中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2014年
12期
925-931
,共7页
卢燕琼%蒋斯%张洁清%宋红林%李力
盧燕瓊%蔣斯%張潔清%宋紅林%李力
로연경%장사%장길청%송홍림%리력
子宫内膜肿瘤%细胞系,肿瘤%血管内皮生长因子A%成纤维细胞生长因子2%丝裂原激活蛋白激酶类%雌二醇
子宮內膜腫瘤%細胞繫,腫瘤%血管內皮生長因子A%成纖維細胞生長因子2%絲裂原激活蛋白激酶類%雌二醇
자궁내막종류%세포계,종류%혈관내피생장인자A%성섬유세포생장인자2%사렬원격활단백격매류%자이순
Endometrial neoplasms%Cell line,tumor%Vascular endothelial growth factor A%Fibroblast growth factor 2%Mitogen-activated protein kinases%Estradiol
目的 探讨雌二醇诱导子宫内膜癌细胞系Ishikawa细胞生成的血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)对有丝分裂原活化蛋白激酶(MAPK)通路中主要基因蛋白的影响.方法 实验分为4组:雌二醇组(E2组,加入1 μmol/L的雌二醇作用30 min),抑制剂组[包括Bibf1120组:加入10 μmol/L的VEGF受体抑制剂——Bibf1120; Ponatinib组:加入2.5 μmol/L的bFGF受体抑制剂——Ponatinib; U0126组:加入10μmol/L的MAPK激酶(MEK)特异性抑制剂——U0126.各组均预处理1 h],抑制剂+E2组(包括Bibf1120+E2组、Ponatinib+E2组和U0126+E2组,各抑制剂预处理1h后,加入1μmol/L的雌二醇作用30 min);对照组(仅加入无血清DMEM培养基).(1)以不同浓度(分别为0.01、0.1、1、10、100μmol/L)雌二醇作用于Ishikawa细胞后,采用蛋白印迹法检测磷酸化细胞外信号调节激酶1/2(p-ERK1/2)蛋白活化水平.(2)采用荧光定量PCR技术检测各组细胞中VEGF、bFGF、MEK1/2、ERK1/2mRNA的表达,蛋白印迹法检测各组细胞中VEGF、bFGF蛋白的表达及磷酸化NEK1/2(p-MEK1/2)、p-ERK1/2蛋白活化水平,流式细胞仪检测各组细胞的细胞周期比例,体外穿膜实验检测各组细胞的迁移能力.结果 (1)不同浓度(分别为0.01、0.1、1、10、100 μmo1/L)的雌二醇作用30 min后Ishikawa细胞中p-ERK1/2蛋白的活化水平分别为0.16±0.03、0.10±0.03、0.41±0.04、0.19±.0.03、0.19±0.03,均明显高于对照组的0.05±0.00 (P＜0.05),且雌二醇浓度为1μmol/L时其活化水平最高.(2)E2组细胞中VEGF、bFGF mRNA及蛋白的表达水平均高于对照组,分别与对照组比较,差异均有统计学意义(P＜0.05);Bibf1120+E2组细胞中VEGF mRNA及蛋白的表达水平分别与E2组比较,差异均有统计学意义(P＜0.05).E2组MEK1/2、ERK1/2mRNA及p-MEK1/2、p-ERK1/2蛋白活化水平均显著高于对照组(P＜0.05);Bibf1120+E2组、Ponatinib+E2组、U0126+E2组细胞中MEK1/2、ERK1/2mRNA及p-MEK1/2、p-ERK1/2蛋白活化水平均低于E2组(P＜0.05).E2组G1期及S期细胞比例分别为(53.6±3.2)％和(29.2±4.2)％,分别与对照组的(65.1±2.6)％和(20.2±1.9)％比较,差异均有统计学意义(P＜0.05);U0126+E2组、Bibf1120+E2组、Ponatinib+E2组G1期细胞比例分别为(66.8±2.6)％、(63.1±2.6)％和(63.3±0.4)％,S期细胞比例分别为(25.4±1.9)％、(25.0±3.8)％和(23.8±0.5)％,分别与E2组比较,差异均有统计学意义(P＜0.05);U0126+E2组分别与Bibf1120+E2组、Ponatinib+E2组比较,差异也均有统计学意义(P＜0.05).E2组穿过微孔的细胞数为(110±17)个,与对照组的(65±8)个比较,差异有统计学意义(P＜0.05);U0126+E2组、Bibf1 120+E2组、Ponatinib+E2组穿过微孔的细胞数分别为(28±4)、(38±5)、(42±6)个,分别与E2组比较,差异均有统计学意义(P＜0.05);U0126+E2组分别与Bibf1 120+E2组、Ponatinib+E2组比较,差异也均有统计学意义(P＜0.05).结论 雌二醇通过非基因转录效应产生的VEGF、bFGF可以激活MAPK通路,促进子宫内膜癌的发生、发展.
目的 探討雌二醇誘導子宮內膜癌細胞繫Ishikawa細胞生成的血管內皮生長因子(VEGF)和堿性成纖維細胞生長因子(bFGF)對有絲分裂原活化蛋白激酶(MAPK)通路中主要基因蛋白的影響.方法 實驗分為4組:雌二醇組(E2組,加入1 μmol/L的雌二醇作用30 min),抑製劑組[包括Bibf1120組:加入10 μmol/L的VEGF受體抑製劑——Bibf1120; Ponatinib組:加入2.5 μmol/L的bFGF受體抑製劑——Ponatinib; U0126組:加入10μmol/L的MAPK激酶(MEK)特異性抑製劑——U0126.各組均預處理1 h],抑製劑+E2組(包括Bibf1120+E2組、Ponatinib+E2組和U0126+E2組,各抑製劑預處理1h後,加入1μmol/L的雌二醇作用30 min);對照組(僅加入無血清DMEM培養基).(1)以不同濃度(分彆為0.01、0.1、1、10、100μmol/L)雌二醇作用于Ishikawa細胞後,採用蛋白印跡法檢測燐痠化細胞外信號調節激酶1/2(p-ERK1/2)蛋白活化水平.(2)採用熒光定量PCR技術檢測各組細胞中VEGF、bFGF、MEK1/2、ERK1/2mRNA的錶達,蛋白印跡法檢測各組細胞中VEGF、bFGF蛋白的錶達及燐痠化NEK1/2(p-MEK1/2)、p-ERK1/2蛋白活化水平,流式細胞儀檢測各組細胞的細胞週期比例,體外穿膜實驗檢測各組細胞的遷移能力.結果 (1)不同濃度(分彆為0.01、0.1、1、10、100 μmo1/L)的雌二醇作用30 min後Ishikawa細胞中p-ERK1/2蛋白的活化水平分彆為0.16±0.03、0.10±0.03、0.41±0.04、0.19±.0.03、0.19±0.03,均明顯高于對照組的0.05±0.00 (P＜0.05),且雌二醇濃度為1μmol/L時其活化水平最高.(2)E2組細胞中VEGF、bFGF mRNA及蛋白的錶達水平均高于對照組,分彆與對照組比較,差異均有統計學意義(P＜0.05);Bibf1120+E2組細胞中VEGF mRNA及蛋白的錶達水平分彆與E2組比較,差異均有統計學意義(P＜0.05).E2組MEK1/2、ERK1/2mRNA及p-MEK1/2、p-ERK1/2蛋白活化水平均顯著高于對照組(P＜0.05);Bibf1120+E2組、Ponatinib+E2組、U0126+E2組細胞中MEK1/2、ERK1/2mRNA及p-MEK1/2、p-ERK1/2蛋白活化水平均低于E2組(P＜0.05).E2組G1期及S期細胞比例分彆為(53.6±3.2)％和(29.2±4.2)％,分彆與對照組的(65.1±2.6)％和(20.2±1.9)％比較,差異均有統計學意義(P＜0.05);U0126+E2組、Bibf1120+E2組、Ponatinib+E2組G1期細胞比例分彆為(66.8±2.6)％、(63.1±2.6)％和(63.3±0.4)％,S期細胞比例分彆為(25.4±1.9)％、(25.0±3.8)％和(23.8±0.5)％,分彆與E2組比較,差異均有統計學意義(P＜0.05);U0126+E2組分彆與Bibf1120+E2組、Ponatinib+E2組比較,差異也均有統計學意義(P＜0.05).E2組穿過微孔的細胞數為(110±17)箇,與對照組的(65±8)箇比較,差異有統計學意義(P＜0.05);U0126+E2組、Bibf1 120+E2組、Ponatinib+E2組穿過微孔的細胞數分彆為(28±4)、(38±5)、(42±6)箇,分彆與E2組比較,差異均有統計學意義(P＜0.05);U0126+E2組分彆與Bibf1 120+E2組、Ponatinib+E2組比較,差異也均有統計學意義(P＜0.05).結論 雌二醇通過非基因轉錄效應產生的VEGF、bFGF可以激活MAPK通路,促進子宮內膜癌的髮生、髮展.
목적 탐토자이순유도자궁내막암세포계Ishikawa세포생성적혈관내피생장인자(VEGF)화감성성섬유세포생장인자(bFGF)대유사분렬원활화단백격매(MAPK)통로중주요기인단백적영향.방법 실험분위4조:자이순조(E2조,가입1 μmol/L적자이순작용30 min),억제제조[포괄Bibf1120조:가입10 μmol/L적VEGF수체억제제——Bibf1120; Ponatinib조:가입2.5 μmol/L적bFGF수체억제제——Ponatinib; U0126조:가입10μmol/L적MAPK격매(MEK)특이성억제제——U0126.각조균예처리1 h],억제제+E2조(포괄Bibf1120+E2조、Ponatinib+E2조화U0126+E2조,각억제제예처리1h후,가입1μmol/L적자이순작용30 min);대조조(부가입무혈청DMEM배양기).(1)이불동농도(분별위0.01、0.1、1、10、100μmol/L)자이순작용우Ishikawa세포후,채용단백인적법검측린산화세포외신호조절격매1/2(p-ERK1/2)단백활화수평.(2)채용형광정량PCR기술검측각조세포중VEGF、bFGF、MEK1/2、ERK1/2mRNA적표체,단백인적법검측각조세포중VEGF、bFGF단백적표체급린산화NEK1/2(p-MEK1/2)、p-ERK1/2단백활화수평,류식세포의검측각조세포적세포주기비례,체외천막실험검측각조세포적천이능력.결과 (1)불동농도(분별위0.01、0.1、1、10、100 μmo1/L)적자이순작용30 min후Ishikawa세포중p-ERK1/2단백적활화수평분별위0.16±0.03、0.10±0.03、0.41±0.04、0.19±.0.03、0.19±0.03,균명현고우대조조적0.05±0.00 (P＜0.05),차자이순농도위1μmol/L시기활화수평최고.(2)E2조세포중VEGF、bFGF mRNA급단백적표체수평균고우대조조,분별여대조조비교,차이균유통계학의의(P＜0.05);Bibf1120+E2조세포중VEGF mRNA급단백적표체수평분별여E2조비교,차이균유통계학의의(P＜0.05).E2조MEK1/2、ERK1/2mRNA급p-MEK1/2、p-ERK1/2단백활화수평균현저고우대조조(P＜0.05);Bibf1120+E2조、Ponatinib+E2조、U0126+E2조세포중MEK1/2、ERK1/2mRNA급p-MEK1/2、p-ERK1/2단백활화수평균저우E2조(P＜0.05).E2조G1기급S기세포비례분별위(53.6±3.2)％화(29.2±4.2)％,분별여대조조적(65.1±2.6)％화(20.2±1.9)％비교,차이균유통계학의의(P＜0.05);U0126+E2조、Bibf1120+E2조、Ponatinib+E2조G1기세포비례분별위(66.8±2.6)％、(63.1±2.6)％화(63.3±0.4)％,S기세포비례분별위(25.4±1.9)％、(25.0±3.8)％화(23.8±0.5)％,분별여E2조비교,차이균유통계학의의(P＜0.05);U0126+E2조분별여Bibf1120+E2조、Ponatinib+E2조비교,차이야균유통계학의의(P＜0.05).E2조천과미공적세포수위(110±17)개,여대조조적(65±8)개비교,차이유통계학의의(P＜0.05);U0126+E2조、Bibf1 120+E2조、Ponatinib+E2조천과미공적세포수분별위(28±4)、(38±5)、(42±6)개,분별여E2조비교,차이균유통계학의의(P＜0.05);U0126+E2조분별여Bibf1 120+E2조、Ponatinib+E2조비교,차이야균유통계학의의(P＜0.05).결론 자이순통과비기인전록효응산생적VEGF、bFGF가이격활MAPK통로,촉진자궁내막암적발생、발전.
Objective To explore the effects of mitogen-activated protein kinase (MAPK) pathway by estradiol induced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in endometrial cancer Ishikawa cells.Methods The experiments were divided into 4 groups:E2 group (Ishikawa cells treated with 1 p mol/L estradiol for 30 minutes); inhibitor group:including Ishikawa cells treated with 10 μmol/L Bibf1 120 (Bibf1 120 group),or treated with 2.5 μmol/L Ponatinib (Ponatinib group),or treated with 10 p mol/L U0126 (U0126 group) for 60 minutes; inhibitor + E2 group:including Ishikawa cells treated with 10 μmol/L Bibf1120 (Bibf1120 + E2 group),or treated with 2.5 μmol/L Ponatinib (Ponatinib + E2 group),or treated with 10 μmol/L U0126 (U0126+ E2 group) for 60 minutes following incubation with 1 μmol/L estradiol for 30 minutes; control group:only adding the culture medium without serum DMEM.(1) Western blot analysis was used to detect phosphorylation extracellular signal-regulated kinase 1/2(p-ERK 1/2) protein expression with stimulation in different concentrations of estradiol (0.01,0.1,1,10,100 μmol/L).(2) Quantitative fluorescent reverse transcription (qRT)-PCR and western blot analysis was used to test the level of mRNA and protein of VEGF,bFGF,MAPK kinase 1/2 (MEK1/2),extracellular signal-regulated kinase 1/2 (ERK1/2),p-ERK1/2 and phosphorylation MEK1/2 (p-MEK1/2).Flow cytometry were used to examine the cell cycle,and transwell chamber assay were used to detect the cell migration in different groups.Results The expression of the p-ERK1/2 protein at 0.01,0.1,1,10,100 μ mol/L were 0.16±0.03,0.10±0.03,0.41 ±0.04,0.19±0.03,0.19±0.03,there were significantly higher than that in control group(0.05±0.00,P＜0.05),and which was more obvious at the concentration of 1 μmol/L estradiol.The expression level of VEGF,bFGF mRNA and protein in E2 group were higher than those in the control group (P＜O.05).VEGF mRNA and protein in Bibf1120+E2 group were higher than those in E2 group.The expression of MEK1/2,ERK1/2 mRNA protein in E2 group were higher than those in control group (P＜0.05).The expression of MEK1/2,ERK1/2 mRNA or p-MEK1/2,p-ERK1/2 protein in Bibf1120 + E2 group,Ponatinib+E2 group or U0126+E2 group were lower than those in E2 group(all P＜0.05).Percentage of G1 phase [(53.6±3.2)％] and S phase[(29.2±4.2)％] in E2 group was significantly different with those in control group respectively(P＜0.05).Percentage of G1 phase[(66.8±2.6)％,(63.1±2.6)％ and (63.3±0.4)％] and S phase [(25.4±1.9)％,(25.0±3.8)％ and(23.8±0.5)％] in U0126+E2 group,Bibf1120+E2 group or Ponatinib +E2 group was also significantly different with those in control group (all P＜0.05); percentage of G1 phase and S phase in U0126+E2 group was significant difference with those in Bibf1120+E2 group or ponatinib+E2 group (P＜0.05).The number of cell colony in E2 group (110± 17) was more than those in control group (65±8) ;the number of cell colony in U0126+E2 group(28±4),Bibf1120+E2 group(38±5) or Ponatinib+E2group(42±6) were significant different with those in E2 group (P＜0.05),the number of cell colony in U0126+E2 group was significant difference with those in Bibf1 120+E2 group or Ponatinib+E2 group (all P＜0.05).The results shown that the abilities of proliferation and cell migration were significantly increased in cells after estradiol stimulation.Conclusion Estradiol inducing the production of VEGF and bFGF could activate MAPK pathway through ER-independent manner,further promote development.
