【作者】 刘娟娟；

【导师】 林蓓；

【作者基本信息】 中国医科大学 ， 妇产科学， 2010， 博士

【摘要】 前言卵巢癌在妇科恶性肿瘤中病死率居首位,卵巢癌的发生发展机制一直是妇科肿瘤基础与临床研究探索的重点,了解卵巢癌生物学行为的控制方法及发生发展的分子机制对于卵巢癌的预防及治疗十分重要。卵巢癌的发生与发展是一个极其复杂的多因素的过程,目前对其发生机理尚不清楚。近年来,糖复合物和肿瘤的关系受到广泛关注。糖复合物是细胞膜的重要组成部分,对细胞-细胞、细胞-分子的识别十分重要,参与信号转导、分子粘附等分子相互作用,与细胞生长、凋亡、运动、分化等生命过程密切相关。细胞癌变后细胞膜上的糖复合物,特别是其糖链部分发生结构和量的变化。卵巢癌主要表现为II型糖链的改变,如Lewis y抗原。研究表明75%的上皮性卵巢癌出现Lewis y不同程度的过量表达,且增高的患者预后不良。我们在前期工作中利用基因转染技术将人α1,2-岩藻糖转移酶(α1,2-fucosyltransferase,α1,2-FT)基因转入卵巢癌细胞系RMG-I,建立了Lewis y稳定高表达卵巢癌细胞系RMG-I-H,发现转染后RMG-I-H细胞对化疗药5-FU的敏感性下降,提示Lewis y抗原具有提高卵巢癌细胞生存能力的作用。细胞表面的受体几乎都是糖蛋白,研究表明,改变糖基转移酶的表达可影响细胞表面受体的糖链结构,继而影响其表达数目及功能。我们在前期工作中利用基因芯片技术检测了α1,2-FT基因转染前后细胞系癌相关基因的表达差异,结果显示转染后细胞中有88种差异性表达基因,差异基因主要表现在细胞增殖、信号转导、蛋白氨基酸磷酸化、转录、凋亡等方面。因此,我们推测Lewis y可能作为信号转导通路上的重要结构参与细胞内信号转导,进而影响卵巢癌细胞的生物学行为。本研究主要在前期工作的基础上,利用已经建立的α1,2-FT及Lewis y稳定高表达细胞模型,探讨Lewis y抗原对卵巢癌细胞增殖的影响及作用机制,为研究卵巢癌的发生发展机制奠定基础,为卵巢癌的早期诊断及靶器官治疗提供理论依据。方法第一部分：RT-PCR方法检测基因转染前后细胞中α1,2-FT mRNA的表达；免疫细胞化学染色法检测细胞中Lewis y抗原的表达；MTT法检测基因转染前后细胞的增殖情况；流式细胞仪分析细胞周期；分别用α-L-岩藻糖苷酶和抗Lewis y抗体处理卵巢癌细胞系,MTT法检测处理后细胞的增殖情况。第二部分：RT-PCR方法检测基因转染前后细胞中EGFR, HER2/neu mRNAs的表达；Western blot方法检测基因转染前后细胞中EGFR, HER2/neu蛋白的表达；用EGFR及HER2/neu特异性抗体进行免疫沉淀,并应用抗Lewis y单克隆抗体为一抗进行Western印迹杂交,检测EGFR, HER2/neu上Lewis y抗原的表达情况；Western blot方法检测细胞中EGFR, HER2/neu蛋白的磷酸化情况。第三部分：Western blot方法检测基因转染前后细胞中PI3K／Akt和Raf/MEK/MAPK信号转导通路上重要信号分子的表达及磷酸化情况；分别用EGFR酪氨酸激酶特异性抑制剂ZD1839和抗Lewis y单克隆抗体处理细胞,Western blot方法检测上述重要信号分子的表达及磷酸化情况；MTT法检测细胞在不同浓度PI3K特异性抑制剂LY294002作用下的存活情况。结果第一部分：RT-PCR结果显示转染后细胞中al,2-FT mRNA表达显著增高。免疫细胞化学染色结果显示Lewis y抗原在转染后细胞中显著高表达。MTT结果显示随着细胞表面Lewis y的增多,卵巢癌细胞增殖加快。α-L-岩藻糖苷酶和抗Lewisy抗体都显著抑制细胞的增殖。细胞周期检测结果显示Lewis y高表达增强DNA合成,促进G0-G1期细胞进入S期和G2-M期,使细胞周期缩短。第二部分：RT-PCR结果显示转染后细胞中HER2/neu mRNA表达显著增高,而EGFR mRNA未见明显改变。免疫共沉淀结合Western blot检测到EGFR蛋白的表达在转染前后细胞中无明显变化,但基因转染后EGFR上Lewis y的相对含量较转染前显著增加；与EGFR相反,基因转染后HER2/neu蛋白的表达较转染前显著增加,但转染前后HER2/neu上Lewis y的相对含量却没有明显的变化。此外,Western blot还检测到基因转染后EGFR和HER2/neu的酪氨酸磷酸化水平都显著升高。第三部分：Western blot结果显示转染后细胞中Akt、ERK1／2总蛋白的表达未发生明显改变,但它们的磷酸化水平都显著增高；分别用EGFR酪氨酸激酶抑制剂ZD1839和抗Lewis y抗体处理后,转染前后细胞中磷酸化Akt、ERK1／2的表达水平都下降,尤其以转染后细胞下降得更明显,且存在于两种细胞中的高低区别也消失。MTT检测结果显示PI3K特异性抑制剂LY294002显著抑制Lewis y高表达卵巢癌细胞的增殖。结论1. Lewis y抗原促进卵巢癌细胞增殖,加快细胞周期进展。2. Lewis y抗原不但是EGFR还是HER2/neu结构上的一部分。3. Lewis y抗原作为EGFR家族结构上的一部分,其表达增加不但激活了EGFR及HER2/neu受体酪氨酸激酶,还进一步激活了EGFR家族下游的PI3K/Akt和Raf/MEK/MAPK信号转导通路,导致细胞核内HER2/neu等基因转录的加速,刺激DNA合成,最终促进细胞跳过G1期限制点进入S期,促进细胞增殖。4. PI3K/Akt信号转导通路在Lewis y抗原促进细胞增殖中起着重要的作用。更多还原

【Abstract】 IntroductionOvarian cancer is the leading cause of death in gynecological cancers, the occurrence and development mechanism of ovarian cancer has been the focus of basic and clinical research in gynecologic cancer, understand the methods of controlling the biological behavior of ovarian cancer and the occurrence and development molecular mechanisms of ovarian cancer is very important for prevention and treatment of ovarian cancer.The occurrence and development of ovarian cancer is an extremely complicated multi-factor process, currently its pathogenesis is not clear. In recent years, the research on the relationship between glycans and tumors has attracted wide attention. Glycans are important components of cell membrane, which play essential roles in cell-cell interaction, cell-molecule recognition, as well as involve in signal transduction and molecule adhesion, therefore closely relate to many important life processes such as cell growth, apoptosis, mobility and differentiation etc. Upon cancerous transformation, cell membrane glycans, especially the carbohydrate chain of them, undergo structural and quantitative changes. The major presentation of ovarian cancer is alteration in type II carbohydrate chains, such as Lewis y antigen. Research shows that 75% of epithelial ovarian cancers have varying degree of Lewis y overexpression, and increased expression is associated with poor prognosis of patients. In our preliminary study, we introduced al,2-fucosyltransferase (al,2-FT) gene into human ovarian cancer cell line RMG-I through gene transfection and established cell model overexpressing Lewis y antigen. It was discovered that the RMG-I-H cells after transfection becomes highly tolerant to the anti-tumor drug,5-FU. It suggested that the Lewis y antigen possessed the function of boosting the survival ability of ovarian carcinoma cells.Most cell surface receptors are glycoproteins, studies showed that changes in glycosyltransferase expression might affect structure of carbohydrate chains on cell surface receptors and therefore impacted the expression and function of those glycoprotein receptors. In previous studies, we tested the differences in oncogene expression before and after al,2-FT gene transfection using gene chips technology. Results showed that:there were 88 differentially expressed genes after cell transfection. Altered genes mainly involved these genes regulating cell proliferation, signal transduction, protein amino acid phosphorylation, transcription, apoptosis and so on. Thus, it is possible that Lewis y may be an important component in signaling transduction pathway participating in signal transduction inside cell and further promoting proliferation of ovarian cancer cells.This study mainly on the basis of the preliminary work, and the use of a1,2-FT and Lewis y stable high expression cell model which has been already established, discuss the effect and molecular mechanism of Lewis y antigen on the proliferation of ovarian cancer cells. The results will lay the ground work for studying occurrence and development of ovarian cancer, provide a theoretical basis for ovarian cancer early diagnosis and treatment of target organ.MethodsThe first part:Reverse transcription-polymerase chain reaction (RT-PCR) was used to test the expression of a1,2-FT mRNA in the pre-and post-transfection cell lines; immunocytochemical staining was performed to detect the expression of Lewis y antigen in the cell lines; MTT assay was carried out to test the proliferation of the pre-and post-transfection cell lines; flow cytometry was used to analysis the cell cycle; the ovarian cancer cell lines were treated with a-L-fucosidase and anti-Lewis y antibody respectively, then MTT assay was carried out to test the proliferation.The second part:RT-PCR was used to test the expression of EGFR, HER2/neu mRNAs in the pre-and post-transfection cell lines; Western blot was carried out to detect the the expression of EGFR, HER2/neu proteins in the pre-and post-transfection cell lines; immunoprecipitaion using specific antibodies of EGFR, HER2/neu combined with Western blot using monoclonal antibody against Lewis y as primary antibody was used to test the expression level of Lewis y on the EGFR, HER2/neu; Western blot was performed to detect the the phosphorylation level of EGFR, HER2/neu proteins in cell lines.The third part:Western blot was carried out to detect the expression and phosphorylation level of PI3K/Akt and Raf/MEK/MAPK signaling pathways related-proteins in the pre-and post-transfection cell lines; the cells were treated with EGFR tyrosine kinase specific inhibitor ZD1839 and anti-Lewis y antibody respectively, then Western blot was used to detect the the expression and phosphorylation level of above mentioned signal proteins; MTT assay was carried out to test the survival of the cells treated with various PI3K inhibitor LY294002.ResultsThe first part:RT-PCR results showed the expression of a1,2-FT mRNA was significantly upregulated in post-transfection cell line RMG-I-H. Immunocytochemical staining results showed the expression of Lewis y antigen was significantly upregulated in RMG-I-H cells. MTT assay results showed the cell proliferation sped up as the Lewis y antigen was increased. Both of a-L-fucosidase and anti-Lewis y antibody inhibited the cell proliferation. Cell cycle test results showed Lewis y overexpression enhanced DNA synthesis and promoted cells in G0-G1 phase to enter S and G2-M phase, leading to shortened cell cycle.The second part:RT-PCR results showed the expression of HER2/neu mRNA was significantly upregulated in post-transfection cell line RMG-I-H, while the mRNA of EGFR was unchanged. Immunoprecipitation combined with Western blot showed the expression of EGFR protein in the transfected cells had no significant change, but the relative content of Lewis y on EGFR afterα1,2-FT gene transfection increased; in opposite to EGFR, although the expression of HER2/neu protein significantly increased afterα1,2-FT gene transfection, but the relative content of Lewis y on HER2/neu had no significant change. Western blot showed the phosphorylation level of EGFR, HER2/neu proteins were all significantly upregulated after transfection.The third part:Western blot showed the expression of Akt and ERK1/2 proteins were not obviously altered inα1,2-FT transfected cells, but the relative phosphorylation of Akt and ERK1/2 were all increased. When the cells were treated with EGFR tyrosine kinase specific inhibitor ZD1839 and anti-Lewis y antibody respectively, phosphorylation of both Akt and ERK1/2 were apparently decreased in non-andα1,2-FT transfected cells, especially theα1,2-FT transfected cells. By contrast, differences in phosphorylation intensity for Akt and ERK1/2 among non-andα1,2-FT transfected cell groups were attenuated in ZD1839 or anti-Lewis y antibody-treated cells. MTT assay results showed LY294002 inhibited the growth of Lewis y-overexpressing cells.Conclusions1. Lewis y antigen promotes the proliferation of the ovarian cancer cells, accelerates cell cycle progress.2. Lewis y antigen is not only an integral part of EGFR, but also a component of HER2/neu.3. Lewis y antigen as an integral part of EGFR family, its overexpression not only activates the tyrosine kinase of EGFR and HER2/neu, but also activates the downstream PI3K/Akt and Raf/MEK/MAPK signal transduction pathways of EGFR family, leads to accelerated gene transcription of HER2/neu in nucleus, enhances DNA synthesis and promotes cells in G0-G1 phase to enter S and G2-M phase, finally promotes the proliferation of the ovarian cancer cells.4. PI3K/Akt signaling pathway plays an important role in Lewis y antigen promoting cell proliferation.更多还原