反义整合素连接激酶寡核苷酸对上皮—间充质转型的调控及抑制喉癌转移的研究

发布时间：2019-05-31 17:45

【摘要】： 研究背景及目的：喉鳞状细胞癌(laryngeal squamous cell carcinoma, LSCC)是头颈部最常见的恶性肿瘤之一,肿瘤浸润和转移是造成喉癌患者死亡的主要原因之一,喉癌发生侵袭转移是一个多环节、多步骤的复杂过程,包括肿瘤细胞粘附、移动、酶降解和血管生成等多种肿瘤细胞的生物学行为,转移的确切机制目前尚不十分明确。整合素连接激酶(integrin-linked kinase,ILK)是一个59KD的细胞内信号蛋白,具有丝氨酸／苏氨酸蛋白激酶活性。ILK不仅在正常发育中起着重要作用,其在肿瘤转移过程中也有着与其它已知的癌基因一样的生物学效应。ILK介导细胞与细胞外基质的连接,调节肌动蛋白细胞骨架的运动,参与细胞粘附的形成,促进肿瘤细胞的粘附和转移。上皮-间充质转型(epithelial-mesenchymal transition, EMT)是多细胞生物形态学发生中的一个基本生理过程。是指上皮细胞向间质细胞转分化的现象,在此过程中,上皮细胞极性丧失,迁移能力增强,同时逐渐获得间质表型。上皮-间充质转型参与了上皮源性肿瘤的发生及发展,尤其在癌的侵袭和转移方面。因此,对EMT在肿瘤形成和侵袭转移中的作用及其调控机制的研究正越来越受到人们的关注。目前,ILK作为一种EMT的诱导因子,在喉癌方面的研究几乎是一片空白。因此,我们以人喉鳞状细胞癌细胞株Hep-2为研究对象,采用反义ILK寡核苷酸对其进行转染,就ILK对人喉鳞状细胞癌Hep-2细胞上皮-间充质转型的影响及其在肿瘤侵袭、转移过程中的作用和机制进行了研究。方法：采用免疫组织化学法研究临床喉鳞状细胞癌标本中ILK的表达与临床病理因素的相关性,并且进一步以人喉鳞状细胞癌细胞株Hep-2为研究对象,采用ILK反义寡核苷酸对其进行转染,应用MTT、流式细胞术分析、Western Blot以及Transwell小室等分子生物学技术和方法,深入研究了ILK对人喉鳞状细胞癌Hep-2细胞上皮-间充质转型相关基因表达的影响及其在肿瘤侵袭、转移过程中的作用和机制。结果：ILK在56例喉鳞状细胞癌患者癌组织标本中的阳性表达率(66.07%)明显高于ILK在28例癌旁组织中的阳性表达率(21.43%),统计学分析结果显示二者存在显著性差异(p0.05)。并且,ILK的高表达与喉癌患者病理分级及是否存在淋巴结转移密切相关。ILK在人喉鳞状细胞癌Hep-2细胞中亦呈高表达,免疫细胞化学显示ILK在人喉鳞状细胞癌Hep-2细胞内的表达以胞质为主,胞核仅见少量弱表达。反义ILK干扰降低了人喉鳞状细胞癌Hep-2细胞的ILK的表达,抑制了细胞的增殖并增加了细胞的凋亡。EMT相关基因的表达亦出现了相应的变化：间充质标志物Vimentin蛋白表达水平降低,上皮性标志物E-cadherin的表达显著增加。这些基因蛋白水平的改变也同时导致了人喉鳞状细胞癌Hep-2细胞异质粘附能力、迁移能力以及侵袭能力的降低。结论：ILK可促进人喉鳞状细胞癌发生转移,反义ILK干扰可诱导人喉鳞状细胞癌Hep-2细胞间充质-上皮细胞转型的发生,并因此而显著降低其侵袭和转移潜力。
[Abstract]:The research background and purpose: Laryngeal square cell carcinoma (LSCC) is one of the most common malignant tumors in the head and neck, and the invasion and metastasis of the tumor is one of the main causes of the death of the patients with laryngeal carcinoma. The invasion and metastasis of the laryngeal carcinoma is a multi-link and multi-step complex process. Including the biological behavior of various tumor cells such as tumor cell adhesion, movement, enzyme degradation and angiogenesis, the exact mechanism of the transfer is not yet very clear. integrin-linked kinase (ILK) is a 59KD intracellular signal protein with serine/ threonine protein The activity of ILK plays an important role not only in the normal development, but also in the process of tumor metastasis. Study effect. ILK mediates the connection of the cell to the extracellular matrix, regulates the movement of the actin cytoskeleton, participates in the formation of cell adhesion, promotes the adhesion of the tumor cells, Epithelium-Mesenchymal transition (EMT) is one of the multi-cell biological morphology. A basic physiological process. It refers to the phenomenon of the differentiation of the epithelial cells to the mesenchymal cells. In this process, the polarity of the epithelial cells is lost, the migration ability is enhanced, and at the same time, The formation and development of epithelial-derived tumors are involved in the transformation of epithelial-mesenchymal transition, especially in the case of cancer. Therefore, the role of EMT in tumor formation and invasion and metastasis and its regulation and control mechanism are becoming more and more important At present, ILK, as an induction factor for EMT, has been studied in the field of laryngeal cancer. The effect of ILK on the transformation of human laryngeal squamous cell carcinoma (Hep-2) cells and its role in the invasion and metastasis of human laryngeal squamous cell carcinoma (Hep-2) was studied by using human laryngeal squamous cell carcinoma cell line Hep-2 as the research object. Methods: The relationship between the expression of ILK and the clinicopathological factors in the clinical specimens of laryngeal squamous cell carcinoma was studied by immunocytochemical method, and the human laryngeal squamous cell carcinoma cell line Hep-2 was used as the research object, and the ILK antisense oligonucleotide was used. The effects of ILK on the expression of human laryngeal squamous cell carcinoma (Hep-2) and the expression of mesenchymal transition-related genes in human laryngeal squamous cell carcinoma (Hep-2) and its tumor invasion were studied by means of MTT, flow cytometry, Western Blot and Transwell. Results: The positive expression rate of ILK in 56 patients with laryngeal squamous cell carcinoma (66.07%) was significantly higher than that of ILK in 28 patients with carcinoma (21.43%). There was a significant difference in the expression of ILK in the patients with laryngeal carcinoma (p0.05). The expression of ILK in the Hep-2 cells of human laryngeal squamous cell carcinoma (Hep-2) and the expression of ILK in the human laryngeal squamous cell carcinoma (Hep-2) were highly correlated with the presence or absence of lymph node metastasis. The expression of ILK in human laryngeal squamous cell carcinoma (Hep-2) cells was reduced by the expression of ILK in human laryngeal squamous cell carcinoma (Hep-2). The proliferation of the cells and the increase of the apoptosis of the cells. The expression of the EMT-related genes also showed a corresponding change: the level of the expression of the mesenchymal marker Vimentin, the epithelial marker E- There was a significant increase in the expression of cadherin. The changes in the level of these genes also led to the heterogeneous adhesion of the Hep-2 cells in the human laryngeal squamous cell carcinoma. Conclusion: ILK can promote the transfer of human laryngeal squamous cell carcinoma, and the anti-sense ILK interference can induce the transformation of human laryngeal squamous cell carcinoma Hep-2 cell-derived mesenchymal-epithelial cells.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.65

【共引文献】