CAJ | 학술논문

目的研究15 Hz l mT的正弦波电磁场对大鼠骨髓间充质干细胞(MSCs)的细胞外信号调节激酶(ERK)和丝裂原激活的蛋白激酶(MAPK) p38的激活规律及相互作用特点,探讨ERK和p38 MAPK信号通路在电磁场促成骨效应中的作用.方法选取第3代体外分离培养的大鼠骨髓间充质干细胞,分为对照组、曝磁组、曝磁+ PD98059组和曝磁+SB202190组四个大组.曝磁组细胞置于带有电磁发生器的培养箱中培养,曝磁+ PD98059组和曝磁+SB202190组细胞分别加入ERK信号通路阻断剂PD98059和p38 MAPK信号通路阻断剂SB202190后再置入带有电磁发生器的培养箱中培养,对照组细胞正常培养.用免疫印迹(Western blot)法检测ERK和p38 MAPK的蛋白表达及磷酸化水平变化.按照细胞碱性磷酸酶(ALP)试剂盒说明书操作步骤对各组细胞ALP活性进行检测,其活性变化可间接反映细胞的分化成骨活性;用噻唑蓝比色法检测各组细胞的增殖活性变化.结果 ①电磁场作用下,骨髓间充质干细胞内p38 MAPK信号通路可被快速激活,曝磁15 min后出现p38磷酸化水平升高(P＜0.05);加用SB202190后,再行电磁场刺激,细胞内p38磷酸化水平仍然维持在较低水平,与曝磁组比较,差异有统计学意义(P＜0.05).②与对照组比较,曝磁5d后细胞内ALP活性显著升高(P＜0.05),SB202190可明显阻断该效应(P＜0.05).③与对照组比较,曝磁3d后骨髓间充质干细胞的增殖活性明显升高(P＜0.05),SB202190不能阻断该效应(曝磁+SB202190组与曝磁组比较,P ＞0.05).④SB202190阻断p38 MAPK信号通路并曝磁5 min后,ERK MAPK磷酸化水平明显强化(P＜0.05);PD98059阻断ERK MAPK信号通路并曝磁30 min后,p38 MAPK磷酸化水平明显强化(P＜0.05).结论 ERK和p38 MAPK信号通路分别参与了电磁场对骨髓间充质干细胞增殖和分化成骨过程的调节,并在电磁场作用下两通路表现出串扰现象.
목적 연구15 Hz l mT적정현파전자장대대서골수간충질간세포(MSCs)적세포외신호조절격매(ERK)화사렬원격활적단백격매(MAPK) p38적격활규률급상호작용특점,탐토ERK화p38 MAPK신호통로재전자장촉성골효응중적작용.방법 선취제3대체외분리배양적대서골수간충질간세포,분위대조조、폭자조、폭자+ PD98059조화폭자+SB202190조사개대조.폭자조세포치우대유전자발생기적배양상중배양,폭자+ PD98059조화폭자+SB202190조세포분별가입ERK신호통로조단제PD98059화p38 MAPK신호통로조단제SB202190후재치입대유전자발생기적배양상중배양,대조조세포정상배양.용면역인적(Western blot)법검측ERK화p38 MAPK적단백표체급린산화수평변화.안조세포감성린산매(ALP)시제합설명서조작보취대각조세포ALP활성진행검측,기활성변화가간접반영세포적분화성골활성;용새서람비색법검측각조세포적증식활성변화.결과 ①전자장작용하,골수간충질간세포내p38 MAPK신호통로가피쾌속격활,폭자15 min후출현p38린산화수평승고(P＜0.05);가용SB202190후,재행전자장자격,세포내p38린산화수평잉연유지재교저수평,여폭자조비교,차이유통계학의의(P＜0.05).②여대조조비교,폭자5d후세포내ALP활성현저승고(P＜0.05),SB202190가명현조단해효응(P＜0.05).③여대조조비교,폭자3d후골수간충질간세포적증식활성명현승고(P＜0.05),SB202190불능조단해효응(폭자+SB202190조여폭자조비교,P ＞0.05).④SB202190조단p38 MAPK신호통로병폭자5 min후,ERK MAPK린산화수평명현강화(P＜0.05);PD98059조단ERK MAPK신호통로병폭자30 min후,p38 MAPK린산화수평명현강화(P＜0.05).결론 ERK화p38 MAPK신호통로분별삼여료전자장대골수간충질간세포증식화분화성골과정적조절,병재전자장작용하량통로표현출천우현상.
Objective To study the effects of electromagnetic field (EMF) on the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways in mesenchymal stem cells (MSCs) and their interaction, and to explore the cellular signal transduction mechanism of the biological effects induced by EMF.Methods The 3rd-passage rat bone marrow MSCs were randomly divided into a control group, an EMF group, an EMF + PD98059 group and an EMF + SB202190 group.Cells in the EMF group were cultured in the electromagnetic field, those in the EMF + PD98059 and EMF + SB202190 groups cultured in the electromagnetic field after PD98059 or SB202190 was added, and those in the control group were cultured normally.Different groups of cells were exposed to electromagnetic fields (15 Hz, 1 mT, sine wave form) for different exposure duration.The activated phosphorylated and non-phosphorylated p38MAPK were measured using Western blotting analysis with their specific corresponding antibodies.The alkaline phosphatase (ALP) activity in cells in different groups was detected according to the instructions of ALP kit.MTT assay was applied to investigate the proliferation of cells.Results Electromagnetic fields could rapidly induce the activation of p38 MAPK (P ＜ 0.05) and the phosphorylation of p38 MAPK elevated after 15 min exposure to EMF.The phosphorylation of p38 MAPK was significantly lower in the EMF + SB202190 group than that of the EMF group.After 5 days of EMF exposure, the ALP activity of cells was significantly improved, and the effect could be inhibited by SB202190.The bone marrow mesenchymal stem cells proliferation increased significantly after being exposed to EMF for 3 days, and it could not be blocked by SB202190.Phosphorylation of ERK and MAPK increased significantly when the p38 MAPK pathway was blocked by SB202190 and exposed to EMF for 5 minutes, and it also increased significantly when the ERK MAPK pathway was blocked by PD98059 and received 30 minutes of EMF exposure.Conclusion EMF can quickly activate ERK and p38 MAPK pathways to induce cell proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.Moreover, in EMF there is a mutual interference between ERK and p38 MAPK pathways.