Shp2在口腔鳞癌中的表达及对其生物学行为的影响

发布时间：2018-05-18 10:04

  本文选题：蛋白酪氨酸磷酸酶Shp2 + 口腔鳞状细胞癌　； 参考：《山东大学》2014年博士论文

【摘要】：目的： 头颈部鳞状细胞癌(Head and neck squamous cell carcinoma, HNSCC)主要影响口腔、舌咽、口咽、喉部及涎腺等,在常见的恶性肿瘤类型中位居第六,而口腔颌面部恶性肿瘤中约80%以上为口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)。尽管近几年我们在OSCC的治疗以及发病机制研究方面取得了一定的进展,但是在过去的几十年中其存活率仍没有明显改善,平均每年确诊的病例中仍有约50%的死亡率。蛋白酪氨酸磷酸酶Shp2(Src homology phosphotyrosyl phosphatase2)由PTPN11基因编码,广泛地表达于机体各种组织和细胞中,其含有2个SH2结构区域,分别为N-SH2、C-SH2,及一个PTP结构区域,其作为细胞因子、生长因子及其他胞外刺激因素的下游信号分子,参与细胞增殖、分化、迁移等诸多细胞重要的生命活动。研究表明,Shp2的编码基因PTPN11存在大量与人类疾病相关的遗传多态性与突变位点,作为重要的节点分子,Shp2在血液系统恶性肿瘤如白血病及实体肿瘤如乳腺癌、肺癌、胃癌、肝癌等的发生发展过程中发挥着重要的调控作用,是其肿瘤治疗的潜在分子靶点。目前国内外关于Shp2在口腔鳞癌中的表达及作用的研究鲜见报道。因此,本实验从临床标本、体外细胞和分子机制三个层面研究Shp2在口腔鳞癌中的表达及对其生物学行为的影响。首先通过临床病例标本检测口腔鳞癌组织及癌旁组织中Shp2的表达差异,并进一步分析其表达水平与患者的临床资料之间的关系,探讨Shp2在口腔鳞癌中的临床意义。其次,应用RNA干扰(RNA interference, RNAi)技术,构建Shp2慢病毒干扰载体,抑制舌鳞状细胞癌细胞SCC-4的Shp2基因表达,探讨Shp2基因沉默后对SCC-4细胞增殖、凋亡及侵袭能力的作用。同时研究Shp2基因沉默后对细胞凋亡相关蛋白的影响,初步阐明Shp2在口腔鳞癌细胞凋亡发生中的分子机制。本研究将为口腔鳞癌临床诊断、预后及治疗的新靶点提供新的思路和理论基础。 方法： 1.收集临床口腔鳞癌组织蜡块及临床有关资料,采用免疫组织化学染色方法观察Shp2蛋白在口腔鳞癌组织中的表达,并对免疫组化结果进行评分,评估Shp2的表达与患者的年龄、性别、烟酒暴露、肿瘤部位、肿瘤大小、临床分期、淋巴转移及病理分级等的相关性。 2.收集18例临床手术中新鲜的口腔鳞癌及相应的癌旁正常组织,采用WesternBlot方法,进一步验证Shp2蛋白在口腔鳞癌组织及癌旁正常组织中的表达差异。 3.于Shp2PTPN11基因编码区选择4个siRNA靶点合成4对shRNA干扰序列,分别与慢病毒载体连接,构建Shp2基因RNA干扰慢病毒载体,筛选出最佳干扰组,显微镜下观察Shp2慢病毒干扰载体感染目的细胞人舌鳞状细胞癌细胞SCC-4的效率。应用Real Time PCR及Western blot检测其干扰效率,筛选出最佳干扰组,建立稳定抑制Shp2基因表达的SCC-4细胞株,为探讨Shp2在口腔鳞癌发生中的作用提供细胞模型。 4.采用四甲基偶氮唑盐(MTT)比色法观察Shp2基因沉默后对SCC-4细胞24h、48h、72h及96h增殖率的影响。5.应用流式细胞技术(Annexin V-APC/7-ADD染色法)及Westen Blot方法研究Shp2基因沉默后对SCC-4细胞凋亡的影响及其凋亡相关蛋白p53、Bax及Bcl-2的变化。6.采用Transwell小室侵袭实验,通过苏木精染色和细胞计数观察Shp2基因沉默后对SCC-4细胞体外侵袭能力的影响。结果：1.免疫组化结果发现,在口腔正常黏膜组织中,Shp2表达阴性或者呈弱表达,而在口腔鳞癌临床样本中高表达；进一步临床资料统计分析表明,在口腔鳞癌中Shp2蛋白的表达与患者的性别、年龄、是否吸烟、饮酒无关,与肿瘤的发生部位和体积大小亦无关。然而,临床分期在III/IV期的肿瘤Shp2的表达较Ⅰ/Ⅱ期的强,在有淋巴转移的病例中,Shp2的表达显著增加,统计学上均有显著性差异(p0.05)。 2.临床新鲜口腔鳞癌组织进一步证明,除一例以外,Shp2在口腔鳞癌组织中的表达明显高于癌旁正常组织中的表达(p0.05),进一步验证了免疫组化结果。 3. Real-time PCR和Western blot检测结果显示Shp2-shRNA3干扰效率最佳,SCC-4细胞中Shp2基因及蛋白表达明显降低,应用RNAi设计的慢病毒干扰载体具有较好的干扰效果。 4.MTT法测定发现,Shp2基因沉默后,96h内SCC-4细胞的生长明显慢于对照组,出现明显的增殖抑制。 5.采用nnexin-V-APC/7-ADD双荧光标记,流式细胞仪检测细胞凋亡发现,转染Shp2慢病毒于扰载体72h后的SCC-4细胞中,细胞凋亡率明显增加,同对照组正常细胞相比,差异具有显著性(p0.05)。Western Blot结果显示,凋亡相关通路蛋白p53、Bax表达增加,Bcl-2表达下降。 6.[ranswell小室侵袭实验结果显示,转染Shp2慢病毒干扰载体72h后的SCC-4细胞的侵袭能力与对照组相比较明显下降,差异具有显著性(p0.05)。 结论： Shp2在口腔鳞癌组织中的表达明显高于癌旁正常组织,过表达的Shp2与口腔鳞癌的临床分期及淋巴转移密切相关；Shp2基因对口腔鳞癌细胞的生长起促进作用,同时可提高肿瘤细胞的侵袭能力；Shp2能抑制口腔鳞癌细胞凋亡,凋亡相关蛋白p53、Bax及bcl-2可能参与其凋亡调控机制。以上结果提示,Shp2与口腔鳞癌的进展和转移有关,在口腔鳞癌可能发挥重要的促癌基因作用,是口腔鳞癌淋巴结转移的危险因子,可作为口腔鳞癌临床诊断、判断预后、诊疗方案制定及肿瘤靶向治疗的潜在生物标记物和分子指标。
[Abstract]:Objective:
Head and neck squamous cell carcinoma (HNSCC) mainly affects oral, glossopharynx, oropharynx, larynx, and salivary glands. It ranks sixth in common malignant tumor types, while more than 80% of oral and maxillofacial malignant tumors are oral squamous cell carcinoma (oral squamous cell carcinoma, OSCC). Some progress has been made in the treatment and pathogenesis of SCC, but the survival rate has not improved significantly over the past few decades, with an average of about 50% of the mortality in the confirmed cases each year. The protein tyrosine phosphatase Shp2 (Src homology phosphotyrosyl phosphatase2) is encoded by the PTPN11 gene and is widely expressed in the machine. In various tissues and cells, it contains 2 SH2 structure regions, which are N-SH2, C-SH2, and a PTP structure region. They are downstream signal molecules of cytokines, growth factors and other extracellular stimuli, participating in cell proliferation, differentiation, migration and many other vital activities. The study shows that the Shp2 encoding gene PTPN11 exists. A large number of genetic polymorphisms and mutation sites associated with human disease, as an important node molecule, play an important regulatory role in the development of hematological malignancies such as leukemia and solid tumors such as breast cancer, lung cancer, gastric cancer, and liver cancer. It is a potential molecular target for the treatment of cancer, and Shp2 is currently in the world. The study of the expression and role of Shp2 in oral squamous cell carcinoma is rarely reported. Therefore, this experiment studies the expression of Shp2 in oral squamous cell carcinoma and its effect on its biological behavior from three levels of clinical specimens, in vitro cell and molecular mechanism. First, the difference of Shp2 expression in oral squamous cell carcinoma and para cancerous tissue is detected by clinical case specimens, and The relationship between the expression level and the clinical data of the patients was further analyzed, and the clinical significance of Shp2 in oral squamous cell carcinoma was discussed. Secondly, the RNA interference (RNA interference, RNAi) technology was used to construct the Shp2 lentivirus interference carrier, to inhibit the Shp2 gene expression of SCC-4 in the squamous cell carcinoma cell of the tongue and to explore the proliferation of SCC-4 cells after the Shp2 gene was silenced. The effect of apoptosis and invasion ability. At the same time, the effect of Shp2 gene silencing on apoptosis related proteins is studied, and the molecular mechanism of Shp2 in the apoptosis of oral squamous cell carcinoma is preliminarily clarified. This study will provide new ideas and theoretical basis for the clinical diagnosis of oral squamous cell carcinoma, and the new target of prognosis and treatment.
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