卵泡刺激素对卵巢癌细胞生物学行为的影响及其相关分子机制
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摘要
卵巢恶性肿瘤是女性生殖器常见的三大恶性肿瘤之一,其中卵巢癌占其85%～90%,虽然手术、化学药物治疗、放射治疗等传统治疗手段不断改进,卵巢癌的治疗效果改善甚微,其5年生存率徘徊于30%～40%,病亡率居妇科恶性肿瘤之首。
临床上卵巢癌好发于绝经和卵巢切除术后妇女,常伴有卵泡刺激素(FSH)和黄体生成素(LH)的升高;而多次妊娠、母乳喂养、口服避孕药和接受激素替代疗法的妇女发病风险较低,FSH和LH也较低。近20年来有关研究也表明FSH对卵巢癌的发生发展起重要作用,但其对卵巢癌细胞生物学行为的影响研究甚少。本课题研究表明FSH与卵巢癌细胞的增殖、凋亡、迁移、侵袭和血管内皮生长因子(VEGF)有关联;通过对FSH调控卵巢癌VEGF的相关分子机制、卵巢癌临床资料和动物实验的研究分析,将有助于卵巢癌的病因研究,为新的治疗药物和方法的产生提供依据。
本课题共分四部分:①卵泡刺激素对卵巢癌细胞增值、凋亡、迁移、侵袭和血管内皮生长因子的影响;②卵泡刺激素调控卵巢癌细胞血管内皮生长因子的相关分子机制;③卵巢癌相关临床资料的分析;④动物实验。
第一部分卵泡刺激素对卵巢癌细胞增值、凋亡、迁移、侵袭和血管内皮生长因子的影响
目的研究FSH对卵巢癌细胞株SKOV-3和ES-2细胞增殖、凋亡、迁移、侵袭和血管内皮生长因子的影响。
方法将FSH以不同浓度(0、10、20、40、80、160mIU/mL)分别作用于1×10~5或2.5×10~6个SKOV-3细胞和6×10~4或1.5×10~6个ES-2细胞,其中FSH作用于SKOV-3细胞的适宜时间为48h,ES-2细胞为24h,二甲基噻唑二苯基四唑溴盐(MTT)比色法分别测定细胞的增殖活性;流式细胞仪分别检测细胞凋亡和细胞周期情况;明胶酶谱法测定细胞培养上清液中基质金属蛋白酶-2(MMP-2)的活性;双抗体夹心酶联免疫吸附试验(ELISA)测定细胞培养上清液中VEGF蛋白的含量;免疫印迹法测定SKOV-3和ES-2细胞内MMP-2和VEGF蛋白表达的变化;用逆转录聚合酶链反应(RT-PCR)半定量法测定细胞MMP-2和VEGFmRNA含量;划痕和侵袭实验检测细胞的迁移侵袭能力。
结果(1)随着FSH浓度的增加,SKOV-3和ES-2细胞的增殖活性均较对照有明显增强,差异均有统计学意义(P＜0.01)。(2)FSH可抑制SKOV-3和ES-2细胞凋亡。(3)FSH作用后,可促进SKOV-3和ES-2细胞周期的改变。(4)FSH可提高SKOV-3和ES-2细胞MMP-2和VEGFmRNA和细胞浆中MMP-2和VEGF蛋白含量及细胞培养上清液中MMP-2活性和VEGF蛋白的表达。(5)FSH可促进SKOV-3和ES-2细胞的迁移侵袭能力。
结论FSH可促进卵巢癌细胞株SKOV-3和ES-2细胞增殖、迁移和侵袭,抑制细胞的凋亡,促进VEGF的表达。
第二部分FSH调控卵巢癌细胞血管内皮生长因子的相关分子机制
目的研究FSH调控卵巢癌细胞血管内皮生长因子的相关分子机制。
方法将FSH以不同浓度(0、10、20、40、80、160mIU/mL)分别作用于2.5×10~6个SKOV-3细胞或1.5×10~6个ES-2细胞,分别检测pAKT/AKT、pERK/ERK、survivin、缺氧诱导因子-1α(HIF-1α)的表达,分别针对AKT、survivin、HIF-1α的mRNA各设计了3条特异性的siRNAs和一条阴性对照non-targetsiRNA,并分别克隆入pRNA质粒载体。将重组质粒转染SKOV-3和或ES-2细胞,用RNA干扰(RNAi)分别抑制细胞中AKT、survivin或HIF-1α的表达,采用Western blot检测干扰的效果;同时应用特异性的PI3K/AKT通路阻断剂LY294002抑制AKT的磷酸化和特异性的MAPK通路阻断剂U0126抑制ERK1/2的磷酸化,在无FSH刺激或FSH刺激的条件下,研究相关因素之间的关系。
结果(1)随着FSH浓度的增加,SKOV-3细胞的survivin和HIF-1α的表达明显增强。(2)在无FSH刺激的条件下,survivin或HIF-1α的RNAi均可使SKOV-3细胞的VEGFmRNA及胞浆蛋白表达下降。在FSH刺激的条件下,仅仅survivin的RNAi仍可使SKOV-3细胞的VEGFmRNA及胞浆蛋白表达下降,HIF-1α的RNAi无明显抑制作用。FSH可能主要是通过survivin促进VEGF表达的。(3)随着FSH浓度的增加,SKOV-3和ES-2细胞的pAKT的表达明显增强,pERK的表达无明显变化。(4)AKT的RNAi或PI3K/AKT通路阻断剂LY294002均可抑制FSH引起的SKOV-3和ES-2细胞中AKT磷酸化的下调,进而引起survivin的表达下降;MAPK通路阻断剂U0126可抑制ERK1/2的磷酸化,但不能有效抑制FSH引起的SKOV-3和ES-2细胞中survivin的上调。
结论FSH可能是通过PI3K/AKT通路促进卵巢癌细胞survivin的表达,进而促进VEGF的表达。
第三部分AKT和pAKT在卵巢癌组织中的表达
目的探讨pAKT/AKT在不同卵巢癌组织中的表达及其与预后的关系。
方法用免疫组织化学的方法分别检测pAKT/AKT在82例不同卵巢癌组织(其中21例卵巢浆液性囊腺瘤、18例卵巢交界性浆液性囊腺瘤、26卵巢浆液性囊腺癌和17卵巢透明细胞癌)中的表达,并用卡方检验和生存分析等了解它们与卵巢癌预后的关系。
结果与卵巢浆液性囊腺瘤相比,卵巢浆液性囊腺癌pAKT和AKT的阳性表达率更高(P＜0.01),pAKT和AKT的表达与肿瘤分化程度、2年和5年生存率有关联,生存分析也表明pAKT和AKT表达阳性的卵巢浆液性囊腺癌患者5年生存率分别为15%(3 of 20)和14.3%(3 of 21),pAKT和AKT表达阴性的卵巢浆液性囊腺癌患者5年生存率分别为66.7%(4 of 6)和80%(4 of 5),均远高于前者。
结论pAKT/AKT的表达可能与卵巢癌的预后有关联,抑制pAKT/AKT的表达可能有助于改善卵巢癌患者的预后。
第四部分卵泡刺激素对卵巢癌细胞在裸鼠体内生长的影响
目的采用体内实验,探讨在皮下注射FSH的条件下,裸鼠皮下移植卵巢癌组织的生长与其的关系。
方法SKOV-3细胞培养方法同第一部分。经连续传代后,将细胞浓度调整为2.5×10~7个/ml,取0.2ml注射于18裸鼠皮下,随机分成3组,每组6只。进行下列处理:将裸鼠随机分成3组,每组6只。进行下列处理:(1)生理盐水组:皮下注射0.1ml生理盐水,每天1次。(2)FSH1低剂量加药组:皮下注射FSH(3IU·只·d~(-1)),每天1次。(3)FSH2高剂量加药组:皮下注射FSH(10IU·只·d~(-1)),每天1次。SKOV-3细胞连续注射29天。ES-2细胞为22天。每三或四天观察荷瘤裸鼠并绘制移植瘤的生长曲线,并称取裸鼠体重。注射完药物后,采用摘除左眼球方法,收集裸鼠血液,采集血浆测FSH浓度,取出皮下移植瘤称重。
结果在体内实验中我们发现注射FSH药物的裸鼠血药浓度可达绝经后水平。各组裸鼠体重无明显变化。低剂量加药组裸鼠移植瘤的体积和重量均高于生理盐水组并有统计学差异,高剂量加药组裸鼠移植瘤的重量高于生理盐水组并有统计学差异,但高剂量加药组裸鼠移植瘤的体积与生理盐水组比较无统计学差异。
结论FSH与卵巢癌的发生、发展有关联。
综上所述,FSH可促进卵巢癌细胞株SKOV-3和ES-2细胞增殖、迁移和侵袭,抑制细胞的凋亡,促进VEGF的表达。FSH可能是通过PI3K/AKT通路促进卵巢癌细胞survivin的表达,进而促进VEGF的表达。pAKT/AKT的表达可能与卵巢癌的发生有关联。体外实验也提示FSH可能与卵巢癌的发生、发展有关联。本课题为进一步研究卵巢癌的发病机制提供了新的思路,为卵巢癌的基因治疗提供了新的靶点。
At present,ovarian malignancies is one of the three gynecological malignancies. Ovarian cancer is the most common histopathological type accounting for 85%-90% of all ovarian malignancies.Despite continuous improvement in surgical modalities, chemotherapeutics and radiotherapetics over the past several years,the 5-year survival rate for ovarian cancer with the most poor prognosis of all gynecological malignancies is about 30%-40%,which has not improved significantly.
Clinically,Ovarian cancer is often found in postmenopausal women with high serum follicle stimulating hormone(FSH) and luteinizing hormone(LH) level.The risk for women who have many pregancies,oral contraceptives,hormone replacement therapy is low while their serum FSH and LH concentration is also low.In the past 20 years,previous studies have proven that FSH plays an importmant role in cancinogenesis.But little is known about the relationship between FSH and ovarian carcinogenesis,neither the roles of FSH on biological behaviours of ovarian cancer cells.The results of our experiments suggest that FSH may be related to proliferation, apoptosis,migration,invasion and the expression of vascular endothelial growth factor(VEGF) in ovarian cancer cell line SKOV-3 and ES-2.Through the research on the molecular mechanism between FSH and VEGF,our clinical data and animal trial, we may provide a new stratery for further studying the carcinogenesis of ovarian cancer.
These experiments were devided into 4 parts,as follows:①The effects of FSH on proliferation,apoptosis,migration,invasion and VEGF;②The roles of FSH on VEGF in ovarian cancer cells;③Expression and AKT and pAKT in ovarian cancer;④The growth of ovarian cancer in vivo induced by FSH riment.
SectionⅠ.The roles of FSH on biological behaviours of ovarian cancer cells Objectives To investigate whether FSH can induce the effects of proliferation,apoptosis,migration,invasion and the expression of VEGF in ovarian cancer cell line SKOV-3 and ES-2 in vitro.
Methods Cultured SKOV-3 and ES-2 were treated by FSH with different concentration.The proliferative effects of the cells were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT) colorimetry.The apoptosis and cell cycle were showed by the flow cytometry.The matrix metalloproteinases-2(MMP-2) protein levels in supernatants were determined by zymography.The VEGF protein levels in supernatants were determined by enzyme-linked immunosorbent assay(ELISA).The protein levels of MMP-2 and VEGF in SKOV-3 and ES-2 cytoplasma were tested by Western blot methods.The expressions of MMP-2 and VEGF mRNA in SKOV-3 and ES-2 were determined by reverse-transcription polymerase chain reaction(RT-PCR).Migration and invasion assay can investigate the migratory and invasive activities.
Results FSH can induce the proliferative effects in ovarian cancer cell line SKOV-3 and ES-2.FSH can suppress the apoptosis and induce the effect of cell cycle in SKOV-3 and ES-2.Expressions of MMP-2 and VEGF mRNA and protein increased with the concentration increasement in SKOV-3 and ES-2.The migratory and invasive activities were also up-regulated by FSH.
Conclusions FSH can induce the effects of proliferation,migration,invasion and the expression of VEGF and suppress the apoptosis in ovarian cancer cell line SKOV-3 and ES-2.
SectionⅡ.The molecular mechanism between FSH and VEGF
Objectives To investigate the molecular mechanism between FSH and VEGF in ovarian cancer cell.
Methods Cultured SKOV-3 and ES-2 were treated by FSH with different concentration.The cells were then harvested and the proteins or mRNA of pAKT/AKT or pERK/ERK or survivin or hypoxia inducible factor-1α(HIF-1α) were extracted for Western blot or RT-PCR or real-time PCR analysis.The nine 19-nucleotide(nt) DNA sequences targeting survivin,HIF-1αand AKT and one non-target siRNA were designed,and cloned into pRNA plasmid vectors.The recombinant plasmids were transfected into SKOV-3 or ES-2 cells.The expression of AKT or surviving or HIF-1αwas knocked down by RNA interference(RNAi) and accessed by Western blot.Moreover,to investigate the molecular mechanism of FSH, the specific inhibitor of PI3K/AKT pathway LY294002 or the specific inhibitor of MAPK pathway U0126 was also used to inhibit the phosphorylation of AKT or ERK1/2 and to study the relations in all these molecules.
Results①FSH can stimulate survivin and HIF-1αexpression in SKOV-3 cells;②RNAi of SURVIVIN or HIF-1αboth blocked the expression of VEGF mRNA and protein,but only RNAi of survivin suppressed the increasement of VEGF mRNA and protein induced by FSH.FSH may increase the expression of VEGF through surviving;③FSH can induce the AKT phosphorylation in SKOV-3 and ES-2 cells, whereas it had no obvious influence on the ERK phosphorylation;④The specific PI3K inhibitor LY294002 or RNAi of AKT antagonized the enhancement effects of FSH on the AKT phosphorylation and survivin expression,whereas the specific ERK inhibitor U0126 only descreased the ERK phosphorylation and had no effect on the survivin expression.
Conclusions FSH stimulation of VEGF expression is mediated through the PI3K/AKT/survivin pathway.
SectionⅢ.Expression of AKT and pAKT in ovarian cancer
Objectives To examine the expressions of pAKT/AKT in ovarian cancer tissues and then explore the relationship between the aberrant expressions of them and the prognosis of ovarian cancer.
Methods The expression of pAKT/AKT was examined using the immunohistochemical method in the eighty-two samples of paraffin-embedded ovarian tumor tissue,including 21 ovarian benign serous cystadenomas,18 ovarian borderline serous cystadenomas,26 ovarian serous cystadenocarcinomas,and 17 ovarian clear cell carcinomas.The x2 analysis and Kaplan-Meier product-limit method was used to analyze the relationship between the aberrant expressions of them and the prognosis of ovarian cancer.
Results Compared with ovarian serous cystadenoma samples,ovarian serous cystadenocarcinoma samples had much higher positive rates for AKT and pAKT protein expression(P＜0.01).Expression of AKT and pAKT was higher in grade 2/3 than that in grade1 in the ovarian serous cystadenocarcinoma specimens(P＜0.01). We also observed strong correlations between the expression of AKT and pAKT and the 2-year or and 5-year survival rates in the ovarian serous cystadenocarcinoma cases(P＜0.01).Our results also show that the 5-year survival rate was only about 14.3%(3 of 21) in patients with ovarian serous cystadenocarcinoma who had AKT expression,whereas it was 80%(4 of 5) in those who did not have AKT expression; the 5-year survival rate was only 15%(3 of 20) in patients with ovarian serous cystadenocarcinoma who had pAKT expression,whereas it was about 66.6%(4 of 6) in those wh6 did not have pAKT expression.
Conclusions These results suggest that the expression of AKT and pAKT is likely to be related to the prognosis of ovarian cancer and that inhibition of AKT and pAKT expression may be able to improve the prognosis of this tumor.
SectionⅣ.The growth of ovarian cancer in vivo induced by FSH
Objectives To investigate the function of FSH on the growth of ovarian cancer in vivo.
Methods SKOV-3 or ES-2 cells were cultured and heterotransplanted into nude mice. The nude mice were treated with FSH(3 IU·d~-1)) or FSH(10 IU·d~(-1)) or physiological saline.The weights of the nude mice and the valumes of the transplantable tumors were measured every three or four days.At last the nude mice were executed and the serum FSH concentrations and the weights of the transplantable tumors were determined.
Results The serum FSH concentrations of the nude mice treated with FSH were higher than those without FSH treatment(P＜0.05).The weights of the nude mice in the experimental and control group have not statistically significant difference.The valumes and weights of the transplantable tumors treated with FSH(3 IU·d~-1)) were both greater than those in the control group(P＜0.05).The weights of the transplantable tumors treated with FSH(10 IU·d~(-1)) were greater than those in the control group(P＜0.05),but the valumes of the transplantable tumors treated with FSH(10 IU·d~(-1)) were statistically nonsignificant.
Conclusions FSH may be related to the formation and development of ovarian cancer in vivo.
In summury,the results demonstrates that FSH can induce the effects of proliferation,migration,invasion and the expression of VEGF and suppress the apoptosis in ovarian cancer cell line SKOV-3 and ES-2.FSH stimulation of VEGF expression is mediated through the PI3K/AKT/survivin pathway.The expression of AKT and pAKT is likely to be related to the prognosis of ovarian cancer and that inhibition of AKT and pAKT expression may be able to improve the prognosis of this tumor.FSH may be related to the formation and development of ovarian cancer in vivo.
临床上卵巢癌好发于绝经和卵巢切除术后妇女,常伴有卵泡刺激素(FSH)和黄体生成素(LH)的升高;而多次妊娠、母乳喂养、口服避孕药和接受激素替代疗法的妇女发病风险较低,FSH和LH也较低。近20年来有关研究也表明FSH对卵巢癌的发生发展起重要作用,但其对卵巢癌细胞生物学行为的影响研究甚少。本课题研究表明FSH与卵巢癌细胞的增殖、凋亡、迁移、侵袭和血管内皮生长因子(VEGF)有关联;通过对FSH调控卵巢癌VEGF的相关分子机制、卵巢癌临床资料和动物实验的研究分析,将有助于卵巢癌的病因研究,为新的治疗药物和方法的产生提供依据。
本课题共分四部分:①卵泡刺激素对卵巢癌细胞增值、凋亡、迁移、侵袭和血管内皮生长因子的影响;②卵泡刺激素调控卵巢癌细胞血管内皮生长因子的相关分子机制;③卵巢癌相关临床资料的分析;④动物实验。
第一部分卵泡刺激素对卵巢癌细胞增值、凋亡、迁移、侵袭和血管内皮生长因子的影响
目的研究FSH对卵巢癌细胞株SKOV-3和ES-2细胞增殖、凋亡、迁移、侵袭和血管内皮生长因子的影响。
方法将FSH以不同浓度(0、10、20、40、80、160mIU/mL)分别作用于1×10~5或2.5×10~6个SKOV-3细胞和6×10~4或1.5×10~6个ES-2细胞,其中FSH作用于SKOV-3细胞的适宜时间为48h,ES-2细胞为24h,二甲基噻唑二苯基四唑溴盐(MTT)比色法分别测定细胞的增殖活性;流式细胞仪分别检测细胞凋亡和细胞周期情况;明胶酶谱法测定细胞培养上清液中基质金属蛋白酶-2(MMP-2)的活性;双抗体夹心酶联免疫吸附试验(ELISA)测定细胞培养上清液中VEGF蛋白的含量;免疫印迹法测定SKOV-3和ES-2细胞内MMP-2和VEGF蛋白表达的变化;用逆转录聚合酶链反应(RT-PCR)半定量法测定细胞MMP-2和VEGFmRNA含量;划痕和侵袭实验检测细胞的迁移侵袭能力。
结果(1)随着FSH浓度的增加,SKOV-3和ES-2细胞的增殖活性均较对照有明显增强,差异均有统计学意义(P＜0.01)。(2)FSH可抑制SKOV-3和ES-2细胞凋亡。(3)FSH作用后,可促进SKOV-3和ES-2细胞周期的改变。(4)FSH可提高SKOV-3和ES-2细胞MMP-2和VEGFmRNA和细胞浆中MMP-2和VEGF蛋白含量及细胞培养上清液中MMP-2活性和VEGF蛋白的表达。(5)FSH可促进SKOV-3和ES-2细胞的迁移侵袭能力。
结论FSH可促进卵巢癌细胞株SKOV-3和ES-2细胞增殖、迁移和侵袭,抑制细胞的凋亡,促进VEGF的表达。
第二部分FSH调控卵巢癌细胞血管内皮生长因子的相关分子机制
目的研究FSH调控卵巢癌细胞血管内皮生长因子的相关分子机制。
方法将FSH以不同浓度(0、10、20、40、80、160mIU/mL)分别作用于2.5×10~6个SKOV-3细胞或1.5×10~6个ES-2细胞,分别检测pAKT/AKT、pERK/ERK、survivin、缺氧诱导因子-1α(HIF-1α)的表达,分别针对AKT、survivin、HIF-1α的mRNA各设计了3条特异性的siRNAs和一条阴性对照non-targetsiRNA,并分别克隆入pRNA质粒载体。将重组质粒转染SKOV-3和或ES-2细胞,用RNA干扰(RNAi)分别抑制细胞中AKT、survivin或HIF-1α的表达,采用Western blot检测干扰的效果;同时应用特异性的PI3K/AKT通路阻断剂LY294002抑制AKT的磷酸化和特异性的MAPK通路阻断剂U0126抑制ERK1/2的磷酸化,在无FSH刺激或FSH刺激的条件下,研究相关因素之间的关系。
结果(1)随着FSH浓度的增加,SKOV-3细胞的survivin和HIF-1α的表达明显增强。(2)在无FSH刺激的条件下,survivin或HIF-1α的RNAi均可使SKOV-3细胞的VEGFmRNA及胞浆蛋白表达下降。在FSH刺激的条件下,仅仅survivin的RNAi仍可使SKOV-3细胞的VEGFmRNA及胞浆蛋白表达下降,HIF-1α的RNAi无明显抑制作用。FSH可能主要是通过survivin促进VEGF表达的。(3)随着FSH浓度的增加,SKOV-3和ES-2细胞的pAKT的表达明显增强,pERK的表达无明显变化。(4)AKT的RNAi或PI3K/AKT通路阻断剂LY294002均可抑制FSH引起的SKOV-3和ES-2细胞中AKT磷酸化的下调,进而引起survivin的表达下降;MAPK通路阻断剂U0126可抑制ERK1/2的磷酸化,但不能有效抑制FSH引起的SKOV-3和ES-2细胞中survivin的上调。
结论FSH可能是通过PI3K/AKT通路促进卵巢癌细胞survivin的表达,进而促进VEGF的表达。
第三部分AKT和pAKT在卵巢癌组织中的表达
目的探讨pAKT/AKT在不同卵巢癌组织中的表达及其与预后的关系。
方法用免疫组织化学的方法分别检测pAKT/AKT在82例不同卵巢癌组织(其中21例卵巢浆液性囊腺瘤、18例卵巢交界性浆液性囊腺瘤、26卵巢浆液性囊腺癌和17卵巢透明细胞癌)中的表达,并用卡方检验和生存分析等了解它们与卵巢癌预后的关系。
结果与卵巢浆液性囊腺瘤相比,卵巢浆液性囊腺癌pAKT和AKT的阳性表达率更高(P＜0.01),pAKT和AKT的表达与肿瘤分化程度、2年和5年生存率有关联,生存分析也表明pAKT和AKT表达阳性的卵巢浆液性囊腺癌患者5年生存率分别为15%(3 of 20)和14.3%(3 of 21),pAKT和AKT表达阴性的卵巢浆液性囊腺癌患者5年生存率分别为66.7%(4 of 6)和80%(4 of 5),均远高于前者。
结论pAKT/AKT的表达可能与卵巢癌的预后有关联,抑制pAKT/AKT的表达可能有助于改善卵巢癌患者的预后。
第四部分卵泡刺激素对卵巢癌细胞在裸鼠体内生长的影响
目的采用体内实验,探讨在皮下注射FSH的条件下,裸鼠皮下移植卵巢癌组织的生长与其的关系。
方法SKOV-3细胞培养方法同第一部分。经连续传代后,将细胞浓度调整为2.5×10~7个/ml,取0.2ml注射于18裸鼠皮下,随机分成3组,每组6只。进行下列处理:将裸鼠随机分成3组,每组6只。进行下列处理:(1)生理盐水组:皮下注射0.1ml生理盐水,每天1次。(2)FSH1低剂量加药组:皮下注射FSH(3IU·只·d~(-1)),每天1次。(3)FSH2高剂量加药组:皮下注射FSH(10IU·只·d~(-1)),每天1次。SKOV-3细胞连续注射29天。ES-2细胞为22天。每三或四天观察荷瘤裸鼠并绘制移植瘤的生长曲线,并称取裸鼠体重。注射完药物后,采用摘除左眼球方法,收集裸鼠血液,采集血浆测FSH浓度,取出皮下移植瘤称重。
结果在体内实验中我们发现注射FSH药物的裸鼠血药浓度可达绝经后水平。各组裸鼠体重无明显变化。低剂量加药组裸鼠移植瘤的体积和重量均高于生理盐水组并有统计学差异,高剂量加药组裸鼠移植瘤的重量高于生理盐水组并有统计学差异,但高剂量加药组裸鼠移植瘤的体积与生理盐水组比较无统计学差异。
结论FSH与卵巢癌的发生、发展有关联。
综上所述,FSH可促进卵巢癌细胞株SKOV-3和ES-2细胞增殖、迁移和侵袭,抑制细胞的凋亡,促进VEGF的表达。FSH可能是通过PI3K/AKT通路促进卵巢癌细胞survivin的表达,进而促进VEGF的表达。pAKT/AKT的表达可能与卵巢癌的发生有关联。体外实验也提示FSH可能与卵巢癌的发生、发展有关联。本课题为进一步研究卵巢癌的发病机制提供了新的思路,为卵巢癌的基因治疗提供了新的靶点。
At present,ovarian malignancies is one of the three gynecological malignancies. Ovarian cancer is the most common histopathological type accounting for 85%-90% of all ovarian malignancies.Despite continuous improvement in surgical modalities, chemotherapeutics and radiotherapetics over the past several years,the 5-year survival rate for ovarian cancer with the most poor prognosis of all gynecological malignancies is about 30%-40%,which has not improved significantly.
Clinically,Ovarian cancer is often found in postmenopausal women with high serum follicle stimulating hormone(FSH) and luteinizing hormone(LH) level.The risk for women who have many pregancies,oral contraceptives,hormone replacement therapy is low while their serum FSH and LH concentration is also low.In the past 20 years,previous studies have proven that FSH plays an importmant role in cancinogenesis.But little is known about the relationship between FSH and ovarian carcinogenesis,neither the roles of FSH on biological behaviours of ovarian cancer cells.The results of our experiments suggest that FSH may be related to proliferation, apoptosis,migration,invasion and the expression of vascular endothelial growth factor(VEGF) in ovarian cancer cell line SKOV-3 and ES-2.Through the research on the molecular mechanism between FSH and VEGF,our clinical data and animal trial, we may provide a new stratery for further studying the carcinogenesis of ovarian cancer.
These experiments were devided into 4 parts,as follows:①The effects of FSH on proliferation,apoptosis,migration,invasion and VEGF;②The roles of FSH on VEGF in ovarian cancer cells;③Expression and AKT and pAKT in ovarian cancer;④The growth of ovarian cancer in vivo induced by FSH riment.
SectionⅠ.The roles of FSH on biological behaviours of ovarian cancer cells Objectives To investigate whether FSH can induce the effects of proliferation,apoptosis,migration,invasion and the expression of VEGF in ovarian cancer cell line SKOV-3 and ES-2 in vitro.
Methods Cultured SKOV-3 and ES-2 were treated by FSH with different concentration.The proliferative effects of the cells were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT) colorimetry.The apoptosis and cell cycle were showed by the flow cytometry.The matrix metalloproteinases-2(MMP-2) protein levels in supernatants were determined by zymography.The VEGF protein levels in supernatants were determined by enzyme-linked immunosorbent assay(ELISA).The protein levels of MMP-2 and VEGF in SKOV-3 and ES-2 cytoplasma were tested by Western blot methods.The expressions of MMP-2 and VEGF mRNA in SKOV-3 and ES-2 were determined by reverse-transcription polymerase chain reaction(RT-PCR).Migration and invasion assay can investigate the migratory and invasive activities.
Results FSH can induce the proliferative effects in ovarian cancer cell line SKOV-3 and ES-2.FSH can suppress the apoptosis and induce the effect of cell cycle in SKOV-3 and ES-2.Expressions of MMP-2 and VEGF mRNA and protein increased with the concentration increasement in SKOV-3 and ES-2.The migratory and invasive activities were also up-regulated by FSH.
Conclusions FSH can induce the effects of proliferation,migration,invasion and the expression of VEGF and suppress the apoptosis in ovarian cancer cell line SKOV-3 and ES-2.
SectionⅡ.The molecular mechanism between FSH and VEGF
Objectives To investigate the molecular mechanism between FSH and VEGF in ovarian cancer cell.
Methods Cultured SKOV-3 and ES-2 were treated by FSH with different concentration.The cells were then harvested and the proteins or mRNA of pAKT/AKT or pERK/ERK or survivin or hypoxia inducible factor-1α(HIF-1α) were extracted for Western blot or RT-PCR or real-time PCR analysis.The nine 19-nucleotide(nt) DNA sequences targeting survivin,HIF-1αand AKT and one non-target siRNA were designed,and cloned into pRNA plasmid vectors.The recombinant plasmids were transfected into SKOV-3 or ES-2 cells.The expression of AKT or surviving or HIF-1αwas knocked down by RNA interference(RNAi) and accessed by Western blot.Moreover,to investigate the molecular mechanism of FSH, the specific inhibitor of PI3K/AKT pathway LY294002 or the specific inhibitor of MAPK pathway U0126 was also used to inhibit the phosphorylation of AKT or ERK1/2 and to study the relations in all these molecules.
Results①FSH can stimulate survivin and HIF-1αexpression in SKOV-3 cells;②RNAi of SURVIVIN or HIF-1αboth blocked the expression of VEGF mRNA and protein,but only RNAi of survivin suppressed the increasement of VEGF mRNA and protein induced by FSH.FSH may increase the expression of VEGF through surviving;③FSH can induce the AKT phosphorylation in SKOV-3 and ES-2 cells, whereas it had no obvious influence on the ERK phosphorylation;④The specific PI3K inhibitor LY294002 or RNAi of AKT antagonized the enhancement effects of FSH on the AKT phosphorylation and survivin expression,whereas the specific ERK inhibitor U0126 only descreased the ERK phosphorylation and had no effect on the survivin expression.
Conclusions FSH stimulation of VEGF expression is mediated through the PI3K/AKT/survivin pathway.
SectionⅢ.Expression of AKT and pAKT in ovarian cancer
Objectives To examine the expressions of pAKT/AKT in ovarian cancer tissues and then explore the relationship between the aberrant expressions of them and the prognosis of ovarian cancer.
Methods The expression of pAKT/AKT was examined using the immunohistochemical method in the eighty-two samples of paraffin-embedded ovarian tumor tissue,including 21 ovarian benign serous cystadenomas,18 ovarian borderline serous cystadenomas,26 ovarian serous cystadenocarcinomas,and 17 ovarian clear cell carcinomas.The x2 analysis and Kaplan-Meier product-limit method was used to analyze the relationship between the aberrant expressions of them and the prognosis of ovarian cancer.
Results Compared with ovarian serous cystadenoma samples,ovarian serous cystadenocarcinoma samples had much higher positive rates for AKT and pAKT protein expression(P＜0.01).Expression of AKT and pAKT was higher in grade 2/3 than that in grade1 in the ovarian serous cystadenocarcinoma specimens(P＜0.01). We also observed strong correlations between the expression of AKT and pAKT and the 2-year or and 5-year survival rates in the ovarian serous cystadenocarcinoma cases(P＜0.01).Our results also show that the 5-year survival rate was only about 14.3%(3 of 21) in patients with ovarian serous cystadenocarcinoma who had AKT expression,whereas it was 80%(4 of 5) in those who did not have AKT expression; the 5-year survival rate was only 15%(3 of 20) in patients with ovarian serous cystadenocarcinoma who had pAKT expression,whereas it was about 66.6%(4 of 6) in those wh6 did not have pAKT expression.
Conclusions These results suggest that the expression of AKT and pAKT is likely to be related to the prognosis of ovarian cancer and that inhibition of AKT and pAKT expression may be able to improve the prognosis of this tumor.
SectionⅣ.The growth of ovarian cancer in vivo induced by FSH
Objectives To investigate the function of FSH on the growth of ovarian cancer in vivo.
Methods SKOV-3 or ES-2 cells were cultured and heterotransplanted into nude mice. The nude mice were treated with FSH(3 IU·d~-1)) or FSH(10 IU·d~(-1)) or physiological saline.The weights of the nude mice and the valumes of the transplantable tumors were measured every three or four days.At last the nude mice were executed and the serum FSH concentrations and the weights of the transplantable tumors were determined.
Results The serum FSH concentrations of the nude mice treated with FSH were higher than those without FSH treatment(P＜0.05).The weights of the nude mice in the experimental and control group have not statistically significant difference.The valumes and weights of the transplantable tumors treated with FSH(3 IU·d~-1)) were both greater than those in the control group(P＜0.05).The weights of the transplantable tumors treated with FSH(10 IU·d~(-1)) were greater than those in the control group(P＜0.05),but the valumes of the transplantable tumors treated with FSH(10 IU·d~(-1)) were statistically nonsignificant.
Conclusions FSH may be related to the formation and development of ovarian cancer in vivo.
In summury,the results demonstrates that FSH can induce the effects of proliferation,migration,invasion and the expression of VEGF and suppress the apoptosis in ovarian cancer cell line SKOV-3 and ES-2.FSH stimulation of VEGF expression is mediated through the PI3K/AKT/survivin pathway.The expression of AKT and pAKT is likely to be related to the prognosis of ovarian cancer and that inhibition of AKT and pAKT expression may be able to improve the prognosis of this tumor.FSH may be related to the formation and development of ovarian cancer in vivo.
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69. Maizels ET, Cottom J, Jones JC, et al. Follicle stimulating hormone (FSH) activates the p38 mitogen-activated protein kinase pathway, including small heat shock protein phosphorylation and cell rounding in immature rat ovarian granulose cells.Endocrinology,1998,139:3353-56.
70.Syed V,Ulinski G,Mok SC,et al.Reproductive hormone-induced,STAT3-mediated interleukin 6 action in normal and malignant human ovarian surface epithelial cell.J Natl Cancer Inst,2002,94:617-29.
71.Yuan ZQ,Sun M,Feldman RI,et al.Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer.Oncogene,2000,19:2324-30.
72.Shayesteh L,Lu Y,Kuo WL,et al.PIR3CA is implicated as an oncogene in ovarian cancer.Nat Genet,1999,21:99-102.
73.Kurose K,Zhou XP,Araki T,et al.Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels,but not associated with p27 and cyclin D1 expression,in primary epithelial ovarian carcinomas.Am J Pathol,2001,158:2097-106.
74.Asselin E,Mills GB,Tsang Bff.XIAP regulates Akt activity and caspase-3-dependent cisplatin-induced apoptosis in human ovarian cancer cells.Cancer Res,2001,61:1862-8.
75.刘东远.卵泡刺激素促进卵巢上皮性癌移植瘤生长.基础医学和临床,2005,25:437-41.
1. Schiffenbauer YS, Abramovitch R, Meir G, et al. Loss of ovarian function promotes angiogenesis in human ovarian carcinoma. Proc Natl Acad Sci U S A, 1997, 94:13203-8.
2. Gnagy S, Ming EE, Devesa SS, et al. Declining ovarian cancer rates in U. S. women in relation to parity and oral contraceptive usa. Epidemiology, 2000,11:102-5.
3. Reimer T, Gerber B, Kunkel S,et al. Estradiol, gonadotropins, and tumor markers in ovarian cyst fluid. Acta Obstet Gynecol Scand, 1997,76:478-83.
4. Kr(a|¨)mer S, Leeker M, J(a|¨)ger W. Gonadotropin levels in ovarian cyst fluids: a predictor of malignancy? Int J Biol Markers, 1998, 13:165-8.
5. Zheng W, Lu JJ, Luo F, et al. Ovarian epithelial tumor growth promotion by follicle-stimulating hormone and inhibition of the effect by luteinizing hormone. Gynecol Oncol, 2000, 76:80-8.
6. Hofstra LS, Mourits MJ, de Vries EG, et al. Combined treatment with goserelin and tamoxifen in patients with advanced chemotherapy resistant ovarian cancer. Anticancer Res, 1999,19:3627-30.
7. Rzepka-Gorska I, Chudecka-Glaz A, Kosmider M, et al. GnRH analogues as an adjuvant therapy for ovarian cancer patients. Int J Gynaecol Obstet, 2003, 81:199-205.
8. Chudecka-G(?)az A, Rzepka-Górska I, Kosmowska B. Gonadotropin (LH, FSH) levels in serum and cyst fluid in epithelial tumors of the ovary. Arch Gynecol Obstet, 2004, 270:151-6.
9. Rzepka-Górska I, Chudecka-G(?)az A, Kosmowska B. FSH and LH serum/tumor fluid ratios and malignant tumors of the ovary. Endocr Relat Cancer, 2004,11:315-21.
10. Arslan AA, Zeleniuch-Jacquotte A, Lundin E, et al. Serum follicle-stimulating hormone and risk of epithelial ovarian cancer in postmenopausal women.Cancer Epidemiol Biomarkers Prev,2003,12:1531-5.
11.丰有吉,张惜阴,葛柏青,等.促性腺激素对人卵巢上皮性癌细胞的增殖作用.中华妇产科杂志,1996,31:166-8.
12.周抗美,朱铭伟,丰有吉.促卵泡激素结合片段对卵巢癌细胞株的作用.中华妇产科杂志,1999,34:726-8.
13.Sicinski P,Donaher JL,Geng Y,et al.Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.Nature,1996,384:470-4.
14.Maruuchi T,Sugiyama T,Kataoka A,et al.Effects of a gonadotropin-releasing hormone agonist on rat ovarian adenocarcinoma cell lines in vitro and in vivo.Jpn J Cancer Res,1998,89:977-83.
15.朱铭伟,吴乾渝,葛柏青,等.卵泡刺激素对卵巢癌细胞的增殖及CA125Ⅱ抗原分泌的作用.中华妇产科杂志,1998,33:225-6.
16.Ohtani K,Sakamoto H,Kikuchi A,et al.Follicle-stimulating hormone promotes the growth of human epithelial ovarian cancer cells through the protein kinase C-mediated system.Cancer Lett,2001,166:207-13.
17.沈铿,刘东远,盛林.卵泡刺激素介导卵巢上皮性癌细胞增殖的初步研究.中华妇产科杂志,2003,38:752-5.
18.Syed V,Ulinski G,Mok SC,et al.Expression of gonadotropin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cells.Cancer Res,2001,61:6768-76.
19.Choi in,Choi KC,Auersperg N,et al.Gonadotropins activate proteolysis and increase invasion through protein kinase A and phosphatidylinositol 3-kinase pathways in human epithelial ovarian cancer cells.Cancer Res,2006,66:3912-20.
20.Ji Q,Liu PI,Chen PK,et al.Follicle stimulating hormone-induced growth promotion and gene expression profiles on ovarian surface epithelial cells.Int J Cancer, 2004,112:803-14.
21.Choi JH, Choi KC, Auersperg N, et al. Overexpression of follicle-stimulating hormone receptor activates oncogenic pathways in preneoplastic ovarian surface epithelial cells. J Clin Endocrinol Metab, 2004, 89:5508-16.
22. Choi JH, Choi KC, Auersperg N, et al. Gonadotropins upregulate the epidermal growth factor receptor through activation of mitogen-activated protein kinases and phosphatidyl-inositol-3-kinase in human ovarian surface epithelial cells. Endocr Relat Cancer, 2005,12:407-21.
23. Li Y, GantaS, Cheng C, et al. FSH stimulates ovarian cancer cell growth by action on growth factor variant receptor. Mol Cell Endocrinol, 2007, 267:26-37.
24. Parrott JA, Doraiswamy V, Kim G, et al. Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer. Mol Cell Endocrinol, 2001,172:213-22.
25.Kraemer S, Jaeger WH, Lang N. Growth regulation effects of gonadotropin induced steroidogenic response in human ovarian cancer. Anticancer Res,2001, 21:2005-10.
26.Ala-Fossi SL, Grenman S, Zhang FP, et al. Ovarian cancer and gonadotropins in vitro: new evidence in favor of independence. Anticancer Res, 1999, 19:4289-95.
27. Basille C, Olivennes F, Le Calvez J, et al. Impact of gonadotrophins and steroid hormones on tumour cells derived from borderline ovarian tumours. Hum Reprod, 2006, 21:3241-5.
28. Ivarsson K, Sundfeldt K, Br(a|¨)nnstr(o|¨)m M , et al. Diverse effects of FSH and LH on proliferation of human ovarian surface epithelial cells. Hum Reprod,2001,16:18-23.
29.Emons G,Ortmann O,Teichert HM,et al.Luteinizing hormone-releasing hormone agonist triptorelin in combination with cytotoxic chemotherapy in patients with advanced ovarian carcinoma.Cancer,1996,78:1452-60.
30.黄春芳,刘东远,徐卫华,等.促卵泡激素对顺铂诱导卵巢癌细胞株凋亡的保护作用.中国医学科学院学报,2003,25:443-6.
31.黄春芳,刘东远,沈铿.促卵泡刺激素抑制顺铂诱导的卵巢癌细胞凋亡.中国医学科学院学报,2003,25:447-50.
32.Choi JH,Choi KC,Auersperg N,et al.Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid by gonadotropins in human immortalized ovarian surface epithelium and ovarian cancer cells.Endocr Relat Cancer,2006,13:641-51.
33.Schiffenbauer YS,Meir G,Maoz M,et al.Gonadotropin stimulation of MLS human epithelial ovarian carcinoma cells augments cell adhesion mediated by CD44 and by alpha(v)-integrin.Gynecol Oncol,2002,84:296-302.
34.Wang J,Luo F,Lu JJ,et al.VEGF expression and enhanced production by gonadotropins in ovarian epithelial tumors.Int J Cancer,2002,97:163-7.
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25.Maruuchi T,Sugiyama T,Kataoka A,et al.Effects of a gonadotropin-releasing hormone agonist on rat ovarian adenocarcinoma cell lines in vitro and in vivo.Jpn J Cancer Res,1998,89:977-83.
26.朱铭伟,吴乾渝,葛柏青,等.卵泡刺激素对卵巢癌细胞的增殖及CA125 Ⅱ抗原分泌的作用.中华妇产科杂志,1998,33:225-6.
27.Syed V,Ulinski G,Mok SC,et al.Expression of gonadotropin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cells.Cancer Res,2001,61:6768-76.
28.Schiffenbauer YS,Meir G,Maoz M,et al.Gonadotropin stimulation of MLS human epithelial ovarian carcinoma cells augments cell adhesion mediated by CD44 and by alpha(v)-integrin.Gynecol Oncol, 2002,84:296-302.
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30.Emons G,Ortmann O,Teichert HM,et al.Luteinizing hormone-releasing hormone agonist triptorelin in combination with cytotoxic chemotherapy in patients with advanced ovarian carcinoma.Cancer,1996,78:1452-60.
31.黄春芳,刘东远,徐卫华,等.促卵泡激素对顺铂诱导卵巢癌细胞株凋亡的保护作用.中国医学科学院学报,2003,25:443-6.
32.黄春芳,刘东远,沈铿.促卵泡刺激素抑制顺铂诱导的卵巢癌细胞凋亡.中国医学科学院学报,2003,25:447-50.
33.Choi JH,Choi KC,Auersperg N,et al.E Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid by gonadotropins in human immortalized ovarian surface epithelium and ovarian cancer cells.Endocr Relat Cancer,2006,13:641-51.
34.Wang J,Luo F,Lu JJ,et al.VEGF expression and enhanced production by gonadotropins in ovarian epithelial tumors.Int J Cancer,2002,97:163-7.
35.Parrott JA,Doraiswamy V,Kim G,et al.Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer.Mol Cell Endocrinol,2001,172:213-22.
36.Kraemer S,Jaeger WH,Lang N.Growth regulation effects of gonadotropin induced steroidogenic response in human ovarian cancer.Anticancer Res,2001,21:2005-10.
37.Ohtani K,Sakamoto H,Kikuchi A,et al.Follicle-stimulating hormone promotes the growth of human epithelial ovarian cancer cells through the protein kinase C-mediated system.Cancer Lett,2001,166:207-13.
38.沈铿,刘东远,盛林.卵泡刺激素介导卵巢上皮性癌细胞增殖的初步研究.中华妇产科杂志,2003,38:752-5.
39.Ji Q,Liu PI,then PK,et al.Follicle stimulating hormone-induced growth promotion and gene expression profiles on ovarian surface epithelial cells.Int J Cancer,2004,112:803-14.
40.Li Y,Ganta S,Cheng C,et al.FSH stimulates ovarian cancer cell growth by action on growth factor variant receptor.Mol Cell Endocrinol,2007,267:26-37.
41.Choi JH,Choi KC,Auersperg N,et al.Gonadotropins upregulate the epidermal growth factor receptor through activation of mitogen-activated protein kinases and phosphatidyl-inositol-3-kinase in human ovarian surface epithelial cells.Endocr Relat Cancer,2005,12:407-21.
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45.Sicinski P,Donaher JL,Geng Y,et al.Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.Nature,1996,384:470-4.
46.Ala-Fossi SL,Grenman S,Zhang FP,et al.Ovarian cancer and gonadotropins in vitro:new evidence in favor of independence.Anticancer Res,1999,19:4289-95.
47.Basille C,Olivennes F,Le Calvez J,et al.Impact of gonadotrophins and steroid hormones on tumour cells derived from borderline ovarian tumours.Hum Reprod,2006,21:3241-5.
48. Ivarsson K, Sundfeldt K, Br(a|¨)nnstr(o|¨)m M , et al. Diverse effects of FSH and LH on p roliferation of human ovarian surface epithelial cells. Hum Reprod,2001,16:18-23.
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68. Gebauer G, Peter AT, Onesime D, et al. Apoptosis of ovarian gaanulosa cells: correlation with the reduced activity of ERK-signaling module. J Cell Biocem, 1999, 75:547-54.
69. Maizels ET, Cottom J, Jones JC, et al. Follicle stimulating hormone (FSH) activates the p38 mitogen-activated protein kinase pathway, including small heat shock protein phosphorylation and cell rounding in immature rat ovarian granulose cells.Endocrinology,1998,139:3353-56.
70.Syed V,Ulinski G,Mok SC,et al.Reproductive hormone-induced,STAT3-mediated interleukin 6 action in normal and malignant human ovarian surface epithelial cell.J Natl Cancer Inst,2002,94:617-29.
71.Yuan ZQ,Sun M,Feldman RI,et al.Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer.Oncogene,2000,19:2324-30.
72.Shayesteh L,Lu Y,Kuo WL,et al.PIR3CA is implicated as an oncogene in ovarian cancer.Nat Genet,1999,21:99-102.
73.Kurose K,Zhou XP,Araki T,et al.Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels,but not associated with p27 and cyclin D1 expression,in primary epithelial ovarian carcinomas.Am J Pathol,2001,158:2097-106.
74.Asselin E,Mills GB,Tsang Bff.XIAP regulates Akt activity and caspase-3-dependent cisplatin-induced apoptosis in human ovarian cancer cells.Cancer Res,2001,61:1862-8.
75.刘东远.卵泡刺激素促进卵巢上皮性癌移植瘤生长.基础医学和临床,2005,25:437-41.
1. Schiffenbauer YS, Abramovitch R, Meir G, et al. Loss of ovarian function promotes angiogenesis in human ovarian carcinoma. Proc Natl Acad Sci U S A, 1997, 94:13203-8.
2. Gnagy S, Ming EE, Devesa SS, et al. Declining ovarian cancer rates in U. S. women in relation to parity and oral contraceptive usa. Epidemiology, 2000,11:102-5.
3. Reimer T, Gerber B, Kunkel S,et al. Estradiol, gonadotropins, and tumor markers in ovarian cyst fluid. Acta Obstet Gynecol Scand, 1997,76:478-83.
4. Kr(a|¨)mer S, Leeker M, J(a|¨)ger W. Gonadotropin levels in ovarian cyst fluids: a predictor of malignancy? Int J Biol Markers, 1998, 13:165-8.
5. Zheng W, Lu JJ, Luo F, et al. Ovarian epithelial tumor growth promotion by follicle-stimulating hormone and inhibition of the effect by luteinizing hormone. Gynecol Oncol, 2000, 76:80-8.
6. Hofstra LS, Mourits MJ, de Vries EG, et al. Combined treatment with goserelin and tamoxifen in patients with advanced chemotherapy resistant ovarian cancer. Anticancer Res, 1999,19:3627-30.
7. Rzepka-Gorska I, Chudecka-Glaz A, Kosmider M, et al. GnRH analogues as an adjuvant therapy for ovarian cancer patients. Int J Gynaecol Obstet, 2003, 81:199-205.
8. Chudecka-G(?)az A, Rzepka-Górska I, Kosmowska B. Gonadotropin (LH, FSH) levels in serum and cyst fluid in epithelial tumors of the ovary. Arch Gynecol Obstet, 2004, 270:151-6.
9. Rzepka-Górska I, Chudecka-G(?)az A, Kosmowska B. FSH and LH serum/tumor fluid ratios and malignant tumors of the ovary. Endocr Relat Cancer, 2004,11:315-21.
10. Arslan AA, Zeleniuch-Jacquotte A, Lundin E, et al. Serum follicle-stimulating hormone and risk of epithelial ovarian cancer in postmenopausal women.Cancer Epidemiol Biomarkers Prev,2003,12:1531-5.
11.丰有吉,张惜阴,葛柏青,等.促性腺激素对人卵巢上皮性癌细胞的增殖作用.中华妇产科杂志,1996,31:166-8.
12.周抗美,朱铭伟,丰有吉.促卵泡激素结合片段对卵巢癌细胞株的作用.中华妇产科杂志,1999,34:726-8.
13.Sicinski P,Donaher JL,Geng Y,et al.Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.Nature,1996,384:470-4.
14.Maruuchi T,Sugiyama T,Kataoka A,et al.Effects of a gonadotropin-releasing hormone agonist on rat ovarian adenocarcinoma cell lines in vitro and in vivo.Jpn J Cancer Res,1998,89:977-83.
15.朱铭伟,吴乾渝,葛柏青,等.卵泡刺激素对卵巢癌细胞的增殖及CA125Ⅱ抗原分泌的作用.中华妇产科杂志,1998,33:225-6.
16.Ohtani K,Sakamoto H,Kikuchi A,et al.Follicle-stimulating hormone promotes the growth of human epithelial ovarian cancer cells through the protein kinase C-mediated system.Cancer Lett,2001,166:207-13.
17.沈铿,刘东远,盛林.卵泡刺激素介导卵巢上皮性癌细胞增殖的初步研究.中华妇产科杂志,2003,38:752-5.
18.Syed V,Ulinski G,Mok SC,et al.Expression of gonadotropin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cells.Cancer Res,2001,61:6768-76.
19.Choi in,Choi KC,Auersperg N,et al.Gonadotropins activate proteolysis and increase invasion through protein kinase A and phosphatidylinositol 3-kinase pathways in human epithelial ovarian cancer cells.Cancer Res,2006,66:3912-20.
20.Ji Q,Liu PI,Chen PK,et al.Follicle stimulating hormone-induced growth promotion and gene expression profiles on ovarian surface epithelial cells.Int J Cancer, 2004,112:803-14.
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