中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2013年
2期
129-133
,共5页
郭瑞霞%雷佳%王新燕%葛新%胡冬梅%马秀英%李留霞%乔玉环
郭瑞霞%雷佳%王新燕%葛新%鬍鼕梅%馬秀英%李留霞%喬玉環
곽서하%뢰가%왕신연%갈신%호동매%마수영%리류하%교옥배
子宫内膜肿瘤%受体,G蛋白偶联%百日咳毒素%1-磷脂酰肌醇3-激酶%原癌基因蛋白质c-akt
子宮內膜腫瘤%受體,G蛋白偶聯%百日咳毒素%1-燐脂酰肌醇3-激酶%原癌基因蛋白質c-akt
자궁내막종류%수체,G단백우련%백일해독소%1-린지선기순3-격매%원암기인단백질c-akt
Endometrial neoplasms%Receptors,G-protein-coupled%Pertussis toxin%1-Phosphatidylinositol 3-kinase%Proto-oncogene proteins c-akt
目的 研究百日咳毒素(PTX)对G蛋白偶联ER(GPER)介导的雌激素激活的子宫内膜癌细胞系Ishikawa和HEC-1A细胞中磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路的影响.方法 采用免疫组化SP法检测Ishikawa和HEC-1A细胞中GPER蛋白的表达.将不同浓度(0、0.1、0.5、1.0 μg/ml)的PTX分别处理Ishikawa和HEC-1A细胞30 min后,再予上述浓度的PTX分别联合17β雌二醇(17β-E2；1×10-6molL)共同处理HEC-1A细胞15 min、Ishikawa细胞30 min,采用蛋白印迹法检测Ishikawa和HEC-1A细胞中GPER、ERα、ERβ蛋白的表达及Akt的活化状态[Akt的活化状态以磷酸化Akt(p-Akt)/Akt表示].结果(1)免疫组化法检测显示,在Ishikawa和HEC-1A细胞中GPER蛋白均在细胞质中呈棕黄色阳性表达.(2)蛋白印迹法检测显示,不同浓度(0、0.1、0.5、1.0μg/ml)的PTX联合17β-E2(1×10-6 mol/L)处理后,在Ishikawa细胞中,p-Akt/Akt分别为0.74±0.54、0.34 ±0.06、0.18±0.03、0.07±0.15,GPER蛋白的表达水平分别为0.872±0.490、0.395 ±0.054、0.145 ±0.014、0.034±0.008,随着PTX浓度的增加,p-Akt/Akt、GPER蛋白的表达水平均明显下降(P＜0.05),且当PTX浓度为1.0μg/ml时下降最显著(F=63.729,P=0.0001;F ＝160.284,P=0.0001)；而ERα和ERβ蛋白的表达水平均无明显变化(P＞0.05).在HEC-1A细胞中,p-Akt/Akt分别为0.73 ±0.09、0.26±0.14、0.11 ±0.03、0,GPER蛋白的表达水平分别为0.927±0.134、0.485±0.022、0.194±0.004、0,随着PTX浓度的增加,p-Akt/Akt、GPER蛋白的表达水平均明显下降(P＜0.05),且当PTX浓度为1.0 μg/ml时均降至0(F=1039.321,P=0.0001；F=109.646,P=0.0001)；而ERα蛋白的表达水平无明显变化(P＞0.05),ERβ蛋白呈阴性表达.结论 在子宫内膜癌HEC-1A和Ishikawa细胞中,FTX阻断GPER后,可抑制雌激素对子宫内膜癌细胞PI3K/Akt信号通路的活化作用.
目的 研究百日咳毒素(PTX)對G蛋白偶聯ER(GPER)介導的雌激素激活的子宮內膜癌細胞繫Ishikawa和HEC-1A細胞中燐脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信號通路的影響.方法 採用免疫組化SP法檢測Ishikawa和HEC-1A細胞中GPER蛋白的錶達.將不同濃度(0、0.1、0.5、1.0 μg/ml)的PTX分彆處理Ishikawa和HEC-1A細胞30 min後,再予上述濃度的PTX分彆聯閤17β雌二醇(17β-E2；1×10-6molL)共同處理HEC-1A細胞15 min、Ishikawa細胞30 min,採用蛋白印跡法檢測Ishikawa和HEC-1A細胞中GPER、ERα、ERβ蛋白的錶達及Akt的活化狀態[Akt的活化狀態以燐痠化Akt(p-Akt)/Akt錶示].結果(1)免疫組化法檢測顯示,在Ishikawa和HEC-1A細胞中GPER蛋白均在細胞質中呈棕黃色暘性錶達.(2)蛋白印跡法檢測顯示,不同濃度(0、0.1、0.5、1.0μg/ml)的PTX聯閤17β-E2(1×10-6 mol/L)處理後,在Ishikawa細胞中,p-Akt/Akt分彆為0.74±0.54、0.34 ±0.06、0.18±0.03、0.07±0.15,GPER蛋白的錶達水平分彆為0.872±0.490、0.395 ±0.054、0.145 ±0.014、0.034±0.008,隨著PTX濃度的增加,p-Akt/Akt、GPER蛋白的錶達水平均明顯下降(P＜0.05),且噹PTX濃度為1.0μg/ml時下降最顯著(F=63.729,P=0.0001;F ＝160.284,P=0.0001)；而ERα和ERβ蛋白的錶達水平均無明顯變化(P＞0.05).在HEC-1A細胞中,p-Akt/Akt分彆為0.73 ±0.09、0.26±0.14、0.11 ±0.03、0,GPER蛋白的錶達水平分彆為0.927±0.134、0.485±0.022、0.194±0.004、0,隨著PTX濃度的增加,p-Akt/Akt、GPER蛋白的錶達水平均明顯下降(P＜0.05),且噹PTX濃度為1.0 μg/ml時均降至0(F=1039.321,P=0.0001；F=109.646,P=0.0001)；而ERα蛋白的錶達水平無明顯變化(P＞0.05),ERβ蛋白呈陰性錶達.結論 在子宮內膜癌HEC-1A和Ishikawa細胞中,FTX阻斷GPER後,可抑製雌激素對子宮內膜癌細胞PI3K/Akt信號通路的活化作用.
목적 연구백일해독소(PTX)대G단백우련ER(GPER)개도적자격소격활적자궁내막암세포계Ishikawa화HEC-1A세포중린지선기순3격매(PI3K)/단백격매B(Akt)신호통로적영향.방법 채용면역조화SP법검측Ishikawa화HEC-1A세포중GPER단백적표체.장불동농도(0、0.1、0.5、1.0 μg/ml)적PTX분별처리Ishikawa화HEC-1A세포30 min후,재여상술농도적PTX분별연합17β자이순(17β-E2；1×10-6molL)공동처리HEC-1A세포15 min、Ishikawa세포30 min,채용단백인적법검측Ishikawa화HEC-1A세포중GPER、ERα、ERβ단백적표체급Akt적활화상태[Akt적활화상태이린산화Akt(p-Akt)/Akt표시].결과(1)면역조화법검측현시,재Ishikawa화HEC-1A세포중GPER단백균재세포질중정종황색양성표체.(2)단백인적법검측현시,불동농도(0、0.1、0.5、1.0μg/ml)적PTX연합17β-E2(1×10-6 mol/L)처리후,재Ishikawa세포중,p-Akt/Akt분별위0.74±0.54、0.34 ±0.06、0.18±0.03、0.07±0.15,GPER단백적표체수평분별위0.872±0.490、0.395 ±0.054、0.145 ±0.014、0.034±0.008,수착PTX농도적증가,p-Akt/Akt、GPER단백적표체수평균명현하강(P＜0.05),차당PTX농도위1.0μg/ml시하강최현저(F=63.729,P=0.0001;F ＝160.284,P=0.0001)；이ERα화ERβ단백적표체수평균무명현변화(P＞0.05).재HEC-1A세포중,p-Akt/Akt분별위0.73 ±0.09、0.26±0.14、0.11 ±0.03、0,GPER단백적표체수평분별위0.927±0.134、0.485±0.022、0.194±0.004、0,수착PTX농도적증가,p-Akt/Akt、GPER단백적표체수평균명현하강(P＜0.05),차당PTX농도위1.0 μg/ml시균강지0(F=1039.321,P=0.0001；F=109.646,P=0.0001)；이ERα단백적표체수평무명현변화(P＞0.05),ERβ단백정음성표체.결론 재자궁내막암HEC-1A화Ishikawa세포중,FTX조단GPER후,가억제자격소대자궁내막암세포PI3K/Akt신호통로적활화작용.
Objective To investigate the influence of pertussis toxin(PTX)on G protein-coupled estrogen receptor(GPER)-mediated activation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt)signaling activated by 17 β-estradiol(17β-E2)in endometrial carcinoma cells.Methods Expressions of GPER protein were detected by immunohistochemical SP method in Ishikawa and HEC-1A cells.Changes of levels of GPER,ERα and ERβ protein and the activation of Akt protein were observed by western blot in the two cells after they were treated by PTX for 30 minutes at different concentrations(0,0.1,0.5,1.0 μg/ml),and then co-stimulated with with 1 × 10-6 mol/L 17β-E2 respectively at different time (Ishikawa 30 minutes,HEC-1A 15 minutes).Results(1)Immunohistochemical SP method showed that GPER was positive stained in cell cytoplasm of Ishikawa and HEC-1A cell.(2)After co-treated with PTX at different concentrations(0,O.1,0.5,1.0 μg/ml)and 10-6 mol/L 17β-E2,in Ishikawa cell,the ratio of pAkt/Akt was 0.74 ±0.54,0.34 ±0.06,0.18 ±0.03,0.07 ±0.15,the gray values of GPER was 0.872 ± 0.490,0.395 ± 0.054,0.145 ± 0.014,0.034 ± 0.008,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which was most obviously when the concentration was 1.0 μg/ml(F =63.729,P =0.0001;F =160.284,P =0.0001);ERα and ERβ protein had no significant change among different groups(P ＞0.05).In HEC-1A cell,the ratio of pAkt/Akt was 0.73 ±0.09,0.26 ±0.14,0.11 ±0.03,0,the Gray values of GPER is 0.927 ±0.134,0.485 ± 0.022,0.194 ± 0.004,0,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which were also completely inhibited when the concentration was 1 μg/ml(F =1039.321,P =0.0001;F =109.646,P =0.0001),ERα protein had no significant differences(P ＞ 0.05)among different groups.ERβ was negatively expressed.Conclusion The results proposed that the activation of PI3K/Akt signaling in Ishikawa and HEC-1A cells could be inhibited after blocking the role of GPER by PTX.
