FSH联合LH上调上皮性卵巢癌侵袭能力的机制研究

发布时间：2018-07-20 16:20

【摘要】：研究背景：卵巢癌居妇女生殖系统恶性肿瘤第2位,其中上皮性卵巢癌（epithelialovarian carcinoma，EOC）的死亡率居妇科恶性肿瘤之首。因此严重威胁了妇女健康和生命。在近20余年内，卵巢癌临床处理出现了三大进展，即手术病理分期、肿瘤细胞减灭术和紫杉醇+铂类作为卵巢上皮癌的一线治疗方案。但卵巢癌早期无典型症状及体征，缺乏早期筛查卵巢恶性肿瘤的值得信赖的检测方法，因此诊断时80%-90%为晚期、复发及转移的患者，治疗极其困难。常规的手术、化疗难以治愈，此外反复手术、化疗严重影响患者生存质量。因此有必要研究其发生发展规律及机制,为寻求有效的治疗方法,改善患者预后奠定基础。促性腺激素是由垂体前叶分泌的糖蛋白激素，分为FSH、LH两种，两者具有相似的化学和结构特征。调节脊椎动物性腺发育，在卵巢、睾丸中以内分泌的方式调节甾体激素的生成及配子的发生。其受体FSHR和LHR是G蛋白偶联受体含有7个跨膜区域具有酪氨酸激酶的作用，通过G蛋白启动细胞内信号传导通路发挥生物效应。上皮间质转化（EMT）定义为上皮细胞通过特定程序转化为具有间质表型细胞的生物学过程。与肿瘤的侵袭、转移、耐药性以及复发率等一系列生物学行为密切相关。其主要的特征有细胞黏附分子(如E-钙黏蛋白)表达的减少、细胞角蛋白细胞骨架转化为波形蛋白（Vimentin）为主的细胞骨架及形态上具有间充质细胞的特征等。目的：观察FSH联合LH对卵巢癌细胞株侵袭能力的影响并探讨相关分子机制方法：(1) Western-blot和免疫组化方法检测卵巢癌细胞株SKOV3、HO8910的FSHR、 LHR表达。 (2)采用CellTiter96Aqueous细胞增殖实验检测不同浓度FSH、LH对SKOV3、HO8910细胞生长的作用。 (3)采用细胞划痕实验检测500mIU/ml的FSH联合LH对卵巢癌细胞株SKOV3、HO8910的迁移能力的影响。 (4)采用Transwell侵袭实验检测500mIU/ml的FSH联合LH对卵巢癌细胞株SKOV3、HO8910的侵袭能力的影响。 (5) Western-blot和实时荧光定量PCR检测卵巢癌细胞株在FSH联合LH处理6h、12h、24h上皮间质转化（EMT）相关分子的表达。结果： ⑴卵巢癌细胞株SKOV3、HO8910均表达FSHR、LHR。 ⑵CellTiter96Aqueous细胞增殖实验显示不同浓度FSH、LH单独/联合均不促进SKOV3和HO8910细胞的增殖。 ⑶Transwell侵袭实验显示，500mIU/ml的FSH和LH联合处理SKOV3、HO8910穿膜细胞数目显著增加，与对照组比较差异有统计学意义（P0.05）。 ⑷细胞划痕实验显示，500mIU/ml的FSH、LH联合处理上调SKOV3和HO8910细胞的迁移能力。 ⑸实时荧光定量PCR检测结果显示，，500mIU/ml的FSH、LH联合处理SKOV3、HO8910细胞，E-钙粘蛋白的mRNA表达下调，Vimentin的mRNA表达上调，差异均有统计学意义（P0.05）。 ⑹Western-blot检测结果显示500mIU/ml的FSH、LH联合处理显著下调SKOV3、HO8910细胞E-cadherin蛋白的表达，上调Vimentin、snail、slug蛋白的表达，差异均有统计学意义（P0.05）。结论： 1）卵巢癌细胞表达FSHR和LHR； 2）FSH和LH联合处理并不促进卵巢癌细胞增殖； 3）FSH和LH联合处理可以增强其迁移和侵袭能力，机制可能与FSH和LH促进EMT发生有关。
[Abstract]:Background of Study :

In recent 20 years , the clinical treatment of ovarian cancer has three major advances , such as pathological stage of operation , tumor cell deactivation and paclitaxel plus platinum as the first - line treatment of ovarian cancer .

Gonadotropin is a glycoprotein hormone secreted by anterior pituitary . It is divided into two types : FSH , LH . It has similar chemical and structural features . It regulates the development of sex gland of vertebrate . It regulates the genesis and gametogenesis of steroid hormone in the ovary and testis . Its receptor FSHR and LHR are G protein coupled receptors . The receptor FSHR and LHR have the function of tyrosine kinase in 7 transmembrane domains .

Epithelial mesenchymal transition ( EMT ) is defined as the biological process of transforming epithelial cells into cells with interstitial phenotype through specific procedures . The main features are the reduction of cell adhesion molecules ( such as E - cadherin ) , the transformation of cytoskeletal framework into the cytoskeletal and morphological features of vimentin , and the characteristics of mesenchymal cells .

Purpose :

To observe the effect of FSH combined with LH on the invasion ability of ovarian cancer cell line and to explore the related molecular mechanism

Methods : ( 1 ) Western - blot and immunohistochemistry were used to detect the FSHR of ovarian cancer cell lines SK3 and HO8910 .

LHR expression .

( 2 ) The effects of FSH and LH on the growth of the cells of SKline and HO8910 were detected by CellTiter96effector cell proliferation assay .

( 3 ) The effect of FSH combined with 500mIU / ml FSH on the migration ability of ovarian cancer cell line SK3 and HO8910 was detected by cell scratch test .

( 4 ) The effects of FSH and LH on the invasion ability of ovarian cancer cell line SK3 and HO8910 were detected by Transwell invasion experiment .

( 5 ) Western - blot and real - time fluorescence quantitative PCR were used to detect the expression of related molecules of epithelial ovarian cancer cell line ( EMT ) at 6h , 12h , 24h after FSH combined LH treatment .

Results :

( 1 ) The ovarian cancer cell lines SK3 and HO8910 express FSHR and LHR .

( 2 ) Cell proliferation experiments showed that both FSH and LH alone did not promote the proliferation of SKOVCs and HO8910 cells .

( 3 ) Transwell invasion experiment showed that 500 mIU / ml of FSH and LH combined with the number of cells in SK3 and HO8910 were significantly increased compared with the control group ( P0.05 ) .

( 4 ) The cell scratch test showed that 500mIU / ml FSH and LH treatment up - regulated the migration ability of SKOVCs and HO8910 cells .

( 5 ) The results of real - time fluorescence quantitative PCR showed that the mRNA expression down - regulation and vimentin expression were down - regulated by 500 mIU / ml FSH and LH , and the mRNA expression of vimentin was up - regulated and the difference was statistically significant ( P0.05 ) .

( 6 ) Western - blot analysis showed that the combined treatment of FSH and LH in 500 mIU / ml significantly downregulated the expression of E - cadherin protein , and up - regulated the expression of E - cadherin in the cells of HO8910 , and the difference was statistically significant ( P0.05 ) .

Conclusion :

1 ) ovarian cancer cells express FSHR and LHR ;

2 ) the combination of FSH and LH does not promote the proliferation of ovarian cancer cells ;

3 ) The combined treatment of FSH and LH can enhance its migration and invasion ability , and the mechanism may be related to FSH and LH for EMT .
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【共引文献】