Western blot检测不同信号通路的信号蛋白及其磷酸化蛋白的变化.结果 IL-4可以大幅提高DC-SIGN在THP-1细胞上的表达,包括mRNA和胞膜蛋白水平.在mRNA、胞质和细胞表面蛋白表达3个水平上,ERK通路阻断剂阻断效果最好,几乎完全阻断了IL-4的诱导效果,JAK-STAT和NF-κB通路阻断剂具有部分阻断效果,而p38通路阻断剂无阻断效果.信号蛋白检测结果显示,IL-4诱导0～120 min内,胞质磷酸化ERK1/2、磷酸化STAT6以及NF-κBp65、NF-κBp50、磷酸化IKB和磷酸化AKT随时间推移浓度逐渐升高,而p38MAPK及其磷酸化蛋白浓度无明显改变.胞核内胞质磷酸化ERK1/2、磷酸化STAT6以及NF-κBp65和NF-κBp50随时间推移浓度逐渐升高.结论 ERK、JAK-STAT和NF-κB通路参与了DC-SIGN启动子的活化,其中以ERK通路为主.
목적 연구IL-4유도THP-1세포표체DC-SIGN적신호조절통로,탐색DC -SIGN표체적신호조공망락.방법 이불파지(PMA)자격THP-1세포24h후가입IL-4작용48 h유도DC-SIGN적표체,병설ERK조단제、NF-κB조단제、JAK-STAT조단제화MAPK조단제처리조.용RT-PCR검측DC-SIGN적mRNA표체,Western blot검측포질내DC-SIGN단백적표체,류식세포술검측세포표면DC-SIGN적표체.령외,제취IL-4유도0、10、20、30、60화120 min적THP-1세포포질화포핵단백,Western blot검측불동신호통로적신호단백급기린산화단백적변화.결과 IL-4가이대폭제고DC-SIGN재THP-1세포상적표체,포괄mRNA화포막단백수평.재mRNA、포질화세포표면단백표체3개수평상,ERK통로조단제조단효과최호,궤호완전조단료IL-4적유도효과,JAK-STAT화NF-κB통로조단제구유부분조단효과,이p38통로조단제무조단효과.신호단백검측결과현시,IL-4유도0～120 min내,포질린산화ERK1/2、린산화STAT6이급NF-κBp65、NF-κBp50、린산화IKB화린산화AKT수시간추이농도축점승고,이p38MAPK급기린산화단백농도무명현개변.포핵내포질린산화ERK1/2、린산화STAT6이급NF-κBp65화NF-κBp50수시간추이농도축점승고.결론 ERK、JAK-STAT화NF-κB통로삼여료DC-SIGN계동자적활화,기중이ERK통로위주.
Objective To detect the signal pathways through which IL-4 regulates expression of DC-SIGN in THP-1 cells.Methods We used phorbol 12-myristate 13-acetate(PMA) differentiated THP-1 cells as the in vitro model of monocyte/macrophage cells to study the signal pathways involved in IL-4 regulated expression of DC-SIGN.DC-SIGN mRNA expression was detected by RT-PCR.Cytoplasmic DC-SIGN protein was tested by Western blot.Flow cytometry was used to detect cell surface expression of DC-SIGN.Cytoplasm and nuclear protein of PMA stimulated THP-1 cells induced by IL-4 for 0,10,20,30,60 and 120 min was extracted and detected by Western blot for signal pathway signaling protein and phosphoprotein.Results We found that a high expression of DC-SIGN could be induced by IL-4 at the levels of mRNA and cell surface protein.Up-regulated expression of DC-SIGN was almost completely blocked by the specific inhibitor of ERK pathway,and partly reduced by the specific inhibitors of JAK-STAT and NF-κB pathways.The activation of the three signaling pathways was directly confirmed by testing the phosphorylation of protein kinase within the cytoplasm and nucleus over time.Conclusion Multiple signaling pathways are involved in IL-4 induced high expression of DC-SIGN on THP-1 cells,in which ERK pathway is the main signal pathway.