【摘要】：喉癌是頭頸部最常見的腫瘤之一,其中以喉鱗狀細(xì)胞癌(laryngeal squamous cell carcinoma,LSCC)為主,約占喉癌的96%~98%。最新研究顯示喉癌的總生存率有下降趨勢(shì),而影響喉癌預(yù)后的關(guān)鍵因素是癌細(xì)胞的侵襲性和轉(zhuǎn)移性,因此進(jìn)一步了解喉癌侵襲轉(zhuǎn)移的相關(guān)分子機(jī)制,可為喉癌患者的預(yù)后評(píng)估及個(gè)體化治療提供必要的依據(jù)。腫瘤的侵襲轉(zhuǎn)移是一個(gè)動(dòng)態(tài)且復(fù)雜的過(guò)程,是多種蛋白分子參與多個(gè)步驟共同作用的結(jié)果。Rho A作為“信號(hào)通路分子開關(guān)”介導(dǎo)了多種信號(hào)轉(zhuǎn)導(dǎo)通路而參與調(diào)節(jié)細(xì)胞的活動(dòng)。近年研究發(fā)現(xiàn)RhoA的表達(dá)水平與腫瘤的發(fā)展密切相關(guān)。Ezrin為膜細(xì)胞骨架連接蛋白,在多種惡性腫瘤中呈高表達(dá),被認(rèn)為是腫瘤轉(zhuǎn)移的關(guān)鍵調(diào)節(jié)因子,在腫瘤的浸潤(rùn)轉(zhuǎn)移中發(fā)揮著重要作用�；罨腞ho A可通過(guò)激活ROCK-1進(jìn)而促進(jìn)Ezrin的磷酸化,增強(qiáng)Ezrin與細(xì)胞骨架的聯(lián)系,從而改變細(xì)胞骨架結(jié)構(gòu)并參與細(xì)胞的運(yùn)動(dòng)過(guò)程。本研究旨在通過(guò)檢測(cè)Ezrin及RhoA在喉鱗癌細(xì)胞中的表達(dá)情況,以了解這些參與腫瘤侵襲轉(zhuǎn)移的基因表達(dá)在喉鱗狀細(xì)胞癌發(fā)生發(fā)展的作用及其相關(guān)性,進(jìn)一步探討喉癌細(xì)胞局部浸潤(rùn)及淋巴結(jié)轉(zhuǎn)移的作用機(jī)制。一、RhoA和Ezrin在新鮮喉癌組織及新鮮喉粘膜組織中的表達(dá)本實(shí)驗(yàn)應(yīng)用免疫組織化學(xué)染色(Immunohistochemistry,IHC)、免疫蛋白印跡(Western blot,WB)及實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(Quantita-ti ve reverse transcription polymerase chain reaction,q RT-PCR)法分別檢測(cè)30例喉鱗狀細(xì)胞癌患者手術(shù)切除新鮮癌組織及30例相對(duì)應(yīng)正常喉粘膜組織中RhoA和Ezrin的蛋白表達(dá)量及mRNA的基因表達(dá)水平,并結(jié)合臨床病理特征進(jìn)行統(tǒng)計(jì)學(xué)分析。應(yīng)用SPSS22統(tǒng)計(jì)分析軟件進(jìn)行統(tǒng)計(jì)學(xué)處理,P0.05時(shí)差異具有統(tǒng)計(jì)學(xué)意義。免疫組化結(jié)果顯示在LSCC組織中RhoA和Ezrin的陽(yáng)性表達(dá)率(60%和70%)明顯高于正常喉粘膜組織(13.3%和16.7%);Western blot結(jié)果顯示RhoA和Ezrin在LSCC組織中的蛋白表達(dá)量均明顯高于正常喉粘膜組織;qRT-PCR結(jié)果顯示RhoA和Ezrin在LSCC組織中的m RNA表達(dá)量也均高于正常喉粘膜組,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。兩者的表達(dá)與淋巴結(jié)的轉(zhuǎn)移、臨床分期密切相關(guān)(P0.05);而與腫瘤分化程度、年齡、性別、吸煙程度無(wú)關(guān)(P0.05)。同時(shí)一致性檢驗(yàn)結(jié)果顯示RhoA和Ezrin在喉鱗狀細(xì)胞癌細(xì)胞中的表達(dá)呈正相關(guān)(Ka ppa=0.638)。二、回顧性分析RhoA和Ezrin在喉鱗癌組織及正常喉粘膜組織中的表達(dá)本實(shí)驗(yàn)應(yīng)用免疫組織化學(xué)染色方法檢測(cè)近10年的喉癌組織石蠟塊68例及正常喉粘膜組織石蠟塊35例中RhoA和Ezrin的表達(dá)情況,并結(jié)合臨床病理特征進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果顯示LSCC組織中RhoA和Ezrin的陽(yáng)性表達(dá)率(80.9%和82.4%)明顯高于正常喉粘膜組織(28.5%和25.7%),差異具有統(tǒng)計(jì)學(xué)意義(P0.05);且兩者的表達(dá)量與淋巴結(jié)的轉(zhuǎn)移、臨床分期密切相關(guān)(P0.05);而與分化程度、年齡、性別、吸煙程度無(wú)關(guān)(P0.05)。一致性檢驗(yàn)結(jié)果顯示RhoA和Ezrin在喉鱗狀細(xì)胞癌細(xì)胞中的表達(dá)呈正相關(guān)(Kappa=0.363),與上述新鮮組織中RhoA和Ezrin的表達(dá)情況基本一致,進(jìn)一步驗(yàn)證結(jié)果的可靠性。綜上所述,RhoA和Ezrin在LSCC組織中不論從細(xì)胞分子水平還是mRNA的基因表達(dá)水平較正常喉粘膜組織均呈高表達(dá),并同時(shí)與喉癌患者淋巴結(jié)轉(zhuǎn)移、臨床分期密切相關(guān),且兩者在喉癌細(xì)胞中的表達(dá)呈正相關(guān),提示兩者可能共同參與喉癌細(xì)胞的浸潤(rùn)轉(zhuǎn)移過(guò)程。免疫組化結(jié)果顯示RhoA分布于細(xì)胞漿及細(xì)胞膜,Ezrin主要分布于細(xì)胞膜,提示Ezrin可能通過(guò)與細(xì)胞膜蛋白結(jié)合使肌動(dòng)蛋白與細(xì)胞膜相連從而改變細(xì)胞骨架結(jié)構(gòu),調(diào)節(jié)細(xì)胞結(jié)構(gòu)及粘附性而參與腫瘤細(xì)胞的浸潤(rùn)轉(zhuǎn)移過(guò)程,兩者之間表達(dá)的一致性提示RhoA可能作為Ezrin上游信號(hào)通路促進(jìn)Ezrin的活化后參與腫瘤的浸潤(rùn)轉(zhuǎn)移過(guò)程。為喉癌的早期診斷和預(yù)后評(píng)估提供新的依據(jù)。
[Abstract]:Laryngeal squamous cell carcinoma (LSCC) is one of the most common tumors in the head and neck, accounting for 96%-98% of the total laryngeal cancer. Recent studies have shown that the overall survival rate of laryngeal cancer is declining, and the key factors affecting the prognosis of laryngeal cancer are the invasiveness and metastasis of cancer cells. Invasion and metastasis of laryngeal carcinoma is a dynamic and complex process, which is the result of multiple protein molecules participating in multiple steps. Rho A, as a "molecular switch of signal pathway", mediates a variety of signal transduction pathways. Recent studies have shown that the expression of Rho A is closely related to the development of tumor. Ezrin, a membrane cytoskeleton connexin, is highly expressed in many malignant tumors. Ezrin is considered to be a key regulator of tumor metastasis and plays an important role in tumor invasion and metastasis. Activated Rho A can activate ROCK-1 by activating ROCK-1. The aim of this study was to investigate the expression of Ezrin and RhoA in laryngeal squamous cell carcinoma (LSCC) cells and to understand the role of these genes involved in tumor invasion and metastasis in the development of LSCC. The expression of RhoA and Ezrin in fresh laryngeal carcinoma tissues and fresh laryngeal mucosa was studied by immunohistochemical staining (IHC), Western blot (WB) and real-time quantitative polymerase chain reaction. Quantita-ti ve reverse transcription polymerase chain reaction (q RT-PCR) was used to detect the expression of RhoA and Ezrin proteins and mRNA in 30 fresh laryngeal squamous cell carcinoma tissues and 30 normal laryngeal mucosa tissues, respectively. The positive expression rates of RhoA and Ezrin in LSCC tissues (60% and 70%) were significantly higher than those in normal laryngeal mucosa tissues (13.3% and 16.7%). The expression of RhoA and Ezrin in LSCC was significantly higher than that in normal laryngeal mucosa (P 0.05). The expression of RhoA and Ezrin was closely related to lymph node metastasis and clinical stage (P 0.05), but not to tumor differentiation, age, sex and smoking (P 0.05). Conformity test showed that the expression of RhoA and Ezrin in laryngeal squamous cell carcinoma cells was positively correlated (Ka ppa=0.638). Second, the expression of RhoA and Ezrin in laryngeal squamous cell carcinoma and normal laryngeal mucosa was retrospectively analyzed. Immunohistochemical staining was used to detect paraffin masses in 68 cases of laryngeal carcinoma and normal laryngeal mucosa in the past 10 years. The expression of RhoA and Ezrin in 35 cases of paraffin-embedded lesions was statistically analyzed. The results showed that the positive rates of RhoA and Ezrin in LSCC tissues (80.9% and 82.4%) were significantly higher than those in normal laryngeal mucosa tissues (28.5% and 25.7%). The difference was statistically significant (P 0.05). The results of consistency test showed that the expression of RhoA and Ezrin in laryngeal squamous cell carcinoma cells was positively correlated (Kappa = 0.363), which was basically consistent with the expression of RhoA and Ezrin in the above fresh tissues, and further verified the reliability of the results. The expression of RhoA and Ezrin in LSCC tissues was higher than that in normal laryngeal mucosa at both cellular and molecular levels. The expression of RhoA and Ezrin was closely related to lymph node metastasis and clinical stage of laryngeal carcinoma. The expression of RhoA and Ezrin in laryngeal carcinoma cells was positively correlated, suggesting that both of them might participate in the invasion and metastasis of laryngeal carcinoma cells. Immunohistochemistry showed that RhoA was distributed in cytoplasm and cell membrane, and Ezrin was mainly distributed in cell membrane, suggesting that Ezrin might participate in the invasion and metastasis of tumor cells by binding to actin and cell membrane proteins to alter cytoskeleton structure, regulate cell structure and adhesion. These results suggest that RhoA may be an upstream signaling pathway of Ezrin to promote the activation of Ezrin and participate in the invasion and metastasis of laryngeal carcinoma.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.65

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