中华围产医学杂志
中華圍產醫學雜誌
중화위산의학잡지
CHINESE JOURNAL OF PERINATAL MEDICINE
2013年
5期
288-293
,共6页
马小燕%沈奇%王晶晶%谢爱兰%朱雪琼
馬小燕%瀋奇%王晶晶%謝愛蘭%硃雪瓊
마소연%침기%왕정정%사애란%주설경
水通道蛋白质3%细胞外信号调节MAP激酶类%羊膜%上皮细胞%羊水过少
水通道蛋白質3%細胞外信號調節MAP激酶類%羊膜%上皮細胞%羊水過少
수통도단백질3%세포외신호조절MAP격매류%양막%상피세포%양수과소
Aquaporin 3%Extracellular signal-regulated MAP kinases%Amnion%Epithelial cells%Oligohydramnios
目的 研究丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)/细胞外信号调节激酶(extracellular signal regulated kinase,ERK)信号转导通路对人羊膜上皮细胞中水通道蛋白3(aquaporin,AQP3)表达的调控作用. 方法 选择2011年1月至11月,在温州医学院附属第二医院产科分娩的不明原因羊水过少和羊水量正常的足月单胎妊娠产妇各10例,均为剖宫产分娩.原代培养羊膜上皮细胞,并用ERK1/2抑制剂U0126进行处理.浓度为0、5、10、20和40 μmol/L的U0126分别作用12 h；再选取使磷酸化ERK1/2表达水平最低的浓度为最佳浓度,作用于细胞0、2、6、12和24 h.采用免疫细胞化学染色检测AQP3蛋白定位,Western印迹检测总ERK1/2、磷酸化ERK1/2和AQP3蛋白表达水平.统计学方法采用t检验和方差分析. 结果 (1)羊水过少组磷酸化ERK1/2和AQP3的表达均低于羊水量正常组(ERK1/2:2.46±0.25与3.46±0.33；AQP3:1.56±0.10与2.34±0.18,t=9.243和13.292,P＜0.01).(2)羊水过少组中U0126不同浓度组间比较,总ERK1/2表达差异无统计学意义(F=0.365,P＞0.05)；5、10、20、40 μmol/L组磷酸化ERK1/2和AQP3表达均低于0μmol/L组(磷酸化ERK1/2:0.96±0.16、1.12±0.13、0.98±0.17、1.02±0.26与2.46±0.25; AQP3:1.10±0.09、1.12±0.08、1.13±0.06、1.11±0.06与1.56±0.10,P＜0.05)；但5μmol/L组与10、20和40 μmol/L组比较,差异均无统计学意义(P＞0.05).羊水过少组U0126作用的最佳浓度为5 μmol/L.该浓度作用2h组磷酸化ERK1/2和AQP3表达均低于0h组(磷酸化ERK1/2:1.27±0.29与2.55±0.12;AQP3:1.44±0.12与2.15±0.09,P＜0.05),但2h组与6、12和24 h组比较,差异均无统计学意义(P＞0.05).最佳作用时间为2h.(3)羊水量正常组和羊水过少组产妇的羊膜上皮细胞中,胞浆及胞膜均有AQP3蛋白阳性染色,但主要位于胞浆.U0126作用后,AQP3蛋白在羊膜上皮细胞中定位无明显改变. 结论 U0126可抑制羊水过少者的羊膜上皮细胞ERK1/2磷酸化及AQP3蛋白表达,提示MAPK/ERK1/2信号转导通路可能对羊膜上皮细胞中AQP3蛋白表达起调控作用,从而影响羊水量的平衡.
目的 研究絲裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)/細胞外信號調節激酶(extracellular signal regulated kinase,ERK)信號轉導通路對人羊膜上皮細胞中水通道蛋白3(aquaporin,AQP3)錶達的調控作用. 方法 選擇2011年1月至11月,在溫州醫學院附屬第二醫院產科分娩的不明原因羊水過少和羊水量正常的足月單胎妊娠產婦各10例,均為剖宮產分娩.原代培養羊膜上皮細胞,併用ERK1/2抑製劑U0126進行處理.濃度為0、5、10、20和40 μmol/L的U0126分彆作用12 h；再選取使燐痠化ERK1/2錶達水平最低的濃度為最佳濃度,作用于細胞0、2、6、12和24 h.採用免疫細胞化學染色檢測AQP3蛋白定位,Western印跡檢測總ERK1/2、燐痠化ERK1/2和AQP3蛋白錶達水平.統計學方法採用t檢驗和方差分析. 結果 (1)羊水過少組燐痠化ERK1/2和AQP3的錶達均低于羊水量正常組(ERK1/2:2.46±0.25與3.46±0.33；AQP3:1.56±0.10與2.34±0.18,t=9.243和13.292,P＜0.01).(2)羊水過少組中U0126不同濃度組間比較,總ERK1/2錶達差異無統計學意義(F=0.365,P＞0.05)；5、10、20、40 μmol/L組燐痠化ERK1/2和AQP3錶達均低于0μmol/L組(燐痠化ERK1/2:0.96±0.16、1.12±0.13、0.98±0.17、1.02±0.26與2.46±0.25; AQP3:1.10±0.09、1.12±0.08、1.13±0.06、1.11±0.06與1.56±0.10,P＜0.05)；但5μmol/L組與10、20和40 μmol/L組比較,差異均無統計學意義(P＞0.05).羊水過少組U0126作用的最佳濃度為5 μmol/L.該濃度作用2h組燐痠化ERK1/2和AQP3錶達均低于0h組(燐痠化ERK1/2:1.27±0.29與2.55±0.12;AQP3:1.44±0.12與2.15±0.09,P＜0.05),但2h組與6、12和24 h組比較,差異均無統計學意義(P＞0.05).最佳作用時間為2h.(3)羊水量正常組和羊水過少組產婦的羊膜上皮細胞中,胞漿及胞膜均有AQP3蛋白暘性染色,但主要位于胞漿.U0126作用後,AQP3蛋白在羊膜上皮細胞中定位無明顯改變. 結論 U0126可抑製羊水過少者的羊膜上皮細胞ERK1/2燐痠化及AQP3蛋白錶達,提示MAPK/ERK1/2信號轉導通路可能對羊膜上皮細胞中AQP3蛋白錶達起調控作用,從而影響羊水量的平衡.
목적 연구사렬원활화단백격매(mitogen-activated protein kinases,MAPK)/세포외신호조절격매(extracellular signal regulated kinase,ERK)신호전도통로대인양막상피세포중수통도단백3(aquaporin,AQP3)표체적조공작용. 방법 선택2011년1월지11월,재온주의학원부속제이의원산과분면적불명원인양수과소화양수량정상적족월단태임신산부각10례,균위부궁산분면.원대배양양막상피세포,병용ERK1/2억제제U0126진행처리.농도위0、5、10、20화40 μmol/L적U0126분별작용12 h；재선취사린산화ERK1/2표체수평최저적농도위최가농도,작용우세포0、2、6、12화24 h.채용면역세포화학염색검측AQP3단백정위,Western인적검측총ERK1/2、린산화ERK1/2화AQP3단백표체수평.통계학방법채용t검험화방차분석. 결과 (1)양수과소조린산화ERK1/2화AQP3적표체균저우양수량정상조(ERK1/2:2.46±0.25여3.46±0.33；AQP3:1.56±0.10여2.34±0.18,t=9.243화13.292,P＜0.01).(2)양수과소조중U0126불동농도조간비교,총ERK1/2표체차이무통계학의의(F=0.365,P＞0.05)；5、10、20、40 μmol/L조린산화ERK1/2화AQP3표체균저우0μmol/L조(린산화ERK1/2:0.96±0.16、1.12±0.13、0.98±0.17、1.02±0.26여2.46±0.25; AQP3:1.10±0.09、1.12±0.08、1.13±0.06、1.11±0.06여1.56±0.10,P＜0.05)；단5μmol/L조여10、20화40 μmol/L조비교,차이균무통계학의의(P＞0.05).양수과소조U0126작용적최가농도위5 μmol/L.해농도작용2h조린산화ERK1/2화AQP3표체균저우0h조(린산화ERK1/2:1.27±0.29여2.55±0.12;AQP3:1.44±0.12여2.15±0.09,P＜0.05),단2h조여6、12화24 h조비교,차이균무통계학의의(P＞0.05).최가작용시간위2h.(3)양수량정상조화양수과소조산부적양막상피세포중,포장급포막균유AQP3단백양성염색,단주요위우포장.U0126작용후,AQP3단백재양막상피세포중정위무명현개변. 결론 U0126가억제양수과소자적양막상피세포ERK1/2린산화급AQP3단백표체,제시MAPK/ERK1/2신호전도통로가능대양막상피세포중AQP3단백표체기조공작용,종이영향양수량적평형.
Objective To investigate the role of mitogen-activated protein kinases (MAPK)/extracellular signal regulated kinase1/2 (ERK1/2) signal transduction pathway in regulating the expression of aquaporin 3 (AQP3) in human amnion epithelial cells.Methods Primary cell culture of human amnion epithelial cells deriving from amnion of term pregnancy with normal amniotic fluid volume (AFV) or isolated oligohydramnios was conducted in the Second Affiliated Hospital of Wenzhou Medical College from January to November 2011.Either group included 10 elective cesarean cases.The primarily cultured cells were treated with different concentrations (0,5,10,20 and 40 mol/L) of ERK inhibitors (U0126) for 12 h,and then the optimal concentration of U0126 which resulted in the lowest expression of phospho~ERK1/2 (p-ERK1/2) was added for different durations(0,2,6,12 and 24 h).Immunocytochemistry was used to detect the localization of AQP3 and Western blot analysis was used to examine the expression of total ERK1/2,p-ERK1/2 and AQP3 in human amnion epithelial cells.Statistical analysis was performed by t-test and one-way ANOVA.Results (1) Compared with those in normal AFV group,p-ERK1/2 and AQP3 expression in human amnion epithelium cells decreased in oligohydramnios group,respectively (p-ERK1/2:3.46 ± 0.33 and 2.46±0.25;AQP3:2.34 ± 0.18 and 1.56±0.10,t=9.243 and 13.292,P＜0.01).(2) In oligohydramnios groups,after treated with different concentrations of U0126,the expressions of total ERK1/2 did not change (F=0.365,P＞0.05).The expression of p-ERK1/2 and AQP3 in 5,10,20,40 μmol/L U0126 (p-ERK1/2:0.96±0.16,1.12±0.13,0.98±0.17 and 1.02±0.26; AQP3:1.10±0.09,1.12±0.08,1.13±0.06 and 1.11±0.06) were all significantly lower than those in 0 μmol/LU0126 group (p-ERK1/2:2.46±0.25; AQP3:1.56±0.10,P＜0.05).However,the expression of p-ERK1/2 and AQP3 showed no significant difference among 5,10,20,and 40μmol/L U0126 groups (P＞0.05).The optimal concentration of U0126 was 5 μmol/L.After treated with 5 μmol/L U0126 for 2 h,the expressions of p-ERK1/2 and AQP3 (1.27±0.29 and 1.44±0.12)were lower than those after treated for 0 h (2.55±0.12 and 2.15±0.09,P＜0.05).However,there was no significant difference among groups treated for 2,6,12 and 24 h.Therefore,the optimal treatment time was 2 h.(3) The expression of AQP3 was distributed in both cell membrane and cytoplasm in amnion epithelial cells with normal amniotic fluid volume or isolated oligohydramnios,but mainly in cytoplasm.U0126 did not change the localization of AQP3 expression.Conclusions U0126 inhibits the phosphorylation of ERK1/2 and expression of AQP3 of women with oligohydramnios,indicating that the MAPK/ERK1/2 signal transduction pathway might regulate the expression of AQP3 in human amnion epithelial cells,and therefore affect the balance of amniotic fluid volume.
