【摘要】：目的：研究趨化因子CCL20在喉鱗癌中的表達及其臨床意義,分析CCL20基因啟動子區(qū)的單核苷酸多態(tài)性位點及其臨床意義,探索趨化因子CCL20對過表達CCR6的喉鱗癌細胞系的體內(nèi)外生物學行為的影響。方法：收集70例行全喉切除術(shù)的喉鱗癌患者腫瘤組織、癌旁組織以及15例聲帶白斑組織(包括其中13例發(fā)生淋巴結(jié)轉(zhuǎn)移喉鱗癌患者的轉(zhuǎn)移淋巴結(jié)和10例無腫瘤轉(zhuǎn)移的淋巴結(jié)的白片),應(yīng)用免疫組化技術(shù)檢測CCL20的表達,分析其與患者臨床病理特征的關(guān)系；收取180例喉鱗癌患者,51例聲帶白斑,116例正常人的外周血,應(yīng)用PCR和測序技術(shù)分析CCL20基因啟動子區(qū)存在的SNP位點,分析其與喉鱗癌,聲帶白斑發(fā)病的聯(lián)系,以及同患者臨床病理特征的關(guān)系；收取36例喉癌患者和42名正常志愿者的外周血,應(yīng)用ELISA技術(shù)檢測喉鱗癌患者和正常人血清中CCL20蛋白的表達水平,分析其與患者臨床病理特征及CCL20啟動子區(qū)SNP位點的關(guān)系；應(yīng)用慢病毒感染構(gòu)建穩(wěn)定表達CCR6的HEp-2-CCR6和HN-8-CCR6喉鱗癌細胞系,并用流式細胞術(shù),細胞爬片免疫組化,鈣流實驗鑒定細胞系CCR6的表達及受體功能情況；應(yīng)用構(gòu)建的過表達CCR6喉鱗癌細胞系,通過CCK-8增殖實驗,劃痕實驗,Transwell遷移和侵襲實驗,以及裸鼠成瘤實驗,研究CCL20對過表達CCR6的喉鱗癌細胞系的生物學功能。結(jié)果：所有癌旁正常組織和聲帶白斑組織,以及66例喉鱗癌組織中檢測到CCL20表達,且表達水平逐漸升高,晚期腫瘤(T3+T4, Ⅲ+Ⅳ期)組織中CCL20的表達高于早期腫瘤(T1+T2,I+II期),發(fā)生淋巴結(jié)轉(zhuǎn)移的患者,喉癌組織CCL20的表達高于未轉(zhuǎn)移者,轉(zhuǎn)移至淋巴結(jié)中的腫瘤細胞高表達CCL20；測序結(jié)果顯示喉鱗癌患者,聲帶白斑患者和正常人CCL20基因的啟動子區(qū)共檢測到7種SNP位點,并分析發(fā)現(xiàn)其中5個SNP位點rs142242947 (-1401CT)、rs140459563 (-1369CT)、rs189913367 (-1298CT)、rs62190018 (-962CA)、rs6749704(-786TC)均與喉鱗癌,聲帶白斑的發(fā)病在整體上無關(guān),但rs62190018(-962CA)攜帶A等位基因的研究對象發(fā)生晚期喉鱗癌(T3+T4)的風險顯著降低,同時發(fā)現(xiàn)rs62190018 (-962CA)基因型為AA或攜帶等位基因A的喉癌患者病情進展為晚期(T3+T4)的風險明顯減小,對患者具有保護意義；rs6749704 (-786TC)位點基因型一旦發(fā)生改變,患者發(fā)生淋巴結(jié)轉(zhuǎn)移和進展為晚期喉癌(III+IV期)是風險顯著增加；ELISA結(jié)果顯示喉鱗癌患者外周血CCL20的表達水平較正常人顯著降低,且發(fā)生淋巴結(jié)轉(zhuǎn)移和晚期喉鱗癌患者CCL20的表達水平更低,而預后相對較好的聲門型喉癌患者外周血CCL20水平顯著高于聲門上型喉癌；rs62190018(-962CA)位點基因型突變?yōu)镃A后,外周血CCL20的表達水平升高,rs6749704(-786TC)位點的一旦發(fā)生突變,外周血CCL20的表達水平則顯著降低；流式細胞術(shù)和細胞爬片免疫組化均證實利用慢病毒感染建立的CCR6過表達穩(wěn)轉(zhuǎn)喉鱗癌細胞系表面表達CCR6蛋白,且鈣流實驗證實其具有生理功能；一定濃度范圍內(nèi),CCL20可增加過表達CCR6喉鱗癌細胞的遷移和侵襲能力,但對其增殖的影響不明顯,且HEp-2-CCR6細胞系在裸鼠體內(nèi)的成瘤能力低于對照組。結(jié)論：喉鱗癌患者腫瘤組織CCL20的高表達可能促進了喉鱗癌細胞的遷移和侵襲能力,參與了喉鱗癌的發(fā)展；SNP rs62190018 (-962CA)基因型為AA或攜帶等位基因A對喉鱗癌患者具有保護意義,而rs6749704(-786TC)一旦突變,將顯著增加喉鱗癌患者發(fā)生淋巴結(jié)轉(zhuǎn)移和病情進展的風險；外周血CCL20的表達水平與CCL20啟動子區(qū)SNP不同的突變形式相關(guān),其表達水平的下降可能參與了喉鱗癌發(fā)生和發(fā)展。
[Abstract]:Objective: To study the expression of the chemokine CCL20 in laryngeal squamous cell carcinoma and its clinical significance. Methods:70 cases of laryngeal squamous cell carcinoma with total laryngectomy were collected, and 15 cases of vocal cord leukoplakia (including 13 cases with lymph node metastasis and 10 non-tumor metastasis lymph nodes) were collected. The expression of CCL20 was detected by immunohistochemistry, and its relationship with the clinicopathological features of the patient was analyzed.180 cases of laryngeal squamous cell carcinoma,51 cases of vocal cord leukoplakia and 116 normal controls were collected, and the SNP sites in the promoter region of the CCL20 gene were analyzed by PCR and sequencing. The relationship between the incidence of the leukoplakia of the vocal cord and the relationship between the clinical and pathological characteristics of the patients was observed, and the peripheral blood of 36 patients with laryngeal carcinoma and 42 normal volunteers was collected, and the expression level of the CCL20 protein in the patients with laryngeal squamous cell carcinoma and the normal human serum was detected by ELISA. The relationship between the clinicopathological features of the patients and the SNP sites of the CCL20 promoter region was analyzed. The Hep-2-CCR6 and HN-8-CCR6 laryngeal squamous cell carcinoma cell lines with stable expression of CCR6 were constructed by lentiviral infection, and the expression of CCR6 and the function of the receptor were identified by flow cytometry, cell-climbing immunohistochemistry and calcium-flow assay. The biological function of the cell line of the laryngeal squamous cell carcinoma of the CCR6 was studied by means of CCK-8 proliferation experiment, scratch test, Transwell migration and invasion experiment, and in nude mice. Results: The expression of CCL20 was detected in all adjacent normal tissues and vocal cord leukoplakia, and in 66 cases of laryngeal squamous cell carcinoma. The expression of CCL20 in the tissues of advanced tumors (T3 + T4, 鈪，

本文編號：2475513