【摘要】：目的:本研究旨在結合口腔鱗狀細胞癌(OSCC)患者臨床病理特征,探討Hedgehog(Hh)信號通路的重要性,并探究其與OSCC血管生成的相關性。方法:1.經知情同意,收集80例病理證實為OSCC的術后組織標本;采取免疫組織化學法檢測Shh、Gli-1、CD34蛋白在80例OSCC患者原發(fā)腫瘤中的表達;依據CD34蛋白表達狀況,計算微血管密度(MVD),并分別統(tǒng)計分析Shh、Gli-1蛋白表達及MVD與臨床病理特征的相關性。2.體外培養(yǎng)SCC4、SCC9、SCC25、Cal27細胞,采用Western Blot方法檢測Shh蛋白在四種OSCC細胞系中的表達;差速離心法提取高表達Shh蛋白細胞系中的微囊泡(MVs),透射電鏡下觀察MVs形態(tài);熒光標記MVs,內皮細胞(ECs)專用培養(yǎng)基中將MVs與人臍靜脈內皮細胞(HUVEC)共培養(yǎng),并在熒光顯微鏡下觀察其相互作用方式。結果:1.免疫組化顯示:a.OSCC組織中Shh、Gli-1蛋白表達及MVD與淋巴結轉移、腫瘤臨床分期顯著相關,與腫瘤直徑大小、患者年齡、性別不相關。b.Spearman相關性分析數(shù)據顯示,Shh蛋白與Gli-1蛋白的表達呈顯著相關關系P=0.008㖞。c.Spearman相關分析和線性回歸數(shù)據顯示,Shh蛋白表達和MVD呈顯著的線性正相關關系。(r=0.696,P0.01)。2.Western Blot顯示:a.Shh蛋白在所檢測OSCC細胞系中均有所表達,其中在Cal27細胞株內呈高表達。b.Cal27細胞系提取的MVs中其Shh蛋白含量較Cal27細胞裂解液中含量明顯增加。3.低溫差速離心法成功分離出Cal27源的微囊泡,并在透射電鏡下觀察到微囊泡穩(wěn)定形態(tài)。4.熒光電子顯微鏡下觀察到共培養(yǎng)的MVs與HUVEC融合,融合后的內皮細胞胞漿中可見綠色熒光物質聚集。結論:1.OSCC中Shh、Gli-1表達及MVD與臨床病理特性中淋巴結轉移、腫瘤TNM分期存在顯著相關性,并且Shh與MVD呈顯著正相關,提示Shh、Gli-1的異常表達可能參與了OSCC的發(fā)生、發(fā)展及血管生成。2.Cal27細胞系提取的MVs中高表達Shh蛋白,體外共培養(yǎng)可見MVs與HUVEC融合,推測OSCC細胞產生的Shh蛋白可能經MVs轉載至腫瘤微環(huán)境,從而調節(jié)原發(fā)腫瘤和/或轉移癌前龕血管網絡形成。
[Abstract]:Objective: This study aims to explore the significance of Hedgehog (Hh) signaling pathway and explore the correlation with OSCC angiogenesis in combination with the clinicopathological features of oral squamous cell carcinoma (OSCC). Methods: 1. after informed consent, 80 pathological specimens of OSCC were collected by pathology and immunohistochemistry was used to detect Shh, Gli-1, and CD34 protein. The expression of the primary tumor in 80 patients with OSCC, the microvessel density (MVD) was calculated according to the expression of CD34 protein, and the expression of Shh, Gli-1 protein and the correlation of MVD with the clinicopathological features of.2. in vitro culture SCC4, SCC9, SCC25, Cal27 cells were used to detect the expression of the protein in the four kinds of cell lines. The microvesicle (MVs) in the high expression Shh protein cell line was extracted by differential centrifugation, and the morphology of MVs was observed under transmission electron microscope. The fluorescence labeled MVs, MVs and human umbilical vein endothelial cells (HUVEC) were co cultured in the special medium of endothelial cell (ECs), and the interaction mode was observed under the fluorescence microscope. Results: 1. immunohistochemistry showed that Shh in a.OSCC tissue Gli-1 protein expression and MVD were associated with lymph node metastasis and tumor clinical staging. The correlation analysis with tumor diameter, age, sex unrelated.B.Spearman correlation analysis showed that the expression of Shh protein and Gli-1 protein was significantly related? P=0.008?.c.Spearman phase analysis and linear regression data, Shh protein expression and MVD There was a significant linear positive correlation. (r=0.696, P0.01).2.Western Blot showed that a.Shh protein was expressed in the detected OSCC cell lines, and the content of Shh protein in MVs expressed in the Cal27 cell line was higher than that in the Cal27 cell lysate, and the content of the Shh protein was significantly increased by the.3. cryogenic differential centrifugation. The microvesicles of L27 source were observed under the transmission electron microscope. The co cultured MVs and HUVEC were observed under the microvesicle stable morphology.4. fluorescence electron microscope. The aggregation of green fluorescent substance was found in the cytoplasm of the fused endothelial cells. Conclusion: Shh, Gli-1 expression, MVD and lymph node metastases in MVD and clinicopathological characteristics in 1.OSCC, and the existence of TNM staging in the tumor. Significant correlation, and there is a significant positive correlation between Shh and MVD, suggesting that the abnormal expression of Shh, Gli-1 may be involved in the occurrence of OSCC, and the development of the.2.Cal27 cell line, and the high expression of Shh protein in the MVs extracted from the vasculogenic.2.Cal27 cell line, and the co culture of MVs and HUVEC in vitro, and presumed that the Shh protein produced by OSCC cells may be reproduced to the tumor microenvironment. Regulation of primary tumor and / or metastasis of precancerous niche network.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.8

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