本文選題：脂質(zhì)運(yùn)載蛋白-2 + 卵巢癌��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:卵巢癌患者早期癥狀隱匿,確診時(shí)已處于晚期,導(dǎo)致卵巢癌患者的死亡率居?jì)D科腫瘤之首,對(duì)婦女生命造成嚴(yán)重威脅。脂質(zhì)運(yùn)載蛋白-2(lipocalin-2,LCN2)是一種25kDa的外分泌型糖蛋白,有研究表明LCN2的表達(dá)量與卵巢癌的惡性程度密切相關(guān)。然而,有關(guān)LCN2在卵巢癌細(xì)胞中的作用及機(jī)制還不明確。本研究將檢測(cè)LCN2的表達(dá)與卵巢癌臨床病理特征的相關(guān)性,以及LCN2對(duì)卵巢癌細(xì)胞增殖活力的影響,并初步探討相關(guān)信號(hào)通路的分子機(jī)制。方法:通過免疫組化方法檢測(cè)LCN2在正常卵巢組織與卵巢腫瘤組織中的表達(dá)程度,統(tǒng)計(jì)評(píng)估LCN2與卵巢癌臨床病理特征的相關(guān)性;應(yīng)用MTT細(xì)胞增殖實(shí)驗(yàn)和平板細(xì)胞克隆形成實(shí)驗(yàn)檢測(cè)過表達(dá)LCN2對(duì)卵巢癌細(xì)胞系(ES2和SKOV3)增殖活力的影響;通過穩(wěn)定轉(zhuǎn)染LCN2質(zhì)粒DNA加藥物篩選的方法構(gòu)建穩(wěn)定過表達(dá)LCN2的卵巢癌細(xì)胞系;通過磷酸蛋白芯片篩選過表達(dá)LCN2后產(chǎn)生的相關(guān)差異表達(dá)的蛋白;應(yīng)用Western Blot和細(xì)胞免疫熒光法驗(yàn)證部分差異蛋白的亞細(xì)胞定位及表達(dá)強(qiáng)度的變化。結(jié)果:1免疫組織化學(xué)結(jié)果:LCN2在正常卵巢組織中呈陰性表達(dá),在卵巢癌組織中有不同程度的陽性表達(dá)。表達(dá)程度量化評(píng)分后,經(jīng)統(tǒng)計(jì)學(xué)分析發(fā)現(xiàn),LCN2在卵巢癌組織中表達(dá)量高于正常卵巢組織并與卵巢癌組織分化程度具有顯著性差異(P0.0001)。2 MTT細(xì)胞增殖實(shí)驗(yàn)結(jié)果:選擇相對(duì)低表達(dá)LCN2的兩個(gè)卵巢癌細(xì)胞系(ES2和SKOV3),瞬時(shí)轉(zhuǎn)染LCN2質(zhì)粒和空白對(duì)照質(zhì)粒48h之后,過表達(dá)LCN2組的細(xì)胞增殖活力高于對(duì)照組(P0.05)。3平板細(xì)胞克隆形成實(shí)驗(yàn)結(jié)果:分別在ES2和SKOV3細(xì)胞中,穩(wěn)定轉(zhuǎn)染等量的LCN2質(zhì)粒和空白對(duì)照質(zhì)粒,經(jīng)G418篩選至克隆形成,結(jié)果顯示過表達(dá)LCN2組克隆形成率明顯多于對(duì)照組(P0.05)。4磷酸蛋白芯片實(shí)驗(yàn)結(jié)果:在卵巢癌細(xì)胞系中,利用已經(jīng)成功構(gòu)建的穩(wěn)定高表達(dá)LCN2細(xì)胞系,磷酸蛋白芯片篩選結(jié)果顯示過表達(dá)LCN2組相對(duì)于對(duì)照組,ERK和GSK3β的磷酸化水平均明顯上調(diào)(P0.05)。5 Western Blot檢測(cè)結(jié)果:首先在四種卵巢癌細(xì)胞系(ES2、SKOV3、CAOV3、OVCAR3)中分別檢測(cè)了LCN2、磷酸化ERK(p-ERK)、ERK、磷酸化GSK3β(p-GSK3β)和GSK3β及β-catenin的蛋白表達(dá)水平,發(fā)現(xiàn)LCN2高表達(dá)的細(xì)胞系(CAOV3和OVCAR3)對(duì)比LCN2低表達(dá)的細(xì)胞系(ES2和SKOV3),磷酸化ERK和磷酸化GSK3β的表達(dá)水平上調(diào)。隨后,在穩(wěn)定過表達(dá)LCN2的ES2和SKOV3細(xì)胞系中再次驗(yàn)證LCN2、磷酸化ERK、ERK、磷酸化GSK3β和GSK3β的蛋白表達(dá)水平,結(jié)果顯示過表達(dá)LCN2上調(diào)了磷酸化ERK、磷酸化GSK3β和β-catenin的表達(dá)(P0.05)。6細(xì)胞免疫熒光結(jié)果:在SKOV3細(xì)胞系中瞬時(shí)轉(zhuǎn)染pEGFPN3和pEGFPN3-LCN2質(zhì)粒DNA,發(fā)現(xiàn)成功轉(zhuǎn)染LCN2組中,LCN2、磷酸化ERK、磷酸化GSK3β及β-catenin的表達(dá)強(qiáng)度相對(duì)于對(duì)照組有明顯增加。結(jié)論:1 LCN2在卵巢癌組織中高表達(dá),并且與組織的分化程度負(fù)相關(guān)。2過表達(dá)LCN2可以促進(jìn)卵巢癌細(xì)胞的增殖活力。3過表達(dá)LCN2上調(diào)磷酸化ERK、磷酸化GSK3β及β-catenin表達(dá)水平,提示LCN2促進(jìn)卵巢癌細(xì)胞的增殖可能經(jīng)ERK/GSK3β信號(hào)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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.31

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本文編號(hào)：1813289