中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2014年
1期
36-41
,共6页
羊膜%上皮细胞%水通道蛋白质3%环AMP依赖性蛋白激酶类%信号传导
羊膜%上皮細胞%水通道蛋白質3%環AMP依賴性蛋白激酶類%信號傳導
양막%상피세포%수통도단백질3%배AMP의뢰성단백격매류%신호전도
Amnion%Epithelial cells%Aquaporin 3%Cyclic AMP-dependent protein kinases%Signal transduction
目的 探讨羊膜上皮细胞水通道蛋白3(AQP3)表达过程中环磷酸腺苷-蛋白激酶A(cAMP-PKA)信号传导通路的调控机制.方法 选择2012年l至11月温州医科大学附属第二医院无并发症、羊水量正常的足月孕妇30例,剖宫产时取羊膜进行上皮细胞培养,并进行如下分组实验:(1)毛喉素组:不同浓度的毛喉素(forskolin,0、2.5、5、50、100 μmol/L)作用羊膜上皮细胞2h,再以最适宜浓度的forskolin作用不同时间(0、1、2、10、20h)；(2)SP-cAMP组:不同浓度的PKA激动剂SP-cAMP(0、2.5、5、50、100 μmol/L)作用羊膜上皮细胞2h,再以最适宜浓度的SP-cAMP作用不同时间(0、1、2、10、20 h)；(3)H-89组:不同浓度的PKA抑制剂H-89(0、5、10、50、100 μmol/L)作用人羊膜上皮细胞2h,再以最适宜浓度H-89作用不同时间(0、1、2、10、20 h).采用ELISA方法检测各组细胞内CAMP水平及PKA活性,免疫细胞化学染色法检测AQP3表达定位,蛋白印迹法检测总cAMP反应元件结合蛋白(CREB)、磷酸化CREB(p-CREB)和AQP3表达水平,采用细胞计数试剂盒(CCK-8)检测细胞增殖率.结果 (1)各组羊膜上皮细胞的胞质及胞膜均有AQP3蛋白表达.(2)各组不同浓度forskolin、SP-cAMP和H-89作用羊膜上皮细胞后,细胞增殖率无明显变化,分别比较,差异均无统计学意义(P＞0.05).(3)不同浓度forskolin作用羊膜上皮细胞2h,总CREB表达水平比较,差异无统计学意义(P＞0.05).cAMP水平、PKA活性、p-CREB和AQP3表达水平分别比较,差异均有统计学意义(P＜0.05),2.5、5、50μmol/L forskolin作用后,上述4种因子均高于0μmol/L(P＜0.05)；而5 μmol/L明显高于2.5、50 μmol/L(P＜0.05).forskolin作用的最适宜浓度为5μmol/L.(4)不同浓度SP-cAMP作用羊膜上皮细胞2h,细胞中cAMP和总CREB表达水平分别比较,差异均无统计学意义(P＞0.05).PKA活性、p-CREB和AQP3表达水平比较,差异均有统计学意义(P＜0.05)；SP-cAMP浓度为5、50 μmol/L时,上述3种因子高于0μmol/L(P＜0.05)；50 μmol/L时明显高于5 μmol/L(P ＜0.05).SP-cAMP作用的最适宜浓度为50 μmol/L.(5)不同浓度H-89作用羊膜上皮细胞2h,细胞中cAMP水平和总CREB表达水平比较,差异均无统计学意义(P＞0.05).PKA活性、p-CREB和AQP3表达水平比较,差异均有统计学意义(P ＜0.05)；H-89浓度为10、50和100μmol/L时,上述3种因子均低于0 μmol/L(P＜0.05),10 μmol/L时明显低于50和100 μmol/L(P＜0.05).H-89作用的最适宜浓度为10 μmol/L.(6)5 μmol/L forskolin联合10 μmol/L H-89作用2h后,羊膜上皮细胞中总CREB表达水平比较,差异无统计学意义(P＞0.05).p-CREB与AQP3表达水平比较,低于5 μmol/L forskolin作用2 h(P ＜0.05),但高于10 μmol/L H-89作用2 h(P ＜0.05).结论 cAMP-PKA信号传导通路对羊膜上皮细胞中AQP3蛋白的表达起调控作用.
目的 探討羊膜上皮細胞水通道蛋白3(AQP3)錶達過程中環燐痠腺苷-蛋白激酶A(cAMP-PKA)信號傳導通路的調控機製.方法 選擇2012年l至11月溫州醫科大學附屬第二醫院無併髮癥、羊水量正常的足月孕婦30例,剖宮產時取羊膜進行上皮細胞培養,併進行如下分組實驗:(1)毛喉素組:不同濃度的毛喉素(forskolin,0、2.5、5、50、100 μmol/L)作用羊膜上皮細胞2h,再以最適宜濃度的forskolin作用不同時間(0、1、2、10、20h)；(2)SP-cAMP組:不同濃度的PKA激動劑SP-cAMP(0、2.5、5、50、100 μmol/L)作用羊膜上皮細胞2h,再以最適宜濃度的SP-cAMP作用不同時間(0、1、2、10、20 h)；(3)H-89組:不同濃度的PKA抑製劑H-89(0、5、10、50、100 μmol/L)作用人羊膜上皮細胞2h,再以最適宜濃度H-89作用不同時間(0、1、2、10、20 h).採用ELISA方法檢測各組細胞內CAMP水平及PKA活性,免疫細胞化學染色法檢測AQP3錶達定位,蛋白印跡法檢測總cAMP反應元件結閤蛋白(CREB)、燐痠化CREB(p-CREB)和AQP3錶達水平,採用細胞計數試劑盒(CCK-8)檢測細胞增殖率.結果 (1)各組羊膜上皮細胞的胞質及胞膜均有AQP3蛋白錶達.(2)各組不同濃度forskolin、SP-cAMP和H-89作用羊膜上皮細胞後,細胞增殖率無明顯變化,分彆比較,差異均無統計學意義(P＞0.05).(3)不同濃度forskolin作用羊膜上皮細胞2h,總CREB錶達水平比較,差異無統計學意義(P＞0.05).cAMP水平、PKA活性、p-CREB和AQP3錶達水平分彆比較,差異均有統計學意義(P＜0.05),2.5、5、50μmol/L forskolin作用後,上述4種因子均高于0μmol/L(P＜0.05)；而5 μmol/L明顯高于2.5、50 μmol/L(P＜0.05).forskolin作用的最適宜濃度為5μmol/L.(4)不同濃度SP-cAMP作用羊膜上皮細胞2h,細胞中cAMP和總CREB錶達水平分彆比較,差異均無統計學意義(P＞0.05).PKA活性、p-CREB和AQP3錶達水平比較,差異均有統計學意義(P＜0.05)；SP-cAMP濃度為5、50 μmol/L時,上述3種因子高于0μmol/L(P＜0.05)；50 μmol/L時明顯高于5 μmol/L(P ＜0.05).SP-cAMP作用的最適宜濃度為50 μmol/L.(5)不同濃度H-89作用羊膜上皮細胞2h,細胞中cAMP水平和總CREB錶達水平比較,差異均無統計學意義(P＞0.05).PKA活性、p-CREB和AQP3錶達水平比較,差異均有統計學意義(P ＜0.05)；H-89濃度為10、50和100μmol/L時,上述3種因子均低于0 μmol/L(P＜0.05),10 μmol/L時明顯低于50和100 μmol/L(P＜0.05).H-89作用的最適宜濃度為10 μmol/L.(6)5 μmol/L forskolin聯閤10 μmol/L H-89作用2h後,羊膜上皮細胞中總CREB錶達水平比較,差異無統計學意義(P＞0.05).p-CREB與AQP3錶達水平比較,低于5 μmol/L forskolin作用2 h(P ＜0.05),但高于10 μmol/L H-89作用2 h(P ＜0.05).結論 cAMP-PKA信號傳導通路對羊膜上皮細胞中AQP3蛋白的錶達起調控作用.
목적 탐토양막상피세포수통도단백3(AQP3)표체과정중배린산선감-단백격매A(cAMP-PKA)신호전도통로적조공궤제.방법 선택2012년l지11월온주의과대학부속제이의원무병발증、양수량정상적족월잉부30례,부궁산시취양막진행상피세포배양,병진행여하분조실험:(1)모후소조:불동농도적모후소(forskolin,0、2.5、5、50、100 μmol/L)작용양막상피세포2h,재이최괄의농도적forskolin작용불동시간(0、1、2、10、20h)；(2)SP-cAMP조:불동농도적PKA격동제SP-cAMP(0、2.5、5、50、100 μmol/L)작용양막상피세포2h,재이최괄의농도적SP-cAMP작용불동시간(0、1、2、10、20 h)；(3)H-89조:불동농도적PKA억제제H-89(0、5、10、50、100 μmol/L)작용인양막상피세포2h,재이최괄의농도H-89작용불동시간(0、1、2、10、20 h).채용ELISA방법검측각조세포내CAMP수평급PKA활성,면역세포화학염색법검측AQP3표체정위,단백인적법검측총cAMP반응원건결합단백(CREB)、린산화CREB(p-CREB)화AQP3표체수평,채용세포계수시제합(CCK-8)검측세포증식솔.결과 (1)각조양막상피세포적포질급포막균유AQP3단백표체.(2)각조불동농도forskolin、SP-cAMP화H-89작용양막상피세포후,세포증식솔무명현변화,분별비교,차이균무통계학의의(P＞0.05).(3)불동농도forskolin작용양막상피세포2h,총CREB표체수평비교,차이무통계학의의(P＞0.05).cAMP수평、PKA활성、p-CREB화AQP3표체수평분별비교,차이균유통계학의의(P＜0.05),2.5、5、50μmol/L forskolin작용후,상술4충인자균고우0μmol/L(P＜0.05)；이5 μmol/L명현고우2.5、50 μmol/L(P＜0.05).forskolin작용적최괄의농도위5μmol/L.(4)불동농도SP-cAMP작용양막상피세포2h,세포중cAMP화총CREB표체수평분별비교,차이균무통계학의의(P＞0.05).PKA활성、p-CREB화AQP3표체수평비교,차이균유통계학의의(P＜0.05)；SP-cAMP농도위5、50 μmol/L시,상술3충인자고우0μmol/L(P＜0.05)；50 μmol/L시명현고우5 μmol/L(P ＜0.05).SP-cAMP작용적최괄의농도위50 μmol/L.(5)불동농도H-89작용양막상피세포2h,세포중cAMP수평화총CREB표체수평비교,차이균무통계학의의(P＞0.05).PKA활성、p-CREB화AQP3표체수평비교,차이균유통계학의의(P ＜0.05)；H-89농도위10、50화100μmol/L시,상술3충인자균저우0 μmol/L(P＜0.05),10 μmol/L시명현저우50화100 μmol/L(P＜0.05).H-89작용적최괄의농도위10 μmol/L.(6)5 μmol/L forskolin연합10 μmol/L H-89작용2h후,양막상피세포중총CREB표체수평비교,차이무통계학의의(P＞0.05).p-CREB여AQP3표체수평비교,저우5 μmol/L forskolin작용2 h(P ＜0.05),단고우10 μmol/L H-89작용2 h(P ＜0.05).결론 cAMP-PKA신호전도통로대양막상피세포중AQP3단백적표체기조공작용.
Objective To investigate the expression of aquaporins-3 (AQP3) in amniotic epithelial cells regulated by cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal pathway and to explore the mechanisms of its expression.Methods The amniotic epithelial cells were collected from 30 patients who underwent elective caesarean sections at term with normal amniotic fluid volume and primarily cultured.The cultured cells were treated with (1) forskolin groups: different concentration (0,2.5,5,50 or 100 μmol/L) of forskolin treated cells for 2 hours,and the optimal concentration of forskolin treated cells with different time (0,1,2,10 or 20 hours) ; (2)SP-cAMP groups: different concentration (0,2.5,5,50 or 100 μmol/L) of SP-cAMP treated cells for 2 hours,and the optimal concentration of SP-cAMP treated cells with different time (0,1,2,10 or 20 hours); (3)H-89 groups: different concentration (0,5,10,50 or 100 μmol/L) of H-89 treated cells for 2 hours,and the optimal concentration of H-89 treated cells with different time (0,1,2,10 or 20 hours).The level of intracellular cAMP and activity of PKA were detected by using ELISA,and immunohistochemistry was used to detect the localization of AQP3,the protein expression of total cAMP-response element binding protein (CREB) and phospho-CREB (p-CREB) and AQP3 were assessed by western blot analysis.Cell proliferation was assessed by cell counting kit-8 (CCK-8)assay.Results (1) The brown staining of AQP3 was detected in both cell membrane and cytoplasm in each group.(2) There was no significant change of the cell proliferation rate among groups with different concentration of forskolin,SP-cAMP and H-89 treatment (P ＞ 0.05).(3) After different concentration of forskolin treated 2 hours,the expression of total CREB had no significant difference among them(P ＞ 0.05).While the expression of cAMP level,PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 2.5 μmol/L,5 μmol/L,50 μmol/L forskolin group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 5 μmol/L forskolin group were higher than that in 2.5 μmol/L and 50 μmol/L (P ＜ 0.05).The optimal forskolin concentration was 5 μmol/L.(4) After different concentration of SP-cAMP treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 5 μμmol/L,50 μmol/L SP-cAMP group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 50 μmol/L SP-cAMP group were higher than that in 5 μmol/L (P ＜0.05).The optimal SP-cAMP concentration was 50 μmol/L (5) After different concentration of H-89 treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were lower in 10 μmol/L,50 μmol/L and 100 μmol/L H-89 group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 10 μmol/L H-89 group were lower than that in 50 μmol/L,100 μmol/L (P ＜ 0.05).The optimal H-89 concentration was 10 μmol/L.(6) p-CREB and AQP3 protein expression were significantly lower in 5 μmol/L forskolin combined 10 μmol/L H-89 incubating 2 hours group when compared with 5 μmol/L forskolin,but higher than that in 10 μmol/L H-89 treated group (P ＜ 0.05).Total CREB was no significant difference among the three groups (P ＞ 0.05).Conclusion cAMP-PKA signal transduction pathway may regulate AQP3 protein expression in human amniotic epithelial cells.
