中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2011年
12期
931-935
,共5页
姜伟%王宜生%丛青%李明江%Bin Ye%徐丛剑
薑偉%王宜生%叢青%李明江%Bin Ye%徐叢劍
강위%왕의생%총청%리명강%Bin Ye%서총검
卵巢肿瘤%肿瘤浸润%血小板活化因子%cAMP反应元件结合蛋白质%基质金属蛋白酶2
卵巢腫瘤%腫瘤浸潤%血小闆活化因子%cAMP反應元件結閤蛋白質%基質金屬蛋白酶2
란소종류%종류침윤%혈소판활화인자%cAMP반응원건결합단백질%기질금속단백매2
Ovarian neoplasms%Neoplasm invasiveness%Platelet activating factor%CyclicAMP response element-binding protein%Matrix metalloproteinase 2
目的 探讨血小板活化因子(PAF)对卵巢癌细胞侵袭的影响及其相关机制,为卵巢癌的治疗提供新的靶点.方法 (1)以100 nmol/L的PAF分别处理卵巢浆液性癌细胞株OVCA429细胞及卵巢黏液性癌细胞株RMUG-L细胞,均以仅加入二甲基亚砜(DMSO)为对照,采用体外侵袭实验检测其穿膜细胞数.(2)以不同浓度(分别为0、0.1、1、10、100、1000 nmol/L)的PAF处理后检测OVCA429细胞中环腺苷酸应答元件结合蛋白(CREB)、磷酸化CREB(p-CREB)、基质金属蛋白酶2(MMP-2)蛋白的表达水平；以100 nmol/L的PAF处理不同时间(分别为0、5min、10 min、30 min、1h及12 h)后检测OVCA429细胞中有丝分裂原活化蛋白激酶p38蛋白(p38 MAPK)、磷酸化p38 MAPK(p-p38 MAPK)、CREB及p-CREB蛋白的表达水平,上述各蛋白的表达水平均采用蛋白印迹法检测.(3) OVCA429细胞在加入PAF( 100 nmol/L)的基础上,分别加入各蛋白的抑制剂,即PAF受体(PAFR)抑制剂-WEB2076(50 μmol/L)、p-p38 MAPK抑制剂-SB203580(10 μmol/L)、CREB结合蛋白(CBP)-CREB相互作用抑制剂-217505(25 μmol/L).实验分为6组:PAF组、PAF+WEB2076组、PAF+ SB203580组、PAF+ 217505组、PAF+ SB203580 +217505组及空白对照组,采用蛋白印迹法检测各组细胞中p-p38 MAPK、p-CREB、MMP-2蛋白的表达强度.结果 (1)体外侵袭实验显示,予PAF处理后,OVCA429细胞的穿膜细胞数[(118±23)个]明显多于其对照细胞[(36±8)个,P＜0.01],而RMUG-L细胞的穿膜细胞数[(45±13)个]与其对照细胞[(53±9)个]比较无明显变化(P＞0.05).(2)不同浓度的PAF处理后,OVCA429细胞中p-CREB、MMP-2蛋白的表达水平呈明显的剂量依赖性(P＜0.01),0.1 nmol/L的PAF即可引起p-CREB、MMP-2蛋白表达水平明显升高,至100 nmol/L时达高峰；而CREB蛋白的表达无明显变化.100nmoL/L的PAF处理不同时间后,OVCA429细胞中p38 MAPK、CREB蛋白表达无明显变化；而p-p38 MAPK、p-CREB蛋白表达水平明显升高(P＜0.01),至处理10 min时达高峰,随后逐渐降低.(3)与空白对照组比较,PAF组细胞中p-p38 MAPK、p-CREB及MMP-2蛋白的表达强度明显增强.与PAF组比较,PAF+ WEB2076组、PAF+SB203580组、PAF+ SB203580+ 217505组细胞中p-p38 MAPK、p-CREB及MMP-2蛋白的表达强度明显减弱,但PAF+ SB203580+ 217505组与PAF+ WEB2076组及PAF+ SB203580组相比无明显差异；PAF +217505组细胞中p-p38 MAPK、p-CREB蛋白的表达强度无明显变化,但其MMP-2蛋白的表达强度明显减弱.结论 PAF可促进卵巢浆液性癌OVCA429细胞的侵袭,这一作用可能是通过p38MAPK激活转录因子CREB,进一步上调MMP-2蛋白的表达而实现的.
目的 探討血小闆活化因子(PAF)對卵巢癌細胞侵襲的影響及其相關機製,為卵巢癌的治療提供新的靶點.方法 (1)以100 nmol/L的PAF分彆處理卵巢漿液性癌細胞株OVCA429細胞及卵巢黏液性癌細胞株RMUG-L細胞,均以僅加入二甲基亞砜(DMSO)為對照,採用體外侵襲實驗檢測其穿膜細胞數.(2)以不同濃度(分彆為0、0.1、1、10、100、1000 nmol/L)的PAF處理後檢測OVCA429細胞中環腺苷痠應答元件結閤蛋白(CREB)、燐痠化CREB(p-CREB)、基質金屬蛋白酶2(MMP-2)蛋白的錶達水平；以100 nmol/L的PAF處理不同時間(分彆為0、5min、10 min、30 min、1h及12 h)後檢測OVCA429細胞中有絲分裂原活化蛋白激酶p38蛋白(p38 MAPK)、燐痠化p38 MAPK(p-p38 MAPK)、CREB及p-CREB蛋白的錶達水平,上述各蛋白的錶達水平均採用蛋白印跡法檢測.(3) OVCA429細胞在加入PAF( 100 nmol/L)的基礎上,分彆加入各蛋白的抑製劑,即PAF受體(PAFR)抑製劑-WEB2076(50 μmol/L)、p-p38 MAPK抑製劑-SB203580(10 μmol/L)、CREB結閤蛋白(CBP)-CREB相互作用抑製劑-217505(25 μmol/L).實驗分為6組:PAF組、PAF+WEB2076組、PAF+ SB203580組、PAF+ 217505組、PAF+ SB203580 +217505組及空白對照組,採用蛋白印跡法檢測各組細胞中p-p38 MAPK、p-CREB、MMP-2蛋白的錶達彊度.結果 (1)體外侵襲實驗顯示,予PAF處理後,OVCA429細胞的穿膜細胞數[(118±23)箇]明顯多于其對照細胞[(36±8)箇,P＜0.01],而RMUG-L細胞的穿膜細胞數[(45±13)箇]與其對照細胞[(53±9)箇]比較無明顯變化(P＞0.05).(2)不同濃度的PAF處理後,OVCA429細胞中p-CREB、MMP-2蛋白的錶達水平呈明顯的劑量依賴性(P＜0.01),0.1 nmol/L的PAF即可引起p-CREB、MMP-2蛋白錶達水平明顯升高,至100 nmol/L時達高峰；而CREB蛋白的錶達無明顯變化.100nmoL/L的PAF處理不同時間後,OVCA429細胞中p38 MAPK、CREB蛋白錶達無明顯變化；而p-p38 MAPK、p-CREB蛋白錶達水平明顯升高(P＜0.01),至處理10 min時達高峰,隨後逐漸降低.(3)與空白對照組比較,PAF組細胞中p-p38 MAPK、p-CREB及MMP-2蛋白的錶達彊度明顯增彊.與PAF組比較,PAF+ WEB2076組、PAF+SB203580組、PAF+ SB203580+ 217505組細胞中p-p38 MAPK、p-CREB及MMP-2蛋白的錶達彊度明顯減弱,但PAF+ SB203580+ 217505組與PAF+ WEB2076組及PAF+ SB203580組相比無明顯差異；PAF +217505組細胞中p-p38 MAPK、p-CREB蛋白的錶達彊度無明顯變化,但其MMP-2蛋白的錶達彊度明顯減弱.結論 PAF可促進卵巢漿液性癌OVCA429細胞的侵襲,這一作用可能是通過p38MAPK激活轉錄因子CREB,進一步上調MMP-2蛋白的錶達而實現的.
목적 탐토혈소판활화인자(PAF)대란소암세포침습적영향급기상관궤제,위란소암적치료제공신적파점.방법 (1)이100 nmol/L적PAF분별처리란소장액성암세포주OVCA429세포급란소점액성암세포주RMUG-L세포,균이부가입이갑기아풍(DMSO)위대조,채용체외침습실험검측기천막세포수.(2)이불동농도(분별위0、0.1、1、10、100、1000 nmol/L)적PAF처리후검측OVCA429세포중배선감산응답원건결합단백(CREB)、린산화CREB(p-CREB)、기질금속단백매2(MMP-2)단백적표체수평；이100 nmol/L적PAF처리불동시간(분별위0、5min、10 min、30 min、1h급12 h)후검측OVCA429세포중유사분렬원활화단백격매p38단백(p38 MAPK)、린산화p38 MAPK(p-p38 MAPK)、CREB급p-CREB단백적표체수평,상술각단백적표체수평균채용단백인적법검측.(3) OVCA429세포재가입PAF( 100 nmol/L)적기출상,분별가입각단백적억제제,즉PAF수체(PAFR)억제제-WEB2076(50 μmol/L)、p-p38 MAPK억제제-SB203580(10 μmol/L)、CREB결합단백(CBP)-CREB상호작용억제제-217505(25 μmol/L).실험분위6조:PAF조、PAF+WEB2076조、PAF+ SB203580조、PAF+ 217505조、PAF+ SB203580 +217505조급공백대조조,채용단백인적법검측각조세포중p-p38 MAPK、p-CREB、MMP-2단백적표체강도.결과 (1)체외침습실험현시,여PAF처리후,OVCA429세포적천막세포수[(118±23)개]명현다우기대조세포[(36±8)개,P＜0.01],이RMUG-L세포적천막세포수[(45±13)개]여기대조세포[(53±9)개]비교무명현변화(P＞0.05).(2)불동농도적PAF처리후,OVCA429세포중p-CREB、MMP-2단백적표체수평정명현적제량의뢰성(P＜0.01),0.1 nmol/L적PAF즉가인기p-CREB、MMP-2단백표체수평명현승고,지100 nmol/L시체고봉；이CREB단백적표체무명현변화.100nmoL/L적PAF처리불동시간후,OVCA429세포중p38 MAPK、CREB단백표체무명현변화；이p-p38 MAPK、p-CREB단백표체수평명현승고(P＜0.01),지처리10 min시체고봉,수후축점강저.(3)여공백대조조비교,PAF조세포중p-p38 MAPK、p-CREB급MMP-2단백적표체강도명현증강.여PAF조비교,PAF+ WEB2076조、PAF+SB203580조、PAF+ SB203580+ 217505조세포중p-p38 MAPK、p-CREB급MMP-2단백적표체강도명현감약,단PAF+ SB203580+ 217505조여PAF+ WEB2076조급PAF+ SB203580조상비무명현차이；PAF +217505조세포중p-p38 MAPK、p-CREB단백적표체강도무명현변화,단기MMP-2단백적표체강도명현감약.결론 PAF가촉진란소장액성암OVCA429세포적침습,저일작용가능시통과p38MAPK격활전록인자CREB,진일보상조MMP-2단백적표체이실현적.
Objective To investigate the effects and possible mechanisms of platelet-activating factor (PAF) on the invasion of ovarian cancer cells and to provide a potential target for ovarian cancer therapy.Methods ( 1 ) Serous type ovarian cancer cell line OVCA429 with platelet-activating factor receptor (PAFR) positive and mucinous type cell line RMUG-L (PAFR negative) were treated with 100 nmol/L of the PAF,cell invasion ability was determined by transwell cell migration assay.(2) For determination of the optimal PAF concentration,ovarian cancer cell OVCA429 was treated by 0,0.1,1,10,100,and 1000 nmol/L of PAF for 10 minutes or 24 hour,respectively.To observe the different time point of protein changes,OVCA429 were treated by 100 nmol/L of PAF for 0,5 minutes,10 minutes,30 minutes,1 hour or 12 hours,respectively.The total proteins of treated cells were extracted according to standard protocol.The expression of p38 mitogen-activated protein kinase (p38 MAPK),phosphorylated p38 MAPK (p-p38 MAPK),transcription factor response element-binding protein (CREB),phosphorylated CREB (p-CREB) and matrix metalloproteinase-2 (MMP-2) were detected by western blot.(3) To verify the pathway involved in the PAF induction of the cancer cell invasion,we repeated the experiments by adding the inhibitors when treating cells with PAF.The inhibitors used were as follows,PAFR inhibitor-WEB2076 (50 μmol/L),pp38 MAPK inhibitor-SB203580 (10 μmol/L),CREB binding protein (CBP)-CREB interaction inhibitor217505(25 μ mol/L).All treated cells were divided into 6 groups:control group,PAF group,PAF + WEB2076 group,PAF + SB203580 group,PAF + 217505 group and PAF + SB203580 + 217505group.Results ( 1 ) By transwell assay,100 nmol/L of PAF could improve the invasion ability of OVCA429 cell significantly [ PAF:( 118 ± 23 ) cells vs.control:(36 ± 8 ) cells,P ＜ 0.0l ],while the same treatment had no effect on RMUG-L cells [PAF:(45 t 13) cells vs.control:(53 ±9) cells,P＞0.05].(2) Even a very low concentration of PAF (0.1 nmol/L) could increase the expression of p-CREB and MMP-2,while the most effective concentration of PAF was 100 nmol/L.The highest p-CREB protein expression was detected at 10 minutes after administration of 100 nmol/L PAF,as well as the expression of p-p38 MAPK protein.Even 12 hours after treatment the p-p38 MAPK protein could be detected,while there was no significant difference in the expression of CREB ( P ＞ 0.05 ).(3) As compared with PAF group,both in PAF + WEB2076 group and PAF + SB203580 group,the expressions of p-p38 MAPK,p-CREB and MMP-2 protein were decreased significantly; in PAF + 217505 group,although the expression of p-p38 MAPK and p-CREB protein was significantly higher than the control group,the expression of MMP-2 protein was significantly lower; in PAF + SB203580 + 217505 group,the expression of these three proteins were also significantly lower,but there was no significant difference as compared with that in the PAF + WEB2076 or PAF + SB203580 group.Conclusion PAF could induce MMP-2 expression and contributed to PAFR positive ovarian cancer cell invasion via activation of CREB by phosphorylating of p38 MAPK.
