【作者】 黎景佳；

【导师】 杨新明；

【作者基本信息】 中南大学 ， 耳鼻咽喉头颈外科学， 2010， 博士

【摘要】 目的：喉鳞状细胞癌(laryngeal squamous cell carcinoma, LSCC)是头颈部常见的恶性肿瘤之一,肿瘤的局部复发、淋巴结转移和远处转移是影响LSCC患者预后的重要因素。研究表明,细胞间粘附力下降或丧失所致的肿瘤细胞从原发灶脱落是肿瘤发生侵袭转移的早期和关键步骤。本部分研究旨在探讨细胞粘附分子上皮型钙粘蛋白(E-cadherin)在LSCC中的表达及与肿瘤临床病理特征、预后之间的关系。方法：采用免疫组化方法检测64例喉癌组织和30例癌旁非肿瘤组织中E-cadherin蛋白表达水平,比较二者表达差异。根据患者年龄、肿瘤临床分型、原发灶大小、淋巴结转移、病理组织学分级将病例分组,应用单因素方差分析研究E-cadherin表达与肿瘤临床病理特征之间的关系,Logistic逐步回归分析法研究与淋巴结转移有关的预测因子。结合临床随访资料,用Kaplan-Meier法绘制生存曲线,Log-rank法比较E-cadherin高表达组和E-cadherin低表达组5年生存率,Cox多因素回归法分析疾病预后的独立预测因素。结果：1.64例LSCC组织E-cadherin平均染色分数169±68分,30例癌旁非肿瘤组织E-cadherin平均染色分数346±38分,肿瘤组织中E-cadherin平均染色分数明显低于癌旁非肿瘤组织(P<0.001)；2.肿瘤组织中E-cadherin表达水平与LSCC淋巴结转移(P<0.001)有明显相关性,而与患者年龄(P=0.66)、肿瘤临床分型(P=0.445)、原发灶大小(P=0.303)和病理分级(P=0.159)无关；3.除已知的因素如肿瘤原发灶大小(P=0.012)、病理组织学分级(P=0.003),E-cadherin表达水平(P<0.001)也是LSCC淋巴结转移的独立预测因子；4.根据LSCC组织中E-cadherin平均染色分数将病例分为E-cadherin高表达组和E-cadherin低表达组,E-cadherin高表达组5年生存率为74.1%,E-cadherin低表达组5年生存率为42.9%,E-cadherin高表达组的5年生存率高于E-cadherin低表达组,差异具有统计学意义(Log-rank, P<0.05);5.多变量Cox风险比例模型分析表明,仅淋巴结转移是LSCC预后的独立预测因子(P=0.002),其余各参数,如肿瘤临床分型、原发灶大小、病理分级和E-cadherin表达水平均不能单独预测疾病的预后。结论：1.E-cadherin在LSCC组织中表达减低,可能参与了喉癌的发生发展；2.E-cadherin表达水平可能作为预测LSCC颈淋巴结隐匿性转移的潜在肿瘤标志物；3.E-cadherin表达水平减低与LSCC复发和疾病生存率减低有关。目的：探讨表皮生长因子受体(epidermal growth factor receptor, EGFR)配体EGF和EGFR小分子酪氨酸激酶抑制剂Erlotinib对体外培养的喉癌Hep-2细胞增殖和细胞周期的影响。方法：分别用不同浓度EGF、Erlotinib干预喉癌Hep-2细胞,MTT实验比较其对细胞生长的影响,并计算细胞生存(增殖)率,筛选EGF、Erlotinib干预浓度,流式细胞仪分析EGF、Erlotinib对喉癌Hep-2细胞周期和凋亡的影响。结果：1.MTT实验：EGF能明显促进喉癌Hep-2细胞的增殖,其对喉癌Hep-2细胞生长的促进作用与EGF干预时间、浓度呈正相关；Erlotinib能有效抑制喉癌Hep-2细胞的增殖,使细胞数量减少、密度减低、细胞生存率下降,Erlotinib对喉癌Hep-2细胞生长的抑制作用与干预时间、浓度呈正相关；2.流式细胞仪分析：EGF使喉癌Hep-2细胞G1期比例减少而S期比例增加(P<0.05),对细胞凋亡无明显影响；Erlotinib使喉癌Hep-2细胞G1期比例增加而S期比例减少(P<0.05),并使喉癌Hep-2细胞凋亡率明显高于对照组,差异有统计学意义(P<0.001)。结论：1.EGF促进喉癌Hep-2细胞周期从G1期向S期转化,促进细胞增殖；2.Erlotinib使喉癌Hep-2细胞周期阻滞于G1期,并能诱导喉癌Hep-2细胞凋亡,抑制细胞增殖。目的：前一部分实验已明确EGF、Erlotinib对喉癌Hep-2细胞生长增殖和细胞周期的影响,并分别筛选出EGF、Erlotinib干预浓度。以此为基础,本部分实验研究EGF、Erlotinib对体外培养的喉癌Hep-2细胞表型、运动迁移、侵袭转移能力的影响。方法：分别用10ng/ml EGF、20μmol/L Erlotinib干预喉癌Hep-2细胞,倒置显微镜下观察细胞表型改变,免疫细胞化学实验和Western blot检测喉癌Hep-2细胞E-cadherin、vimentin等相关蛋白表达和定位改变,并用划痕愈合实验和Transwell侵袭实验观察细胞运动迁移能力、侵袭转移能力变化。结果：1.倒置显微镜观察细胞表型改变：EGF干预的喉癌Hep-2细胞彼此分散,失去细胞间连接,形态向长梭形、纺锤形的间质细胞表型转变；Erlotinib干预的细胞形态变圆或变方,成团生长,细胞之间连接紧密,细胞的上皮特征更明显；2.免疫细胞化学实验：EGF使喉癌Hep-2细胞E-cadherin表达下调,细胞膜表达极低；Erlotinib使喉癌Hep-2细胞E-cadherin表达上调,部分细胞E-cadherin出现细胞膜表达；3. Western blot:EGF干预喉癌Hep-2细胞后,E-cadherin表达下调,Vimentin和p-EGFR表达上调,EGFR表达无明显改变；Erlotinib干预喉癌Hep-2细胞后,E-cadherin表达上调,Vimentin和p-EGFR表达下调,而EGFR表达改变不明显；4.划痕愈合实验：EGF能明显促进划痕愈合,使细胞侵袭运动能力增强；Erlotinib使划痕愈合延迟,能部分抑制喉癌Hep-2细胞的侵袭运动能力,实验组24h细胞迁移距离与对照组比较差异均有统计学意义(P<0.05);5. Transwell侵袭实验：EGF使喉癌Hep-2细胞侵袭转移能力增强,干预24h后穿过Transwell小室的每200倍视野平均细胞数为58.5±10.3个,与对照组比较差异有统计学意义(P<0.05)；Erlotinib能抑制喉癌Hep-2细胞的侵袭转移能力,干预24h后穿过Transwell小室的每200倍视野平均细胞数为18.7±4.8个,与对照组比较差异有统计学意义(P<0.05)。结论：1.在喉癌Hep-2细胞中可通过调控EGFR信号传导通路来调节E-cadherin的表达,改变细胞表型和侵袭转移能力；2.小分子量EGFR酪氨酸激酶抑制剂Erlotinib能使喉癌Hep-2细胞E-cadherin表达上调,细胞运动迁移和侵袭转移能力减弱。更多还原

【Abstract】 Objective:Laryngeal squamous cell carcinoma (LSCC) is one of the commonest tumors in the head and neck, locoregional recurrence, cervical lymph nodes metastases and distant metastases are the factors that significantly affect the prognosis in LSCC patients. An important step in the development of tumor metastasis is the detachment of malignant cells from their original site, which results from the reduced cell-cell adhesiveness. The aim of this study was to investigate the clinical significance of E-cadherin expression in laryngeal squamous cell carcinoma.Methods:The level of E-cadherin expression in 64 tumor tissues and 30 adjacent non-tumor laryngeal tissues were determined by immunohistochemistry. Patients were classified according to age, primary site, T stage, lymph node metastases and histological differentiation. Staining scores were averaged for each group, and mean scores were compared within groups stratified with respect to clinicopathological parameters by one-way ANOVA test. Survival curves were calculated using the Kaplan-Meier method. Differences in 5-year survival rate were analysed by the log-rank test. Multivariate Cox proportional hazards models were used to examine the relative impact of either variable on the disease prognosis.Results:1. The mean staining score for sixty-four carcinomas was 169±68, and the thirty non-tumor laryngeal tissues had a mean staining score of 346±38, the mean score in tumor tissues was significantly lower than the non-tumor laryngeal tissues(P<0.001);2. The expression of E-cadherin were significantly correlated with lymph node metastases (P＜0.001). The differences of E-cadherin expression between the different age (p=0.66), primary site (P=0.445), T stage (P=0.303) and histological differentiation (P=0.159) group were not statistically significant;3. In addition to other known factors such as T-stage and histological grade, E-cadherin staining score was also the statistically significant, independent predictors of lymph node metastases;4. We classified patients as E-cadherin low-expression group and E-cadherin high-expression group according to the mean staining score. The 5-year survival rate in E-cadherin high-expression group were significantly increased than that of E-cadherin low-expression group (Log-rank, P<0.05);5. The results of multivariate Cox proportional hazards model confirmed that only the presence of cervical lymph node metastases was a statistically significant, independent predictor of prognosis (P=0.002). Other parameters, such as primary sit, T-stage, histological grade, E-cadherin expression, cannot predicate disease prognosis separately.Conclusion:1. The expression of E-cadherin is decreased in the tumor tissues, which indicated that E-cadherin may have been involved in the tumor occurrence and development.2. Expression of E-cadherin is an independent predictor of lymph node metastases in laryngeal cancer, it might be a tumor marker for occult lymph node metastases.3. Decreased E-cadherin expression is associated with recurrence and decreased disease survival rate in laryngeal squamous cell carcinoma. Objective:To investigate the impact of epidermal growth factor receptor (EGFR) ligand EGF and small molecule tyrosine kinase inhibitor Erotinib on the proliferation and cell cycle in human laryngeal cancer cell line Hep-2.Methods:Human laryngeal cancer cell line Hep-2 was treated with EGF and Erlotinib, respectively. Then, cell proliferation in different groups was measured by MTT assay. The influence of EGF and Erlotinib on the cell cycle and apoptosis were analysed by Flow Cytometer.Results:1. MTT assay:EGF promoted proliferation of Hep-2 cells as the extension of treatment concentration and time; Erlotinib inhibited proliferation of Hep-2 cells, which induced the decreases in cell number, density and survival rate.2. Flow Cytometer:Hep-2 cells treated with EGF exhibited the decreases in phase G1 and increases in phase S (P<0.05), while the cell apoptosis was not influenced; Treatment of Erlotinib resulted in the increases in phase G1 and decreases in phase S (P＜0.05), and cell apoptosis rate was significantly increased than the control group (P＜0.001).Conclusion:1.EGF accelerates the cell cycle transition from G1 phase to S phase, and promotes Hep-2 cell proliferation.2. Treatment of Erlotinib results in the G1 phase arrest of cell cycle, and induces Hep-2 cell apoptosis, ultimately inhibits Hep-2 cell proliferation. Objective:To investigate the impact of epidermal growth factor receptor (EGFR) ligand EGF and small molecule tyrosine kinase inhibitor Erotinib on the phenotype, cell motility, invasion and metastases in human laryngeal cancer cell line Hep-2.Methods:Human laryngeal cancer cell line Hep-2 was treated with 10ng/ml EGF and 20μmol/L Erlotinib, respectively. The cell phenotype changes were observed using inverted microscope. Changes in the localization and expression of E-cadherin, vimentin were examined by immunocytochemistry and Western blot. Wound healing assay and Transwell invasion assay were used to detect cell motility, invasion and metastases, respectively.Results:1. In the presence of EGF treatment, Hep-2 cells changed its shape to fusiform fibroblastoid phenotype and its colony formation from compact to sparse, while Erlotinib induced the cell morphological changes to round or square, cell grew into cluster;2. Immunocytochemistry:EGF resulted in the down-regulation of E-cadherin protein, especially in the cell membrane; while Erlotinib could up-regulate the E-cadherin expression, in some cell there were recurrent positive membrane expression;3. Western blot:EGF induced down-regulation of E-cadherin expression, and up-regulation of vimentin, p-EGFR in Hep-2 cells; Erlotinib could lead to up-regulation of E-cadherin, down-regulation of vimentin and p-EGFR. Both the two interference had less impact on EGFR expression;4. Wound healing assay:EGF promoted the wound healing, resulted in the increased cell motility and movement; Erlotinib delayed the wound healing, and partly inhibited cell motility and movement. The 24h distance of cell migration in the two experimental group were significantly different than the control group (P<0.05);5. Transwell invasion assay:EGF promoted the invasion and metastases of Hep-2 cells, the invasive numbers of Hep-2 cells with EGF treatment at 24h time point were 58.5±10.3 (P<0.05); Erlotinib inhibited the invasion and metastases of Hep-2 cells, the invasive numbers of Hep-2 cells with Erlotinib treatment at 24h time point were 18.7±4.8, which was significantly less than the control group (P＜0.05).Conclusion:1.EGFR signal modulation regulates E-cadherin expression and cell phenotype, motility, invasion and metastases in human laryngeal cancer cell line Hep-2. 2. Treatment of small molecule tyrosine kinase inhibitor erotinib up-regulates E-cadherin expression, decreases cell motility and movement, inhibits cell invasion and metastases in Hep-2 cells.更多还原