中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2014年
11期
811-815
,共5页
宋红林%梁少凤%张洁清%李力
宋紅林%樑少鳳%張潔清%李力
송홍림%량소봉%장길청%리력
子宫内膜肿瘤%雌二醇%丝苏氨酸蛋白激酶%核因子κB%细胞增殖%细胞运动
子宮內膜腫瘤%雌二醇%絲囌氨痠蛋白激酶%覈因子κB%細胞增殖%細胞運動
자궁내막종류%자이순%사소안산단백격매%핵인자κB%세포증식%세포운동
Endometrial neoplasms%Estradiol%AKT%NF-κ B%Cell proliferation%Cell movement
目的 探讨雌二醇在子宫内膜癌Ishikawa细胞中是否通过丝苏氨酸蛋白激酶(AKT)介导核因子κB (NF-κB)通路产生细胞因子,及其对细胞增殖及迁移的影响.方法 Western blot法检测雌二醇作用Ishikawa细胞后AKT活化情况,以及AKT和ER抑制剂对雌二醇活化AKT的影响,TransAM NF-κB p65检测不同浓度雌二醇及1×10-6 mol/L雌二醇作用不同时间后的NF-κB活性水平.经雌二醇(雌二醇组)、ER抑制剂(ER组)、AKT抑制剂(AKT组)和NF-κB抑制剂(NF-κB组)分别预处理Ishikawa细胞后,采用荧光定量PCR检测细胞内血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF) mRNA表达,Western blot法检测VEGF、bFGF蛋白的变化,流式细胞仪检测细胞周期的变化,羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)法检测细胞增殖能力,Transwell检测细胞迁移能力.结果 Western blot检测结果显示,雌二醇、AKT和ER抑制剂作用Ishikawa细胞后AKT活性水平分别为0.090±0.075、0.013 ±0.036、0.042±0.008,对照组为0.053±0.036;雌二醇组的AKT活性值比对照组、ER组及AKT组升高,差异均有统计学意义(P＜0.05).TransAM检测显示,雌二醇浓度为1×10-6mol/L时,Ishikawa细胞NF-κB活性为0.77±0.20,较对照组(0.51±0.16)明显升高,差异有统计学意义(P＜0.05);1×10-6mol/L雌二醇作用30 min及1h时,Ishikawa细胞NF-κB活性水平最高(P＜0.05).AKT组NF-κB活性为0.54±0.27,低于1×10-6mol/L雌二醇组(P＜0.05).雌二醇组VEGF、bFGF mRNA的表达分别为6.34±0.45和1.58 ±0.12,明显高于对照组的0.83±0.03和0.34±0.02(均P＜0.01);雌二醇组VEGF、bFGF蛋白的表达均明显高于对照组、ER组、AKT组和NF-κB组(均P＜0.05).雌二醇作用Ishikawa细胞后,细胞增殖数明显增多,G0/G1期比例明显低于对照组(均P＜0.01).结论 雌二醇可能经AKT激活NF-κB通路产生VEGF和bFG因子,促进Ishikawa细胞的增殖与迁移能力.
目的 探討雌二醇在子宮內膜癌Ishikawa細胞中是否通過絲囌氨痠蛋白激酶(AKT)介導覈因子κB (NF-κB)通路產生細胞因子,及其對細胞增殖及遷移的影響.方法 Western blot法檢測雌二醇作用Ishikawa細胞後AKT活化情況,以及AKT和ER抑製劑對雌二醇活化AKT的影響,TransAM NF-κB p65檢測不同濃度雌二醇及1×10-6 mol/L雌二醇作用不同時間後的NF-κB活性水平.經雌二醇(雌二醇組)、ER抑製劑(ER組)、AKT抑製劑(AKT組)和NF-κB抑製劑(NF-κB組)分彆預處理Ishikawa細胞後,採用熒光定量PCR檢測細胞內血管內皮生長因子(VEGF)、堿性成纖維細胞生長因子(bFGF) mRNA錶達,Western blot法檢測VEGF、bFGF蛋白的變化,流式細胞儀檢測細胞週期的變化,羥基熒光素二醋痠鹽琥珀酰亞胺脂(CFSE)法檢測細胞增殖能力,Transwell檢測細胞遷移能力.結果 Western blot檢測結果顯示,雌二醇、AKT和ER抑製劑作用Ishikawa細胞後AKT活性水平分彆為0.090±0.075、0.013 ±0.036、0.042±0.008,對照組為0.053±0.036;雌二醇組的AKT活性值比對照組、ER組及AKT組升高,差異均有統計學意義(P＜0.05).TransAM檢測顯示,雌二醇濃度為1×10-6mol/L時,Ishikawa細胞NF-κB活性為0.77±0.20,較對照組(0.51±0.16)明顯升高,差異有統計學意義(P＜0.05);1×10-6mol/L雌二醇作用30 min及1h時,Ishikawa細胞NF-κB活性水平最高(P＜0.05).AKT組NF-κB活性為0.54±0.27,低于1×10-6mol/L雌二醇組(P＜0.05).雌二醇組VEGF、bFGF mRNA的錶達分彆為6.34±0.45和1.58 ±0.12,明顯高于對照組的0.83±0.03和0.34±0.02(均P＜0.01);雌二醇組VEGF、bFGF蛋白的錶達均明顯高于對照組、ER組、AKT組和NF-κB組(均P＜0.05).雌二醇作用Ishikawa細胞後,細胞增殖數明顯增多,G0/G1期比例明顯低于對照組(均P＜0.01).結論 雌二醇可能經AKT激活NF-κB通路產生VEGF和bFG因子,促進Ishikawa細胞的增殖與遷移能力.
목적 탐토자이순재자궁내막암Ishikawa세포중시부통과사소안산단백격매(AKT)개도핵인자κB (NF-κB)통로산생세포인자,급기대세포증식급천이적영향.방법 Western blot법검측자이순작용Ishikawa세포후AKT활화정황,이급AKT화ER억제제대자이순활화AKT적영향,TransAM NF-κB p65검측불동농도자이순급1×10-6 mol/L자이순작용불동시간후적NF-κB활성수평.경자이순(자이순조)、ER억제제(ER조)、AKT억제제(AKT조)화NF-κB억제제(NF-κB조)분별예처리Ishikawa세포후,채용형광정량PCR검측세포내혈관내피생장인자(VEGF)、감성성섬유세포생장인자(bFGF) mRNA표체,Western blot법검측VEGF、bFGF단백적변화,류식세포의검측세포주기적변화,간기형광소이작산염호박선아알지(CFSE)법검측세포증식능력,Transwell검측세포천이능력.결과 Western blot검측결과현시,자이순、AKT화ER억제제작용Ishikawa세포후AKT활성수평분별위0.090±0.075、0.013 ±0.036、0.042±0.008,대조조위0.053±0.036;자이순조적AKT활성치비대조조、ER조급AKT조승고,차이균유통계학의의(P＜0.05).TransAM검측현시,자이순농도위1×10-6mol/L시,Ishikawa세포NF-κB활성위0.77±0.20,교대조조(0.51±0.16)명현승고,차이유통계학의의(P＜0.05);1×10-6mol/L자이순작용30 min급1h시,Ishikawa세포NF-κB활성수평최고(P＜0.05).AKT조NF-κB활성위0.54±0.27,저우1×10-6mol/L자이순조(P＜0.05).자이순조VEGF、bFGF mRNA적표체분별위6.34±0.45화1.58 ±0.12,명현고우대조조적0.83±0.03화0.34±0.02(균P＜0.01);자이순조VEGF、bFGF단백적표체균명현고우대조조、ER조、AKT조화NF-κB조(균P＜0.05).자이순작용Ishikawa세포후,세포증식수명현증다,G0/G1기비례명현저우대조조(균P＜0.01).결론 자이순가능경AKT격활NF-κB통로산생VEGF화bFG인자,촉진Ishikawa세포적증식여천이능력.
Objective The aim of this study was to explore whether estradiol induces the expression of VEGF and bFGF in the endometrial cancer Ishikawa cells by activation of NF-κB via AKT pathway,and its effect on cell proliferation.Methods Western blot was used to detect the AKT protein expression in Ishikawa cells after stimulation with estradiol,and the effect of AKT inhibitor or ER inhibitor on the activation of AKT.TransAM kit was used to detect the NF-κB p65 activity.qPCR and Western blot were used to detect the expression of VEGF and bFGF mRNA and proteins in the Ishikawa cells after estradiol treatment (E2 group),and pretreated with AKT inhibitor (AKT group) or ER inhibitor (ER group) or NFκB inhibitor (NF-κB group),following the estradiol treatment.Flow cytometry and CFSE (carboxyfluorescein diacetate,succinimidyl ester) staining were used to examine the cell proliferation.Transwell was used to detect the migration ability of Ishikawa cells.Results Expression of p-AKT protein in the Ishikawa cells was markedly higher than that in the control group (P ＜ 0.05).Expressions of p-AKT protein in the AKT and ER groups were significantly decreased than that in the E2 group (P ＜ 0.05).The NF-κB activity was highest after stimulation with 1 × 10-6 mol/L estradiol for 30 min to 1 h.AKT inhibitor significantly reduced the NF-κB activity (P ＜ 0.05).The expressions of VEGF and bFGF mRNA and proteins in the E2 group were significantly increased than that in the control group (P ＜ 0.05),and their expression in the AKT,ER and NF-κB groups were significantly decreased than that in the E2 group (P ＜ 0.05).The proliferation and migration abihties of the Ishikawa cells were significantly increased after estradiol stimulation.Conclusions Estradiol induces the production of VEGF and bFGF through activating NF-κB via AKT pathway,and enhances the proliferation and migration ability of cancer cells.
