CAJ | 학술논문

目的观察骨髓间充质干细胞(BMSC)在体外模拟的急性肾损伤(AKI)微环境中的迁移特点,明确红细胞生成素(EPO)干预对BMSC迁移的影响,并探讨其可能机制.方法肾小管上皮细胞(RTEC)于低氧-复氧(HR)环境中各培养12h获得HR-RETC,体外模拟AKI.采用Transwell体系将BMSC与RTEC共培养.实验分7组:对照组(①组单纯BMSC培养)、BMSC-RTEC共培养组(②组)、BMSC-HR-RTEC共培养+不同浓度EPO干预组(③组～⑦组,EPO浓度:0、1、5、10、50 IU/ml).共培养48 h后检测各组BMSC迁移数量,Western印迹法分别检测下室RTEC内的基质细胞衍生因子1(SDF-1)表达及上室BMSC的磷酸化(p)MAPK、MAPK水平,ELISA法检测RTEC培养上清中的SDF-1浓度.结果 BMSC向HR-RTEC培养室的迁移数量增加,EPO干预可放大此效应,以10 IU/ml时最强[与③组比较,(46.67±7.37)个/HP比(19.00±2.37)个/HP,P＜0.05].③组HR-RTEC的细胞内SDF-1表达水平(0.37±0.01比0.19±0.01,P＜0.05)及培养上清中SDF-1浓度[(61.64±4.88) μg/L比(35.26±8.78) μg/L,P＜0.05]均高于未处理RTEC(②组),EPO干预可进一步增加其水平,以10 IU/ml时最强[(⑥组与③组比较:(173.53±14.66) μg/L比(61.64±4.88) μg/L,P＜0.05],并伴有BMSC内MAPK磷酸化增强.结论体外模拟的AKI微环境对BMSC有定向趋化作用,EPO干预可增强其趋化效果,其机制可能与SDF-1水平增加及SDF-1-CXCR4轴下游的信号蛋白MAPK的磷酸化有关.
목적 관찰골수간충질간세포(BMSC)재체외모의적급성신손상(AKI)미배경중적천이특점,명학홍세포생성소(EPO)간예대BMSC천이적영향,병탐토기가능궤제.방법 신소관상피세포(RTEC)우저양-복양(HR)배경중각배양12h획득HR-RETC,체외모의AKI.채용Transwell체계장BMSC여RTEC공배양.실험분7조:대조조(①조단순BMSC배양)、BMSC-RTEC공배양조(②조)、BMSC-HR-RTEC공배양+불동농도EPO간예조(③조～⑦조,EPO농도:0、1、5、10、50 IU/ml).공배양48 h후검측각조BMSC천이수량,Western인적법분별검측하실RTEC내적기질세포연생인자1(SDF-1)표체급상실BMSC적린산화(p)MAPK、MAPK수평,ELISA법검측RTEC배양상청중적SDF-1농도.결과 BMSC향HR-RTEC배양실적천이수량증가,EPO간예가방대차효응,이10 IU/ml시최강[여③조비교,(46.67±7.37)개/HP비(19.00±2.37)개/HP,P＜0.05].③조HR-RTEC적세포내SDF-1표체수평(0.37±0.01비0.19±0.01,P＜0.05)급배양상청중SDF-1농도[(61.64±4.88) μg/L비(35.26±8.78) μg/L,P＜0.05]균고우미처리RTEC(②조),EPO간예가진일보증가기수평,이10 IU/ml시최강[(⑥조여③조비교:(173.53±14.66) μg/L비(61.64±4.88) μg/L,P＜0.05],병반유BMSC내MAPK린산화증강.결론 체외모의적AKI미배경대BMSC유정향추화작용,EPO간예가증강기추화효과,기궤제가능여SDF-1수평증가급SDF-1-CXCR4축하유적신호단백MAPK적린산화유관.
Objective To investigate the migration of bone-marrow mesenchymal stem cells (BMSCs) under acute kidney injury (AKI) microenvironment in vitro and the effect of erythropoietin (EPO) intervention,and to explore its underlying mechanism.Methods Renal tubular epithelial cells (RTECs) were cultured in hypoxia/re-oxygenation (HR) condition for 12 h,respectively,in order to establish HR-RTEC.BMSCs and RTECs were co-cultured by Transwell system and were divided into 7 groups:control group (group①,only BMSC cultured),BMSC-RTEC co-culturing group (group ②),BMSC-HR-RTEC co-culturing + EPO intervention groups (group ③to group ⑦,EPO concentration:0,1,5,10,50 IU/ml).All the groups were cultured for 48 h and the number of migrating BMSCs was detected.Western blotting was applied for the detection of SDF-1 expression in RTECs and pMAPK and MAPK levels in BMSCs.SDF-1 concentration in the RTECs culture supernatant was tested by ELISA.Results The number of BMSCs migrating to the low chamber where HR-RTECs were cultured was increased,and EPO intervention further enhanced this migration which reached the peak at the concentration of 10 IU/ml [Compared with group③,(46.67±7.37) cells vs (19.00±2.37) cells,P ＜ 0.05].Intracellular expression level and the secreated level of SDF-1 in HR-RTECs in group③ were higher than those in RTECs of group② [0.37±0.01 vs 0.19±0.01,P ＜ 0.05; (61.64±4.88) μg/L vs (35.26±8.78) μg/L,P ＜ 0.05].EPO intervention increased above SDF-1 levels and reached the peak at the concentration of 10 IU/ml [group⑥ vs group③:(173.53± 14.66) μg/L vs (61.64±4.88) μg/L,P＜0.05],accompanied with enhanced phosphorylation of MAPK in BMSCs.Conclusions AKI microenvironment has obvious chemotaxis effect on BMSCs,and EPO intervention can strengthen this effect.The increased SDF-1 level and enhanced phosphorylation of MAPK,the downstream signal protein of SDF-1/CXCR4 axis,are the possible mechanism for EPO performance.