口腔鳞状细胞癌Shh、Gli-1表达及微血管密度与临床病理相关性的实验研究

发布时间：2018-07-16 18:59

【摘要】：目的:本研究旨在结合口腔鳞状细胞癌(OSCC)患者临床病理特征,探讨Hedgehog(Hh)信号通路的重要性,并探究其与OSCC血管生成的相关性。方法:1.经知情同意,收集80例病理证实为OSCC的术后组织标本;采取免疫组织化学法检测Shh、Gli-1、CD34蛋白在80例OSCC患者原发肿瘤中的表达;依据CD34蛋白表达状况,计算微血管密度(MVD),并分别统计分析Shh、Gli-1蛋白表达及MVD与临床病理特征的相关性。2.体外培养SCC4、SCC9、SCC25、Cal27细胞,采用Western Blot方法检测Shh蛋白在四种OSCC细胞系中的表达;差速离心法提取高表达Shh蛋白细胞系中的微囊泡(MVs),透射电镜下观察MVs形态;荧光标记MVs,内皮细胞(ECs)专用培养基中将MVs与人脐静脉内皮细胞(HUVEC)共培养,并在荧光显微镜下观察其相互作用方式。结果:1.免疫组化显示:a.OSCC组织中Shh、Gli-1蛋白表达及MVD与淋巴结转移、肿瘤临床分期显著相关,与肿瘤直径大小、患者年龄、性别不相关。b.Spearman相关性分析数据显示,Shh蛋白与Gli-1蛋白的表达呈显著相关关系龴P=0.008㖞。c.Spearman相关分析和线性回归数据显示,Shh蛋白表达和MVD呈显著的线性正相关关系。(r=0.696,P0.01)。2.Western Blot显示:a.Shh蛋白在所检测OSCC细胞系中均有所表达,其中在Cal27细胞株内呈高表达。b.Cal27细胞系提取的MVs中其Shh蛋白含量较Cal27细胞裂解液中含量明显增加。3.低温差速离心法成功分离出Cal27源的微囊泡,并在透射电镜下观察到微囊泡稳定形态。4.荧光电子显微镜下观察到共培养的MVs与HUVEC融合,融合后的内皮细胞胞浆中可见绿色荧光物质聚集。结论:1.OSCC中Shh、Gli-1表达及MVD与临床病理特性中淋巴结转移、肿瘤TNM分期存在显著相关性,并且Shh与MVD呈显著正相关,提示Shh、Gli-1的异常表达可能参与了OSCC的发生、发展及血管生成。2.Cal27细胞系提取的MVs中高表达Shh蛋白,体外共培养可见MVs与HUVEC融合,推测OSCC细胞产生的Shh蛋白可能经MVs转载至肿瘤微环境,从而调节原发肿瘤和/或转移癌前龛血管网络形成。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8

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