脂质运载蛋白-2对人类卵巢癌细胞增殖能力的影响以及相关分子机制的研究

发布时间：2018-04-28 02:00

  本文选题：脂质运载蛋白-2 + 卵巢癌　； 参考：《河北医科大学》2017年硕士论文

【摘要】：目的:卵巢癌患者早期症状隐匿,确诊时已处于晚期,导致卵巢癌患者的死亡率居妇科肿瘤之首,对妇女生命造成严重威胁。脂质运载蛋白-2(lipocalin-2,LCN2)是一种25kDa的外分泌型糖蛋白,有研究表明LCN2的表达量与卵巢癌的恶性程度密切相关。然而,有关LCN2在卵巢癌细胞中的作用及机制还不明确。本研究将检测LCN2的表达与卵巢癌临床病理特征的相关性,以及LCN2对卵巢癌细胞增殖活力的影响,并初步探讨相关信号通路的分子机制。方法:通过免疫组化方法检测LCN2在正常卵巢组织与卵巢肿瘤组织中的表达程度,统计评估LCN2与卵巢癌临床病理特征的相关性;应用MTT细胞增殖实验和平板细胞克隆形成实验检测过表达LCN2对卵巢癌细胞系(ES2和SKOV3)增殖活力的影响;通过稳定转染LCN2质粒DNA加药物筛选的方法构建稳定过表达LCN2的卵巢癌细胞系;通过磷酸蛋白芯片筛选过表达LCN2后产生的相关差异表达的蛋白;应用Western Blot和细胞免疫荧光法验证部分差异蛋白的亚细胞定位及表达强度的变化。结果:1免疫组织化学结果:LCN2在正常卵巢组织中呈阴性表达,在卵巢癌组织中有不同程度的阳性表达。表达程度量化评分后,经统计学分析发现,LCN2在卵巢癌组织中表达量高于正常卵巢组织并与卵巢癌组织分化程度具有显著性差异(P0.0001)。2 MTT细胞增殖实验结果:选择相对低表达LCN2的两个卵巢癌细胞系(ES2和SKOV3),瞬时转染LCN2质粒和空白对照质粒48h之后,过表达LCN2组的细胞增殖活力高于对照组(P0.05)。3平板细胞克隆形成实验结果:分别在ES2和SKOV3细胞中,稳定转染等量的LCN2质粒和空白对照质粒,经G418筛选至克隆形成,结果显示过表达LCN2组克隆形成率明显多于对照组(P0.05)。4磷酸蛋白芯片实验结果:在卵巢癌细胞系中,利用已经成功构建的稳定高表达LCN2细胞系,磷酸蛋白芯片筛选结果显示过表达LCN2组相对于对照组,ERK和GSK3β的磷酸化水平均明显上调(P0.05)。5 Western Blot检测结果:首先在四种卵巢癌细胞系(ES2、SKOV3、CAOV3、OVCAR3)中分别检测了LCN2、磷酸化ERK(p-ERK)、ERK、磷酸化GSK3β(p-GSK3β)和GSK3β及β-catenin的蛋白表达水平,发现LCN2高表达的细胞系(CAOV3和OVCAR3)对比LCN2低表达的细胞系(ES2和SKOV3),磷酸化ERK和磷酸化GSK3β的表达水平上调。随后,在稳定过表达LCN2的ES2和SKOV3细胞系中再次验证LCN2、磷酸化ERK、ERK、磷酸化GSK3β和GSK3β的蛋白表达水平,结果显示过表达LCN2上调了磷酸化ERK、磷酸化GSK3β和β-catenin的表达(P0.05)。6细胞免疫荧光结果:在SKOV3细胞系中瞬时转染pEGFPN3和pEGFPN3-LCN2质粒DNA,发现成功转染LCN2组中,LCN2、磷酸化ERK、磷酸化GSK3β及β-catenin的表达强度相对于对照组有明显增加。结论:1 LCN2在卵巢癌组织中高表达,并且与组织的分化程度负相关。2过表达LCN2可以促进卵巢癌细胞的增殖活力。3过表达LCN2上调磷酸化ERK、磷酸化GSK3β及β-catenin表达水平,提示LCN2促进卵巢癌细胞的增殖可能经ERK/GSK3β信号通路。
[Abstract]:Objective: the early symptoms of ovarian cancer are insidious, and the diagnosis of ovarian cancer is in the late stage. The mortality of ovarian cancer patients is the first and serious threat to women's life. The lipid carrier protein -2 (Lipocalin-2, LCN2) is a kind of 25kDa exocrine glycoprotein. The study shows that the expression of LCN2 is closely related to the malignant degree of ovarian cancer. However, the role and mechanism of LCN2 in ovarian cancer cells is not clear. This study will examine the correlation between the expression of LCN2 and the clinicopathological features of ovarian cancer, as well as the effect of LCN2 on the proliferation of ovarian cancer cells, and discuss the molecular mechanism of the related signaling pathways preliminarily. The method of immunohistochemistry: the detection of LCN2 in normal eggs by immunohistochemical method. The correlation between the expression of LCN2 and the clinicopathological features of ovarian cancer; the effect of LCN2 on the proliferation of ovarian cancer cell lines (ES2 and SKOV3) by MTT cell proliferation test and cell clone formation test, and the method of stable transfection of LCN2 plasmid DNA plus drug screening To construct the ovarian cancer cell line that stably overexpressed LCN2; screen the related differentially expressed proteins after the expression of LCN2 through the phosphoprotein chip; use Western Blot and cell immunofluorescence to verify the subcellular localization and expression intensity of partial differential proteins. Results: 1 immunohistochemical results: LCN2 in normal ovarian tissue Negative expression was expressed in ovarian cancer tissue. After quantitative evaluation, the expression of LCN2 in ovarian cancer tissues was higher than that of normal ovarian tissue, and the differentiation degree of ovarian cancer tissue was significantly different (P0.0001).2 MTT cell proliferation experiment results: selecting relatively low expression LC Two ovarian cancer cell lines (ES2 and SKOV3) of N2, after transient transfection of LCN2 plasmid and blank control plasmid 48h, the proliferation activity of overexpressed LCN2 group was higher than that of the control group (P0.05).3 flat cell clone formation experimental results: the LCN2 plasmids and blank control plasmids were stably transfected in ES2 and SKOV3 cells, respectively, and screened by G418 to clone them. The results showed that the formation rate of the overexpressed LCN2 group was more than that of the control group (P0.05).4 phosphate protein chip experimental results: in the ovarian cancer cell line, the stable high expression LCN2 cell line has been successfully constructed, the phosphoric acid protein chip screening results showed that the LCN2 group was expressed in the LCN2 group relative to the control group, and the phosphorylation level of ERK and GSK3 beta was clear. The results of P0.05.5 Western Blot detection were first detected in the four ovarian cancer cell lines (ES2, SKOV3, CAOV3, OVCAR3), respectively, of LCN2, phosphorylated ERK (p-ERK), ERK, phosphorylated beta and beta and beta protein expression levels. And SKOV3), the expression level of phosphorylated ERK and phosphorylated GSK3 beta was up-regulated. Subsequently, the expression of LCN2, phosphorylated ERK, ERK, phosphorylated GSK3 beta and GSK3 beta were re validated in the ES2 and SKOV3 cell lines stably over expressed LCN2, and the results showed that the overexpression LCN2 was up to phosphorylated, phosphorylated beta and beta cells exemptled. The results of immunofluorescence: transient transfection of pEGFPN3 and pEGFPN3-LCN2 plasmid DNA in the SKOV3 cell line. It was found that the expression of LCN2, phosphorylated ERK, phosphorylated GSK3 beta and beta -catenin were significantly increased in the LCN2 group. Conclusion: 1 LCN2 is highly expressed in ovarian cancer tissue and negatively related to the degree of tissue differentiation. CN2 can promote the proliferation of ovarian cancer cells.3 overexpression LCN2 up regulation of phosphorylated ERK, phosphorylated GSK3 beta and beta -catenin expression level, suggesting that LCN2 promotes the proliferation of ovarian cancer cells through the ERK/GSK3 beta signaling pathway.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

【参考文献】

中国期刊全文数据库 前2条

1 王晔;包玉倩;;脂质运载蛋白-2的研究新进展[J];复旦学报(医学版);2009年06期

2 李恩民,许丽艳,蔡唯佳,熊华淇,沈忠英,曾毅;SHEEC食管癌细胞中NGAL基因的功能[J];生物化学与生物物理学报;2003年03期

，

本文编号：1813289