CCL20在喉鳞癌发生发展中的生物学功能的研究

发布时间：2019-05-12 16:26

【摘要】：目的：研究趋化因子CCL20在喉鳞癌中的表达及其临床意义,分析CCL20基因启动子区的单核苷酸多态性位点及其临床意义,探索趋化因子CCL20对过表达CCR6的喉鳞癌细胞系的体内外生物学行为的影响。方法：收集70例行全喉切除术的喉鳞癌患者肿瘤组织、癌旁组织以及15例声带白斑组织(包括其中13例发生淋巴结转移喉鳞癌患者的转移淋巴结和10例无肿瘤转移的淋巴结的白片),应用免疫组化技术检测CCL20的表达,分析其与患者临床病理特征的关系；收取180例喉鳞癌患者,51例声带白斑,116例正常人的外周血,应用PCR和测序技术分析CCL20基因启动子区存在的SNP位点,分析其与喉鳞癌,声带白斑发病的联系,以及同患者临床病理特征的关系；收取36例喉癌患者和42名正常志愿者的外周血,应用ELISA技术检测喉鳞癌患者和正常人血清中CCL20蛋白的表达水平,分析其与患者临床病理特征及CCL20启动子区SNP位点的关系；应用慢病毒感染构建稳定表达CCR6的HEp-2-CCR6和HN-8-CCR6喉鳞癌细胞系,并用流式细胞术,细胞爬片免疫组化,钙流实验鉴定细胞系CCR6的表达及受体功能情况；应用构建的过表达CCR6喉鳞癌细胞系,通过CCK-8增殖实验,划痕实验,Transwell迁移和侵袭实验,以及裸鼠成瘤实验,研究CCL20对过表达CCR6的喉鳞癌细胞系的生物学功能。结果：所有癌旁正常组织和声带白斑组织,以及66例喉鳞癌组织中检测到CCL20表达,且表达水平逐渐升高,晚期肿瘤(T3+T4, Ⅲ+Ⅳ期)组织中CCL20的表达高于早期肿瘤(T1+T2,I+II期),发生淋巴结转移的患者,喉癌组织CCL20的表达高于未转移者,转移至淋巴结中的肿瘤细胞高表达CCL20；测序结果显示喉鳞癌患者,声带白斑患者和正常人CCL20基因的启动子区共检测到7种SNP位点,并分析发现其中5个SNP位点rs142242947 (-1401CT)、rs140459563 (-1369CT)、rs189913367 (-1298CT)、rs62190018 (-962CA)、rs6749704(-786TC)均与喉鳞癌,声带白斑的发病在整体上无关,但rs62190018(-962CA)携带A等位基因的研究对象发生晚期喉鳞癌(T3+T4)的风险显著降低,同时发现rs62190018 (-962CA)基因型为AA或携带等位基因A的喉癌患者病情进展为晚期(T3+T4)的风险明显减小,对患者具有保护意义；rs6749704 (-786TC)位点基因型一旦发生改变,患者发生淋巴结转移和进展为晚期喉癌(III+IV期)是风险显著增加；ELISA结果显示喉鳞癌患者外周血CCL20的表达水平较正常人显著降低,且发生淋巴结转移和晚期喉鳞癌患者CCL20的表达水平更低,而预后相对较好的声门型喉癌患者外周血CCL20水平显著高于声门上型喉癌；rs62190018(-962CA)位点基因型突变为CA后,外周血CCL20的表达水平升高,rs6749704(-786TC)位点的一旦发生突变,外周血CCL20的表达水平则显著降低；流式细胞术和细胞爬片免疫组化均证实利用慢病毒感染建立的CCR6过表达稳转喉鳞癌细胞系表面表达CCR6蛋白,且钙流实验证实其具有生理功能；一定浓度范围内,CCL20可增加过表达CCR6喉鳞癌细胞的迁移和侵袭能力,但对其增殖的影响不明显,且HEp-2-CCR6细胞系在裸鼠体内的成瘤能力低于对照组。结论：喉鳞癌患者肿瘤组织CCL20的高表达可能促进了喉鳞癌细胞的迁移和侵袭能力,参与了喉鳞癌的发展；SNP rs62190018 (-962CA)基因型为AA或携带等位基因A对喉鳞癌患者具有保护意义,而rs6749704(-786TC)一旦突变,将显著增加喉鳞癌患者发生淋巴结转移和病情进展的风险；外周血CCL20的表达水平与CCL20启动子区SNP不同的突变形式相关,其表达水平的下降可能参与了喉鳞癌发生和发展。
[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，

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