目的 探讨在雌激素剥夺状态下表皮生长因子(EGF)、表皮生长因子受体(EGFR)、磷酸化细胞外信号调节激酶1/2(p-ERK1/2)在内异症发病中的作用.方法 采用细胞培养基快速雌激素剥夺法和对内异症模型裸鼠切除双侧卵巢(去势)的方法,建立体外和体内两种雌激素剥夺的实验研究模型.(1)体外实验:对体外培养的雌激素剥夺状态下的异位子宫内膜细胞按以下不同的处理方法分为4组:①EGF组:加入不同浓度(0.01、0.1、1、10、50、100 ng/ml)的EGF培养细胞72 h(以浓度为10 ng/ml的EGF培养72 h的结果代表EGF组的结果)；②EGF+ PD98059组:以5×10-2 mol/LPD98059[细胞外信号调节激酶(ERK)抑制剂]培养细胞24 h后,再以10 ng/ml EGF +5×10-2 mol/LPD98059培养细胞72 h；③EGF+ ICI182780组:以10-6 mol/L ICI182780(ER抑制剂)培养细胞24 h后,再以10 ng/ml EGF+ 10-6 mol/L ICI182780培养细胞72 h；④空白对照组:不进行任何处理.用四甲基偶氮唑盐比色法(MTT)检测处理后各组异位子宫内膜细胞的增殖活性[以吸光度(A)值表示]；蛋白印迹法检测EGF组、EGF+PD98059组异位子宫内膜细胞中p-ERK1/2的蛋白表达.(2)体内实验:利用随机数字表法将64只内异症模型裸鼠随机分成对照组和去势组,每组各32只,去势组在造模3周后切除双侧卵巢.于造模后的4,6,8,10周,采用蛋白印迹法检测两组裸鼠腹腔病灶中EGF、EGFR、p-ERK1/2的蛋白表达水平.结果 (1)细胞增殖活性:EGF组经不同浓度(0.01、0.1、1、10、50、100 ng/ml)的EGF作用72 h后,细胞增殖活性依次为0.310±0.010、0.340±0.020、0.670±0.010、0.980±0.030、1.360 ±0.020、1.670±0.020,随EGF浓度的增加,细胞增殖活性逐渐增加.EGF+PD98059组细胞增殖活性为0.680±0.030,与EGF组(以浓度为10 ng/ml的EGF培养72 h的结果代表EGF组的结果)比较,差异有统计学意义(P＜O.01)；EGF+ICI182780组为0.330±0.030,空白对照组为0.310±0.030,两组比较,差异无统计学意义(P＞0.05).(2)蛋白表达水平:①体外实验中,EGF组p-ERK1/2的蛋白表达水平为0.670±0.020,空白对照组为0.600±0.010,两组比较,差异有统计学意义(P＜0.05)；EGF+PD98059组为0.610±0.020,与空白对照组比较,差异无统计学意义(P＞0.05).②体内实验中,在造模后4、6、8周,去势组和对照组裸鼠腹腔异位病灶中EGF蛋白表达水平分别为(0.530±0.015和0.610±0.015)、(0.400±0.029和0.620±0.018)、(0.560±0.026和0.630±0.021)；EGFR蛋白表达水平分别为(0.500±0.030和0.640±0.030)、(0.470±0.020和0.630±0.020)、(0.510±0.030和0.610±0.020)；p-ERK1/2蛋白表达水平分别为(0.500±0.020和0.580±0.020)、(0.490±0.020和0.580±0.020)、(0.570±0.020和0.590±0.020)；两组在各个时间点EGF、EGFR、p-ERK1/2蛋白表达水平比较,差异均有统计学意义(P＜0.01,P＜0.01,P＜0.05)；在造模后10周,去势组和对照组EGF的蛋白表达水平为(0.620±0.020和0.620±0.020),EGFR为(0.610±0.020和0.610±0.020),p-ERK1/2为(0.590±0.010和0.600±0.020),两组比较,差异均无统计学意义(P＞0.05).结论 在雌激素剥夺状态下,EGF可以通过激活ERK信号通路,刺激异位子宫内膜细胞的增殖,但需依赖ER的存在；异位子宫内膜病灶中EGF、EGFR、pERK1/2蛋白表达水平的抑制随雌激素剥夺时间的延长而逐渐减弱.
目的 探討在雌激素剝奪狀態下錶皮生長因子(EGF)、錶皮生長因子受體(EGFR)、燐痠化細胞外信號調節激酶1/2(p-ERK1/2)在內異癥髮病中的作用.方法 採用細胞培養基快速雌激素剝奪法和對內異癥模型裸鼠切除雙側卵巢(去勢)的方法,建立體外和體內兩種雌激素剝奪的實驗研究模型.(1)體外實驗:對體外培養的雌激素剝奪狀態下的異位子宮內膜細胞按以下不同的處理方法分為4組:①EGF組:加入不同濃度(0.01、0.1、1、10、50、100 ng/ml)的EGF培養細胞72 h(以濃度為10 ng/ml的EGF培養72 h的結果代錶EGF組的結果)；②EGF+ PD98059組:以5×10-2 mol/LPD98059[細胞外信號調節激酶(ERK)抑製劑]培養細胞24 h後,再以10 ng/ml EGF +5×10-2 mol/LPD98059培養細胞72 h；③EGF+ ICI182780組:以10-6 mol/L ICI182780(ER抑製劑)培養細胞24 h後,再以10 ng/ml EGF+ 10-6 mol/L ICI182780培養細胞72 h；④空白對照組:不進行任何處理.用四甲基偶氮唑鹽比色法(MTT)檢測處理後各組異位子宮內膜細胞的增殖活性[以吸光度(A)值錶示]；蛋白印跡法檢測EGF組、EGF+PD98059組異位子宮內膜細胞中p-ERK1/2的蛋白錶達.(2)體內實驗:利用隨機數字錶法將64隻內異癥模型裸鼠隨機分成對照組和去勢組,每組各32隻,去勢組在造模3週後切除雙側卵巢.于造模後的4,6,8,10週,採用蛋白印跡法檢測兩組裸鼠腹腔病竈中EGF、EGFR、p-ERK1/2的蛋白錶達水平.結果 (1)細胞增殖活性:EGF組經不同濃度(0.01、0.1、1、10、50、100 ng/ml)的EGF作用72 h後,細胞增殖活性依次為0.310±0.010、0.340±0.020、0.670±0.010、0.980±0.030、1.360 ±0.020、1.670±0.020,隨EGF濃度的增加,細胞增殖活性逐漸增加.EGF+PD98059組細胞增殖活性為0.680±0.030,與EGF組(以濃度為10 ng/ml的EGF培養72 h的結果代錶EGF組的結果)比較,差異有統計學意義(P＜O.01)；EGF+ICI182780組為0.330±0.030,空白對照組為0.310±0.030,兩組比較,差異無統計學意義(P＞0.05).(2)蛋白錶達水平:①體外實驗中,EGF組p-ERK1/2的蛋白錶達水平為0.670±0.020,空白對照組為0.600±0.010,兩組比較,差異有統計學意義(P＜0.05)；EGF+PD98059組為0.610±0.020,與空白對照組比較,差異無統計學意義(P＞0.05).②體內實驗中,在造模後4、6、8週,去勢組和對照組裸鼠腹腔異位病竈中EGF蛋白錶達水平分彆為(0.530±0.015和0.610±0.015)、(0.400±0.029和0.620±0.018)、(0.560±0.026和0.630±0.021)；EGFR蛋白錶達水平分彆為(0.500±0.030和0.640±0.030)、(0.470±0.020和0.630±0.020)、(0.510±0.030和0.610±0.020)；p-ERK1/2蛋白錶達水平分彆為(0.500±0.020和0.580±0.020)、(0.490±0.020和0.580±0.020)、(0.570±0.020和0.590±0.020)；兩組在各箇時間點EGF、EGFR、p-ERK1/2蛋白錶達水平比較,差異均有統計學意義(P＜0.01,P＜0.01,P＜0.05)；在造模後10週,去勢組和對照組EGF的蛋白錶達水平為(0.620±0.020和0.620±0.020),EGFR為(0.610±0.020和0.610±0.020),p-ERK1/2為(0.590±0.010和0.600±0.020),兩組比較,差異均無統計學意義(P＞0.05).結論 在雌激素剝奪狀態下,EGF可以通過激活ERK信號通路,刺激異位子宮內膜細胞的增殖,但需依賴ER的存在；異位子宮內膜病竈中EGF、EGFR、pERK1/2蛋白錶達水平的抑製隨雌激素剝奪時間的延長而逐漸減弱.
목적 탐토재자격소박탈상태하표피생장인자(EGF)、표피생장인자수체(EGFR)、린산화세포외신호조절격매1/2(p-ERK1/2)재내이증발병중적작용.방법 채용세포배양기쾌속자격소박탈법화대내이증모형라서절제쌍측란소(거세)적방법,건입체외화체내량충자격소박탈적실험연구모형.(1)체외실험:대체외배양적자격소박탈상태하적이위자궁내막세포안이하불동적처리방법분위4조:①EGF조:가입불동농도(0.01、0.1、1、10、50、100 ng/ml)적EGF배양세포72 h(이농도위10 ng/ml적EGF배양72 h적결과대표EGF조적결과)；②EGF+ PD98059조:이5×10-2 mol/LPD98059[세포외신호조절격매(ERK)억제제]배양세포24 h후,재이10 ng/ml EGF +5×10-2 mol/LPD98059배양세포72 h；③EGF+ ICI182780조:이10-6 mol/L ICI182780(ER억제제)배양세포24 h후,재이10 ng/ml EGF+ 10-6 mol/L ICI182780배양세포72 h；④공백대조조:불진행임하처리.용사갑기우담서염비색법(MTT)검측처리후각조이위자궁내막세포적증식활성[이흡광도(A)치표시]；단백인적법검측EGF조、EGF+PD98059조이위자궁내막세포중p-ERK1/2적단백표체.(2)체내실험:이용수궤수자표법장64지내이증모형라서수궤분성대조조화거세조,매조각32지,거세조재조모3주후절제쌍측란소.우조모후적4,6,8,10주,채용단백인적법검측량조라서복강병조중EGF、EGFR、p-ERK1/2적단백표체수평.결과 (1)세포증식활성:EGF조경불동농도(0.01、0.1、1、10、50、100 ng/ml)적EGF작용72 h후,세포증식활성의차위0.310±0.010、0.340±0.020、0.670±0.010、0.980±0.030、1.360 ±0.020、1.670±0.020,수EGF농도적증가,세포증식활성축점증가.EGF+PD98059조세포증식활성위0.680±0.030,여EGF조(이농도위10 ng/ml적EGF배양72 h적결과대표EGF조적결과)비교,차이유통계학의의(P＜O.01)；EGF+ICI182780조위0.330±0.030,공백대조조위0.310±0.030,량조비교,차이무통계학의의(P＞0.05).(2)단백표체수평:①체외실험중,EGF조p-ERK1/2적단백표체수평위0.670±0.020,공백대조조위0.600±0.010,량조비교,차이유통계학의의(P＜0.05)；EGF+PD98059조위0.610±0.020,여공백대조조비교,차이무통계학의의(P＞0.05).②체내실험중,재조모후4、6、8주,거세조화대조조라서복강이위병조중EGF단백표체수평분별위(0.530±0.015화0.610±0.015)、(0.400±0.029화0.620±0.018)、(0.560±0.026화0.630±0.021)；EGFR단백표체수평분별위(0.500±0.030화0.640±0.030)、(0.470±0.020화0.630±0.020)、(0.510±0.030화0.610±0.020)；p-ERK1/2단백표체수평분별위(0.500±0.020화0.580±0.020)、(0.490±0.020화0.580±0.020)、(0.570±0.020화0.590±0.020)；량조재각개시간점EGF、EGFR、p-ERK1/2단백표체수평비교,차이균유통계학의의(P＜0.01,P＜0.01,P＜0.05)；재조모후10주,거세조화대조조EGF적단백표체수평위(0.620±0.020화0.620±0.020),EGFR위(0.610±0.020화0.610±0.020),p-ERK1/2위(0.590±0.010화0.600±0.020),량조비교,차이균무통계학의의(P＞0.05).결론 재자격소박탈상태하,EGF가이통과격활ERK신호통로,자격이위자궁내막세포적증식,단수의뢰ER적존재；이위자궁내막병조중EGF、EGFR、pERK1/2단백표체수평적억제수자격소박탈시간적연장이축점감약.
Objective To study the role of epidermal growth factor (EGF),epidermal growth factor receptor(EGFR),extracellular signal-regulated kinase 1/2 (p-ERK1/2) in the pathogenesis of endometriosis under estrogen deprivation conditions.Methods The estrogen was quickly-stripped in medium and the female nude mice were castrated by bilateral oophorectomy to build estrogen deprivation in vitro and in vivo experimental models,respectively.(1) In vitro experiments:according to different treatments the estrogen deprived ectopic endometrial cells were classified into 4 groups:①EGF group:the ectopic endometrial cells were cultured for 72 hours with different concentrations of EGF (0.01,0.1,1,10,50,100 ng/ml),the results of EGF group were represented by the result of cells treated by 10 ng/ml EGF cultured for 72 hours ; ②EGF + PD98059 group:the ectopic endometrial cells were cultured for 72 hours with 5 × 10-2 mol/L PD98059 (inhibitor of ERK),followed by a cultivation for 72 hours treated by 10 ng/ml EGF + 5 × 10-2 mol/L PD98059 ; ③EGF + ICI182780 group:the ectopic endometrial cells were cultured for 72 hours with 10-6 mol/L ICI182780 [inhibitor of estrogen receptor(ER)],followed by a cultivation for 72 hours treated by 10 ng/ml EGF + 10-6 mol/L ICI182780; ④Blank control group:the ectopic endometrial cells were cultured with no treatment.The proliferation activity of ectopic endometrial cells in all groups after treatment were examined by methyl thiazolyl tetrazolium (MTT) method represented by absorbance value (A).The expression of p-ERK1/2 protein were detected by western blot.(2) In vivo experiments:64 female nude mice were randomly divided into control and castration groups (both n =32) using random number chart.The mice in castration group were castrated by bilateral oophorectomy on 3 weeks after the endometriosis model was established.The levels of EGF,EGFR,p-ERK1/2 protein in ectopic lesions of both groups were measured on 4,6,8 and 10 weeks after the endometriosis model was established by western blot.Results (1) The proliferation activity of ectopic endometrial cells:the proliferation activity of ectopic endometrial cells treated by different concentrations of EGF (0.01,0.1,1,10,50,100 ng/ml) for 72 hours were 0.310 ± 0.010,0.340 ± 0.020,0.670 ± 0.010,0.980 ± 0.030,1.360 ± 0.020,1.670 ± 0.020,respectively,the proliferation activity was increased along with of EGF concentrations.The proliferation activity was 0.680 ± 0.030 at EGF + PD98059 group,the differences exhibited significant difference when compared with that at EGF group with 100 ng/ml for 72 hours(P ＜0.01).The proliferation activity of EGF + ICI182780 and blank control groups were 0.330 ±0.030 and 0.310 ±0.030,respectively,which did not reached statistical differences(P ＞ 0.05).(2) The expression of EGF,EGFR,pERK1/2 protein:① In vitro experiments:the levels of p-ERK1/2 protein in EGF and blank control groups were 0.670 ± 0.020 and 0.600 + 0.010,respectively,which reached statistical differences (P ＜ 0.05).The level of p-ERK1/2 protein in EGF + PD98059 group was 0.610 ± 0.020,which exhibited significant differences with that at blank control group(P ＞ 0.05).② In vivo experiments:at 4,6 and 8 weeks after the endometriosis models were established,the expression of EGF protein in the ectopic lesions of castration group and control group were (0.530±0.015 versus 0.610 ±0.015),(0.400 ±0.029 versus 0.620 ±0.018),(0.560 ±0.026versus 0.630 ± 0.021),respectively,the levels of EGFR protein were (0.500 ± 0.030 versus 0.640 ±0.030),(0.470 ± 0.020 versus 0.630 ± 0.020),(0.510 ± 0.030 versus 0.610 ± 0.020) respectively,and the level of p-ERK1/2 protein were (0.500 ± 0.020 versus 0.580 ± 0.020),(0.490 ± 0.020 versus 0.580 ±0.020),(0.570 ±0.020 versus 0.590 0.020),respectively.The difference of EGF,EGFR,pERK1/2 protein expression levels between two groups did not exhibited significant difference(P ＜ 0.01,P ＜0.01,P ＜0.05).At 10 weeks after the endometriosis models were established,the levels of EGF protein in castration group and control group were both 0.620 ± 0.020,the levels of EGFR protein were both 0.610 ±0.020,and the level of p-ERK1/2 protein were 0.590 ±0.010 and 0.600 ± 0.020.No statistical difference (P ＞0.05) was found between those two groups (P ＞ O.05).Conclusions EGF could stimulate the proliferation of ectopic endometrial cells by activating the ERK pathway under estrogen deprivation conditions.The inhibition of EGF signaling system in ectopic lesions was alleviated along with the prolongation of the period of estrogen deprivation.
