【作者】 刘勇；

【导师】 田勇泉；

【作者基本信息】 中南大学 ， 耳鼻咽喉科学， 2012， 博士

【摘要】 第一章EphA2在头颈鳞癌中的表达及其临床意义目的：检测促红细胞生成素产生肝细胞受体A2(EphA2) mRNA及蛋白在头颈部鳞状细胞癌(头颈鳞癌)组织及细胞株中的表达,并探讨其表达与头颈鳞癌临床病理特征和预后之间的关系。方法：采用免疫组织化学技术检测EphA2蛋白在98例头颈鳞癌石蜡组织标本中的表达,并统计分析EphA2蛋白与头颈鳞癌临床病理特征和预后之间的关系；另外,运用RT-PCR及Western blot检测EphA2mRNA及蛋白在13例配对头颈鳞癌组织及癌旁黏膜组织和头颈鳞癌细胞株中的表达。结果：EphA2mRNA及蛋白在头颈鳞癌细胞株及组织中的表达均上调,且EphA2蛋白的表达与头颈鳞癌的肿瘤发生位置(P=0.018)、T分级(T1+T2/T3+T4)(P=0.016)、临床分期(Ⅰ期+1Ⅱ期/Ⅲ期+ⅣV期)(P=0.001)、转移(P=0.002)及复发(P=0.002)密切相关；Kaplan-Meier生存分析结果显示EphA2高表达组与低表达组5年无瘤生存率分别为27.8%与64.1%,5年总生存率分别为38.9%和82.1%,差异具有统计学意义(P均<0.05)；多因素Cox比例风险回归模型进一步显示,EphA2表达水平为头颈鳞癌预后的独立影响因素(P=0.006)。结论：EphA2在头颈鳞癌组织及细胞中表达显著升高,且其高表达与头颈鳞癌患者的复发、转移及预后密切相关。这些结果均提示EphA2可能在头颈鳞癌的发生、发展中可能发挥了重要作用,并对头颈鳞癌复发、转移及预后具有评估价值。第二章EphA2调控头颈鳞癌细胞生长及侵袭转移的体外实验研究目的：研究表明EphA2在多种实体瘤中高表达,且我们前期研究表明EphA2蛋白在头颈鳞癌组织中显著上调,并与头颈鳞癌患者的临床分期、复发、转移及预后密切相关。因此,该部分将沉默EphA2在头颈鳞癌细胞株中的表达,并观察EphA2基因沉默后对头颈鳞癌细胞体外生长、凋亡、运动、侵袭能力生物学行为的影响。方法：利用慢病毒介导的EphA2shRNA沉默头颈鳞癌Tu686细胞中EphA2的表达,RT-PCR及Western blot技术检测EphA2基因的沉默效果；嘌呤霉素筛选EphA2基因稳定沉默的Tu686细胞；通过CCK-8法、流式细胞技术、划痕实验及Transwell侵袭实验观察EphA2沉默后对Tu686细胞的体外生长能力、细胞周期分布、凋亡率、迁移及侵袭能力的影响。结果：慢病毒介导的EphA2shRNA成功抑制了Tu686细胞中EphA2的表达。与对照组Tu686及Tu686EPhA2RNAi-细胞比较,实验组Tu686EPhA2RNAi+细胞增殖能力减弱,处于G0/G1期的细胞比率显著增多(75.04±1.08vs56.09±1.58,55.04±1.38),而处于S期(18.33±0.77vs32.54±1.70,32.66±0.99)和G2/M期(6.64±1.00vs12.30±0.76,11.37±0.87)的细胞比率显著减少(P均<0.01),而凋亡率无显著统计学差异(P>0.05)。划痕48h后Tu686EPhA2RNAi+细胞愈合率仅(46.7±7.6)%,而对照组Tu686和Tu686EPhA2RNAi-细胞的创面愈合率则达到(88.7±4.5)%与(86.0±2.6)%,具有显著统计学差异(P<0.01)。Transwell侵袭实验发现48小时后穿过Transwell聚碳酸酯膜的Tu686EPhA2RNAi+细胞较两对照组明显减少(123±13vs288±20,303±11)(P<0.01)。结论：EphA2表达抑制后能减弱头颈鳞癌Tu686细胞的体外增殖、迁移及侵袭能力,提示其在头颈鳞癌的恶性进展中发挥了重要作用。第三章EphA2调控头颈鳞癌细胞生长及侵袭转移的体内实验研究目的：进一步从体内实验证实组织及细胞水平的实验结果,并利用所获得的移植瘤组织标本对EphA2引起头颈鳞癌颈淋巴结转移的作用机制进行初步探讨。方法：利用慢病毒介导的shRNA沉默EphA2在高转移性头颈鳞癌细胞株M2中的表达；嘌呤霉素筛选稳定克隆,RT-PCR及Western blot技术检测EphA2沉默效果；构建头颈鳞癌高转移动物模型,通过HE染色验证其成瘤及颈淋巴结转移情况；免疫组织化学技术检测移植瘤微血管密度；Western blot技术检测移植瘤标本中EphA2及血管内皮生长因子(VEGF)的表达情况。结果：筛选出EphA2基因稳定沉默的M2细胞,并成功构建头颈鳞癌高转移动物模型,成瘤率达100%。实验组M2EphA2RN Ai+细胞移植瘤较对照组M2及M2EPhA2RNAi-细胞移植瘤体积减少63.5%-69.4%,重量减轻51.8%-60.0%,颈淋巴结转移率亦减少(21.4%vs66.7%,58.3%；P<0.05)。Western blot显示实验组移植瘤组织中EphA2及VEGF蛋白显著下调,免疫组织化学技术发现实验组瘤体微血管密度显著减少(P<0.01)。结论：EphA2沉默后抑制头颈鳞癌细胞在动物体内的生长及转移；并能明显减少头颈鳞癌组织内的肿瘤新生血管,且该肿瘤新生血管的抑制可能与促血管生成因子VEGF的减少有关。更多还原

【Abstract】 Charpter1Clinical significance of EphA2expression in squamous cell carcinoma of the head and neckObjective:To evaluate the expression of EphA2in tissue specimens and cell lines of squamous cell carcinoma of the head and neck (SCCHN), and further correlate EphA2expression with clinicopathological parameters and prognosis in SCCHN.Methods:EphA2expression in98primary SCCHN tissue specimens was analyzed by immunohistochemistry and correlated with clinicopathological parameters. Additionally,13paired SCCHN tissues and6SCCHN cell lines were evaluated for EphA2expression by RT-PCR and immunoblotting.Results:Both of the mRNA and protein of EphA2overexpressed in SCCHN tissues and SCCHN cell lines. More importantly, high EphA2expression was signifcantly associated with tumor site (P=0.018), T classifcation (P=0.016), clinical stage (P=0.001), recurrence (P=0.002), and lymph node metastasis (P=0.002), respectively. Kaplan-Meier survival analysis demonstrated that patients with high EphA2expression had both poorer disease-free survival and overall survival than patients with low EphA2expression (64.1%vs27.8%;82.1%vs38.9%; both P<0.05). Multivariate Cox regression analysis revealed that EphA2overexpression was an independent prognostic factor for patients with SCCHN (P=0.006).Conclusion:This study demonstrated that EphA2protein expression was obviously increased in SCCHN tissue samples and cell line, and EphA2protein overexpression was associated with tumor recurrence, metastasis and poorer prognosis in SCCHN patients. These results suggested that EphA2might play a critical role in the initiation and progression of SCCHN, implicating EphA2as a valuable marker for the prediction of recurrence, metastasis and prognosis in SCCHN. Charpter2Investigation of the regulation of EphA2on the proliferation, migration and ivasion of the squamous cell carcinoma of the head and neck in vitroObjective:EphA2, a member of the receptor tyrosine kinases, is overexpressed in a variety of solid tumors and cancer cell lines. Our previous study has demonstrated that EphA2is overexpressed in SCCHN, and EphA2protein overexpression was associated with tumor recurrence, metastasis and poorer prognosis in SCCHN patients, suggesting the biological significance of EphA2in SCCHN progression. However, the mechanisms of EphA2and the consequence of its down-regulation in SCCHN have not been fully elucidated in vitro.Methods:EphA2shRNA lentiviral particles were used to knockdown EphA2gene expression in SCCHN cell lines Tu686, RT-PCR and Western blotting were used to validate the gene silencing efficiency of EphA2in SCCHN cell lines. Stable clones were obtained by puromycin screening. CCK-8assay, fluorescence-activated cell sorting analysis, invasion and migration assay were carried out to observe the effects of EphA2inhibition on the proliferation, cell cycle, apoptosis, migration and invasion in vitro.Results:EphA2shRNA lentiviral particles efficiently decreased the mRNA and protein expression of EphA2in SCCHN cell lines Tu686. Tu686EphA2RNAi+cell grew slowly than both control groups (Tu686and Tu686EphA2RNAi-). The percentage of Tu686EphA2RNAi+cells in G0/G1phase was significantly increased (75.04±1.08vs56.09±1.58,55.04±1.38)(P<0.01), whereas the percentage of cells in S phase (18.33±0.77vs32.54±1.70,32.66±0.99) and G2/M phase (6.64±1.00vs12.30±0.76,11.37±0.87) were obviously decreased (both P<0.01). However, there was no significant difference in apoptotic rate between differently treated groups(P>0.05). Moreover, inhibited EphA2expression led to significantly decreased migration [Tu686EphA2RNAi+group, wound closure rate (46.7±7.6)%vs Tu686, Tu686EphA2RNAi-control groups, wound closure rate (88.7±4.5)%and (86.0±2.6)%), P<0.01] and invasion (123±13vs288±20,303±11; P<0.01) of SCCHN Tu686cells. Conclusion:Knockdown the expression of EphA2led to the inhibition of tumor growth, migration and invasion in vitro, suggetting that EphA2contributes to the aggressive behavior of SCCHN, indicating inhibition of EphA2may be a promising targeted approach to block SCCHN progression. Charpter3Investigation of the regulation of EphA2on the ivasion and metastasis of the squamous cell carcinoma of the head and neck in vivoObjective:To further confirm the above results we obtained in vivo, and initially explore the mechanisms of EphA2mediating cervical lymph nodes metastasis of SCCHN in xenografted tumors from nude mouse.Methods:EphA2shRNA lentiviral particles were used to knockdown EphA2gene expression in SCCHN cell lines M2with high lymph nodes metastasis rate, RT-PCR and Western blotting were used to validate the gene silencing efficiency of EphA2in SCCHN cell lines. Stable clones, obtained by puromycin screening, were used to establish SCCHN metastatic xenograft mouse model. Hematoxylin-eosin staining were applied to identify cervical lymph nodes metastasis of SCCHN in xenografted tumors. Western blot was used to investigate the protein expression of EphA2and VEGF. Immunohistochemistry was used to observe microvessel density (CD31).Results:EphA2shRNA lentiviral particles efficiently decreased the mRNA and protein expression of EphA2in SCCHN cell lines M2, which were further successfully utilized to establish SCCHN metastatic xenograft mouse model. Xenografted tumors in group of M2EphA2RNAi+decreased63.5%～69.4%in volume and51.8%～60.0%in weight compared with xenografted tumors in groups of M2and M2EphA2RNA1-. More importantly, the cervical lymph nodes metastasis rate in M2EphA2RNAi+was also declined (21.4%vs66.7%,58.3%)(P<0.05). Decreased protein expression of EphA2and VEGF and microvessel density were observed in group of M2EphA2RNAi+Conclusion:Knockdown the expression of EphA2led to the inhibition of tumor growth and metastasis in SCCHN nude mouse model. More importantly, SCCHN angiogenesis was also impeded, which might be associated with the decreased expression of VEGF.更多还原