【作者】 陈斌；

【导师】 周梁；

【作者基本信息】 复旦大学 ， 耳鼻咽喉科学， 2014， 博士

【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 (-1401C>T)、rs140459563 (-1369C>T)、rs189913367 (-1298C>T)、rs62190018 (-962C>A)、rs6749704(-786T>C)均与喉鳞癌,声带白斑的发病在整体上无关,但rs62190018(-962C>A)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 (-962C>A)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 (-786T>C)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(-962C>A)位点基因型突变为CA后,外周血CCL20的表达水平升高,rs6749704(-786T>C)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 (-962C>A)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(-786T>C)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。更多还原

【Abstract】 Objective:To evaluate the expression of chemokine CCL20 expression in laryngeal squmaous cell carcinoma(LSCC)tissue and peripheral blood, explore their correlation with the clinicopathological features of LSCC. To identify the SNPs located in CCL20 promoter, and investigate the association between CCL20 promoter polymorphism, serum CCL20 levels, and risk of laryngeal squamous cell carcinoma (LSCC) and vocal leukoplakia in Chinese Han Population. To explore the effects of CCL20 on CCR6 over-expressed LSCC cell lines in vivo and in vitro.Methods:LSCC and adjacent normal tissue samples from 70 LSCC patients underwent total laryngectomy, and 15 vocal cord leukoplakia tissue samples were collected (including 13 metastatic and 10 non-metastatic lymph nodes sections from LSCC patients), CCL20 expression and its association with clinicopathological feathers were evaluated by immunohistochemistry. CCL20 promoter SNPs were indentified and genotyped in 180 patients with LSCC,51 patients with vocal cord leukoplakia, and 116 healthy controls by Sanger sequencing, and the association between CCL20 promoter polymorphism and the risk of LSCC or vocal cord leukoplakia was analyzed. Serum CCL20 expression level of 36 LSCC patients and 42 healthy controls were measured by ELISA, and its relationship with clinicopathological features and CCL20 promoter region SNP was evaluated. Stable CCR6 overexpressing HEp-2 and HN-8 were established by lentivirus infection. The expression and signal transduction function of CCR6 were identified and verified by flow cytometry, immunohistochemistry, and calcium flux measurement. The effects of CCL20 on CCR6 overexpressing LSCC cell lines were investigated by CCK-8 proliferation assay, wound healing assay, transwell migration and invasion assays, and nude mouse tumorigenicity assay.Results:CCL20 was detected in all adjacent normal tissue and vocal cord leukoplakia tissue, and 66 LSCC tissue by IHC. CCL20 expression level of LSCC tissue was elevated in advanced stage patients (T3+T4, Ⅲ+Ⅳ) and patients with metastatic lymph nodes compared with initial stage patients (T1+T2, Ⅰ+Ⅱ) and patients with non-metastatic lymph nodes.Besides, higher expression level of CCL20 in metastatic LSCC cells in lymph nodes was observed compared with that of primary tumor.7 SNPs were identied from the subjects. All 5 SNPs (2 SNPs were exclude because of low frequence), including rs142242947 (-1401C>T), rs140459563 (-1369C>T)、 rs189913367 (-1298C>T)、rs62190018 (-962C>A)、rs6749704 (-786T>C),were not associated with the risk of LSCC or vocal cord leukoplakia. The same results were found between their SNP haplotypes and the risk of LSCC or vocal cord leukoplakia. However, subjects carrying A allele in rs62190018 (-962C>A) had a significant lower risk of suffering advanced LSCC (T3+T4). Besides, LSCC patients with AA genotype or A allele in rs62190018 (-962C>A) had much lower risk to Progress to advanced stage (T3+T4), while those with TC or CC genotypes or C allele in rs6749704 (-786T>C) had a significant higher risk to progress to advanced stage (T3+T4) and lymph nodes metastasis. ELISA results indicated a decrease of serum CCL20 in LSCC patients compared with healthy controls, and the level of serum CCL20 got lower in those LSCC with lymph nodes metastasis. Besides, CA genotype in rs62190018 (-962C>A) indicated an higher expression level of serum CCL20,while TC or CC genotypes or C allele in rs6749704 (-786T>C) associated with a significant lower level of serum in LSCC patients. The expression of CCR6 protein on the surface of LSCC cell lines was demonstrated by FCM and IHC, and it can be activated by CCL20. Within a certain range of concentrations, CCL20 could enhance the migration and invasion ability of CCR6 overexpressing LSCC cells, while had little effect on the proliferation of LSCC. Besides, compared with control group, HEp-2-CCR6 cell lines had a lower tumorigenicity in nude mice in vivo.Conclusion:High expression of CCL20 in patients with LSCC may promote the migration and invasion of LSCC cells and facilitate the progression of LSCC. LSCC patients with AA genotypes or A allele in rs62190018 (-962C>A) had much lower risk to progress to advaned stage of LSCC, while those with TC or CC genotypes or A allele in rs62190018 (-962C> A) had significant higher risk to progress to advanced stage (T3+T4) and lymph nodes metastasis. Serum CCL20 level had a relationship with SNP rs62190018 (-962C>A and rs62190018 (-962C>A), down-regulation of serum CCL20 may favour the progression of LSCC in vitro and in vivo.更多还原