CAJ | 학술논문

目的解析环孢素A调控滋养细胞侵袭与迁移信号转导的机制.方法应用酶联免疫检测环孢素A作用于滋养细胞后丝裂原活化蛋白激酶(MAPK)活性,Transwell分析滋养细胞侵袭与迁移能力,细胞内共转染荧光素酶活性分析活化T细胞核因子(NFAT)的转录活性,并观察U0126对环孢素A刺激的滋养细胞体外侵袭与迁移能力的影响.结果滋养细胞经不同浓度(0.0001～1.0 μmol/L)环孢素A作用48 h后,均可促进滋养细胞侵袭能力;并呈明显的剂量依赖关系.当环孢素A达10μm0l/L时,这种促进作用减弱.环孢素A以时间和剂量依赖方式诱导滋养细胞MAPK激酶的活化,在1.0μmol/L环孢素A作用30 min达峰值.U0126以剂量依赖方式抑制环孢素A诱导的滋养细胞侵袭与迁移.环孢素A可抑制钙离子载体(离子霉素)+佛波醇乙酯活化的JAR细胞NFAT转录活性并逆转离子霉素+PMA抑制的滋养细胞体外侵袭迁移能力;且环孢素A作用强于NFAT抑制剂,两者没有协同效应.环孢素A活化MAPK/ERK1/2信号与抑制Ca2+/钙调节神经磷酸酶/NFAT信号无交互影响.结论环孢素A通过激活MAPK/ERK1/2及抑制Ca2+/钙调节神经磷酸酶/NFAT信号通路,促进人滋养细胞的体外侵袭与迁移,从而有助于胎盘形成与胚胎发育.
목적 해석배포소A조공자양세포침습여천이신호전도적궤제.방법 응용매련면역검측배포소A작용우자양세포후사렬원활화단백격매(MAPK)활성,Transwell분석자양세포침습여천이능력,세포내공전염형광소매활성분석활화T세포핵인자(NFAT)적전록활성,병관찰U0126대배포소A자격적자양세포체외침습여천이능력적영향.결과 자양세포경불동농도(0.0001～1.0 μmol/L)배포소A작용48 h후,균가촉진자양세포침습능력;병정명현적제량의뢰관계.당배포소A체10μm0l/L시,저충촉진작용감약.배포소A이시간화제량의뢰방식유도자양세포MAPK격매적활화,재1.0μmol/L배포소A작용30 min체봉치.U0126이제량의뢰방식억제배포소A유도적자양세포침습여천이.배포소A가억제개리자재체(리자매소)+불파순을지활화적JAR세포NFAT전록활성병역전리자매소+PMA억제적자양세포체외침습천이능력;차배포소A작용강우NFAT억제제,량자몰유협동효응.배포소A활화MAPK/ERK1/2신호여억제Ca2+/개조절신경린산매/NFAT신호무교호영향.결론 배포소A통과격활MAPK/ERK1/2급억제Ca2+/개조절신경린산매/NFAT신호통로,촉진인자양세포적체외침습여천이,종이유조우태반형성여배태발육.
Objective To investigate the signal pathway by which cyclosporine A(CsA)improves the invasiveness and migration of the first trimester human trophoblast cells. Methods Human first trimester trophoblast cells obtained during artificial abortion and human chorioepithelioma cells of the line JAR were cultured and put into the upper chamber of Transwell cell invasion system.Dimethyl sulfoxide (DMSO)or CsA of the concentrations of 0.0001,0.001,0.01,0.1 and 1.0 μmol/L respectively were added into the upper chamber for 48 h. Some of the upper chambers underwent pretreatment of U0126,a specific inhibitor of MAPK/ERK1/2,for 20 min.The invasiveness and migratory capabilities of the human trophoblast cells was examined.ELISA was used to detect the MAP kinase(MAPK)activity of the human trophoblasts in response to CsA stimulation.JAR cells were inoculated in 96-well plate and co-transferred with p-nuclear factor of activated T cells(pNFAT)-luciferase and internal control plasmid pRL-sV40 for 24 h,and then stimulated with CsA,ionomycin,and phorbol myristate acetate for 4 h;luciferase reporter system was used to detect the activity of the luciferase so as to calculate the activity of NFAT.Results The invasiveness and migration of the human trophoblast cells were enhanced by CsA in a dose-dependent manner,peaked at the CsA concentration of 1.0μmol/L,and then decreased.Activation of MAPK/ERK1/2 in the trophoblasts was observed as early as 10 min after the stimulation of CsA(1.0 μmol/L), and peaked 30 min after the stimulation. Moreover,the CsA-induced ERK1/2 activation remained at a higher level for at least 1 h,and then decreased significantly 2 h following the CsA treatment.Pretreatment with U0126 inhibited the invasion and migration of the trophoblasts.Activation of Ca2+/calcineurin/NFAT by ionomycin inhibited the invasion and migration of human trophoblasts,which were restored by CsA as well as NFAT inhibitor.The promotion of the invasion and migration of human trophoblasts by CsA was stronger than that by the NFAT inhibitor,and no synergy was seen between these 2 factors.U0126 blocked the MAPK/ERK1/2 signal pathways,but not influenced the NFAT transcription activity.Conclusion CsA promotes the invasiveness and migration of human trophoblast cells by activating the MAPK/ERK1/2 and inhibitingCa2+/calcineurin/NFAT signal pathways independently.