【作者】 周俊；

【导师】 张苏展； 陈益定；

【作者基本信息】 浙江大学 ， 肿瘤学， 2013， 博士

【摘要】 研究背景乳腺癌仍然是女性患病率最高的肿瘤,占女性肿瘤死亡病因的第二位。绝经后乳腺癌的发病率在我国仍然处于各年龄段的高峰期。女性绝经后,体内激素水平发生了重大的变化,随着卵巢的衰竭,雌激素(Estradiol.E2)分泌水平的下降,下丘脑以及垂体失去了E2的负反馈,将分泌更多的卵泡刺激素(Follicle-stimulating hormone, FSH)以及黄体生成素(luteinizing Hormone,LH)。女性生命的1/3将在高促性腺激素水平中度过。抗雌激素以及其受体治疗是目前乳腺癌内分泌治疗的主要手段,但是仍然有许多乳腺癌患者不能从内分泌治疗中获益。除雌激素外,研究发现许多的内分泌激素与乳腺癌的发病以及进展相关,包括泌乳素(Prolactin, PRL),人绒毛膜促性腺激素(Human Chorionic Gonadotropin, hCG),LH等。这些激素协同或者直接影响乳腺癌的发生以及进展。近些年的研究发现,FSH参与了女性绝经后疾病的发生,但是FSH在乳腺癌发病中的研究尚没有详细的研究。有研究发现乳腺癌患者血清FSH水平与肿瘤的无复发生存率以及总生存率负相关。并且也有研究发现血清FSH水平与乳腺癌组织的Her-2表达相关。FSH通过其特异性受体(Follicle-stimulating hormone receptor, FSHR)介导细胞增殖,分化。FSHR一直被认为只在特异性组织中表达,即女性颗粒细胞以及男性睾丸支持细胞。近些年的研究发现,FSHR可以在众多的肿瘤细胞中表达包括卵巢癌,前列腺癌,子宫内膜癌,骨骼中表达。FSHR参与了这些肿瘤的增殖与转移。FSHR在骨骼细胞中表达被认为是绝经后女性骨质疏松的主要原因,而非是经典的雌激素途径。在最近的研究中发现,FSHR在许多实体肿瘤的血管内皮细胞中表达,这些表达在肿瘤与正常组织交界处。乳腺癌体外细胞系以及乳腺癌组织是否表达FSHR,以及FSH能否通过FSHR促进乳腺癌的发病仍然不明确。实验目的：选取绝经后病人对研究对象,研究血清FSH以及LH在女性乳腺癌病人和非肿瘤普通妇科炎症疾病对照人群中的差异性；研究血清FSH以及LH水平与绝经后乳腺癌细胞ER, PR, Her-2以及Ki67表达的相关性；研究FSH以及LH与乳腺癌临床分期,分级以及淋巴结转移的关系；研究FSHR是否在乳腺癌体外细胞系以及癌组织中表达。体外实验研究FSH是否促进乳腺癌细胞的增殖与转移,及其具体的分子机制；体内实验研究FSH是否会促进乳腺癌细胞的增殖。实验方法：第一部分：通过收取187名绝经后乳腺癌患者的病例资料以及内分泌数据,收集了同时期374名非肿瘤普通妇科炎症疾病的绝经后患者的内分泌检查数据。非肿瘤人群的构成主要为妇科的炎症性疾病,如阴道炎,宫颈炎等。建立数据库,统计分析FSH以及LH在绝经后乳腺癌患者与绝经后健康人群中的差异。分析FSH以及LH与乳腺癌的ER,PR,Her-2Ki67的表达,以及临床分期,分级,以及淋巴结转移的相关性。第二部分：体外实验研究FSH是否促进乳腺癌细胞的增殖与转移。使用MTT检测FSH对乳腺癌细胞系Bcap-37, MDA-MB-231的促增殖作用。使用Western Blot检测FSH刺激以上两株细胞引起的EGFR,ERK1/2, AKT等关键信号通路分子的激活改变。使用EGFR, ERK1/2等信号通路的抑制剂后,研究FSH促进增殖的主要作用信号通路。通过划痕试验研究FSH对Bcap-37,MDA-MB-231细胞系的迁移能力影响,使用基质胶Transwell实验研究FSH对以上两株细胞的侵袭能力影响,并通过Western Blot检测相应的分子机制。第三部分：体内实验研究FSH是否促进乳腺癌细胞系Bcap-37,MDA-MB-231移植瘤的增殖。通过对Balb/c裸鼠去势手术建立绝经后的动物模型。接种乳腺癌细胞系Bcap-37,MDA-MB-231到Balb/c裸鼠皮下。实验分为4组：假手术组,去势对照组,去势后使用GnRha组,去势后使用GnRHa然后使用FSH组。假手术组和去势对照组用于比较去势对肿瘤增殖的影响,GnRHa组使用GnRHa抑制裸鼠体内的FSH以及LH分泌,排除垂体分泌激素的影响。GnRHa加用FSH组,在GnRHa之后使用FSH使裸鼠体内的FSH达到100IU/L左右的绝经后水平。第四部分：检测FSHR在乳腺癌体外细胞系以及乳腺癌组织中的表达。使用RT-PCR,以及Reverse-PCR检测Bcap-37,MDA-MB-231等乳腺癌细胞系的FSHR表达情况。同时检测FSHR在17例乳腺癌临床样本中的表达情况。实验结果：第一部分：a)绝经后乳腺癌患者血清FSH, LH比对照人群高(61.83±1.475vs.48.13士1.256IU/L；27.83±0.74vs.23.88±0.6499IU/L)。b)绝经后乳腺癌患者血清FSH水平与肿瘤Ki67,Her-2表达正相关；LH水平与Ki67表达正相关。c)血清FSH,LH浓度与绝经后乳腺癌的肿瘤ER,PR,肿瘤的的临床分型,分级以及淋巴结转移情况没有相关性。第二部分：a)FSH可以促进入乳腺癌细胞系Bcap-37,MDA-MB-231的体外增殖,可以促进Bcap-37细胞的迁移。b)FSH通过EGFR/ERK1/2通路促进Bcap-37, MDA-MB-231细胞的增殖。第三部分：a)成功构建了绝经后的Balb/c nu/nu小鼠动物模型。b)FSH(≈100IU/L)可以促进乳腺癌细胞系Bcap-37.MDA-MB-231移植瘤的体内增殖。第四部分：Bcap-37,MDA-MB-231, MCF-7, T-47D, SK-BR-3, BT-474乳腺癌永生化细胞系以及17例乳腺癌病人癌组织无FSHR表达。实验结论：a)绝经后乳腺癌患者血清FSH和LH浓度较绝经后对照人群高；FSH水平与乳腺癌组织的Ki67,Her-2表达正相关；与乳腺癌的临床分型,分期,以及淋巴结转移不相关。b)FSH在体外通过EGFR/ERK1/2促进乳腺癌细胞系Bcap-37的增殖与转移,以及MDA-MB-231细胞的增殖。c) FSH (-100IU/L)可以促进乳腺癌细胞Bcap-37,MDA-MB-231裸鼠移植瘤的体内增殖。d)乳腺癌细胞系(Bcap-37,MDA-MB-231,Mcf-7,T-47D,SKBR-3,BT-474)以及乳腺癌组织样本中不表达FSHR。e) FSH在乳腺癌细胞中的具体作用靶点需要进一步的研究。更多还原

【Abstract】 BackgroundBreast cancer is one of the most common cancers in women and one of the main causes of cancer death worldwide. The hormone level is different in the postmenopausal women when compared to the premenopausal one, as the failure of the ovary. The pituitary secretes more gonadotropin hormones including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) into blood without the feedback of estrogen. It is estimated that women will live in a high gonadotropin hormone level in a third of their all lifetime.Estradiol and its receptor play a critical role in breast cancer endocrinal treatment, however not all of the patients benefit from hormone therapy. It has been found that many other hormones participate in the pathogenesis of breast cancer, such as prolactin, human chorionic gonadotropin, LH. It has been found that serum FSH was related with the recurrence free survival and overall survival rate of breast cancer and high serum level of FSH cause short overall survival rate.It has also been found serum FSH of the breast cancer has a positive relationship with the Her-2expression. However, it is not clear that the FSH participate into the pathogenesis of postmenopausal breast cancer.It is recognized that the follicle stimulating hormone receptor (FSHR) was expressed only in the granulosa and sertoli cells. However, it appeared in many other tissues such as ovarian cancer, prostate cancer, and endometrial cancer in recent researchs. It involved in the proliferation, metastasis and apoptosis of these cancer cells. Remarkably, FSH played as the main reason of osteoporosis in the postmenopausal women published at "Cell" lead a deep understanding of FSH in postmenopausal diseases. Recently, it was found that FSHR is selectively expressed on the surface of the blood vessels of a wide range of tumors. It is not clear whether the breast cancer cell expressed the FSHR.It is still a mystery that FSH enrolled in the postmenopausal breast cancer pathogenesis concerning the high FSH level in postmenopausal women.ObjectiveTo study the difference of serum gonadotropin hormone level between breast cancer and control group in postmenopausal women; To investigate the internal relations of FSH and the tumor clinical classfication,lymph node metastasis, and the status of ER,PR,Her-2and Ki67; To study the expression of FSHR in breast cancer cell line and breast cancer tissues; To study whether FSH induce proliferation and metastasis of breast cancer cell line and the exact mechanism in vitro and in vivo.MethodPart1:Clinical datas of187postmenopausal women diagnosed of breast cancer and374postmenopausal compared group, who suffer from normal gynecological inflammatory such as cervicitis,were collected for statistic analysis.Part2:Two breast cancer cell line (Bcap-37and MDA-MB-231) have been used to study whether the FSH induce proliferation and metastasis in vitro.To detect the cell proliferation in vitro,we use the MTT method; Western Blot has been used to detected the cell pathway changing after the treatment of FSH. The scratch test and Matrix transwell were used to detect the migration and invasion of the two cell line treated with FSH.Part3:Study the promoting proliferation effect of FSH in vivo.We established a postmenopausal animal model of Balb/c nu/nu mouse by ovariectomized (OVX). The experiment was divided into four groups:Sham, OVX control, using GnRha after OVX, using GnRHa followed FSH after OVX.Part4:Study of the expression of FSHR in breast cancer cell lines and tissues. RT-PCR and Reverse-PCR has used to investigate the expression of FSHR in these breast cancer cell lines:Bcap-37, MDA-MB-231, Mcf-7, T-47D, SK-BR-3and BT-474. The expression of FSHR in17samples of breast cancer tissue was also been detected by RT-PCR.ResultsPart1:The serum level of FSH,LH in postmenopausal breast cancer were higher than control group (61.83±1.475vs.48.13±1.256IU/L;27.83±0.74vs.23.88±0.6499IU/L).FSH has a positive correlation with Ki67and Her-2expression in postmenopausal breast cancer, while LH only have positive correlation with Ki67expression. No significant association has been found between gonadotropins and breast cancer clinical classification, clinical grade, lymph node metastasis, ER and PR status.Part2:It has been found that FSH could promote the Bcap-37and MDA-MB-231cell proliferation in vitro. The FSH induce the proliferation and metastasis of the two cell line by activated EGFR/ERK1/2signal pathway.Part3:A high concentration of FSH (-100IU/L) could promote the proliferation of breast cancer cell line Bcap-37and MDA-MB-231in vivo. Part4:The FSHR was not expressed in all of the breast cancer cells tested and breast cancer tissue.ConclusionPostmenupausal breast cancer patient favored a high serum gonadotropin level compared with normal gynecological inflammatory patients.The serum FSH level has a positive correlation with the Her-2and Ki67expression in postmenopausal breast cancer,while LH has a positive corelation with Ki67expression. The FSH cound promote the proliferation and metastasis of breast cancer cell line Bcap-37and MDA-MB-231by activated the EGFR/ERK1/2pathway in vitro.A high concentration of FSH (-100IU/L) may also promote the proliferation of breast cancer cell line in vivo. However, we haven’t found the expression of FSHR in breast cancer cell line been tested and breast cancer tissue. Further studies are needed to charlify the exact mechanism between FSH and postmenopausal breast cancer.更多还原