【作者】 韩松瑛；

【导师】 林贞花； 金锡亨；

【作者基本信息】 延边大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 目的:应用RNAi,Western blot,Real-time PCR,MS-PCR和免疫组化等方法研究Twist-1基因蛋白表达在食管鳞状细胞癌(Esophageal Squamous Cell Carcinoma,ESCC)中的临床病理学意义。材料和方法:食管鳞状细胞癌(ESCC)167例及正常食管组织34例共201例病理组织标本选自韩国三星医疗中心病理科1995-2008年的存档蜡块标本。所有标本均制作成组织芯片(TMA,tissue microarray)。另外,我们选择近几年已发表论文中最常使用的几种Twist抗体,应用RNAi技术、Western blot和免疫细胞化学方法在10种胃癌细胞株中进行Twist-1蛋白表达的对比检测,从而筛选出特异性最好的抗体应用于上述食管病变蜡块标本的免疫组化染色,同时进行Real-time PCR和Twist-1基因启动子区甲基化(Methylation Specific PCR,MS-PCR)检测,探讨Twist-1基因和蛋白在ESCC患者中的表达情况,并分析其临床病理学意义及其机制。结果:与其它Twist抗体比较,单克隆抗体Twist2C1a(ab50887)具有背景染色少、抗原特异性高的特点,因此是比较可靠的Twist-1抗体。单克隆抗体Twist2C1a(ab50887)蛋白免疫组化染色结果表明,Twist-1蛋白阳性表达部位主要是食管鳞癌细胞核,Twist-1蛋白在食管鳞癌中的阳性表达率较高(86/167,51.5%),而在正常食管鳞状上皮组织中则较低(9/34,26.5%),且差异具有显著性(p=0.008＜0.01)。对ESCC患者的临床病理学数据的分析结果表明,Twist-1蛋白过表达预示食管鳞癌患者的不良预后,即5年生存率明显降低,在Cox-比例风险模型统计单变量(RR=3.019,p=0.000)和多变量(RR=3.131,p=0.000)分析中均有意义。但与患者年龄、性别、肿瘤大小、分期、有无淋巴结转移、癌细胞分化情况以及放化疗等不相关(p＞0.05)。另外,根据免疫组化染色结果选取Twist-1蛋白高表达和低表达食管鳞癌蜡块组织各7例以及正常食管鳞状上皮蜡块组织5例,应用Real-time PCR检测标本的mRNA表达水平,结果表明各病例中的mRNA表达水平与免疫组化结果相一致,即免疫组化Twsit-1蛋白过表达病例,其mRNA表达水平也明显增高。同样,MS-PCR结果也证明,Twist-1蛋白低表达病例表现为Twist-1基因启动子区的高度甲基化,而Twist-1蛋白强阳性病例表现为Twist-1基因启动子区无甲基化或甲基化程度明显较弱。结论:单克隆抗体Twist2C1a(ab50887)的抗原特异性要优于其它Twist抗体,可用于各种病变组织中Twist-1蛋白的检测,而且Twist-1蛋白检测可以作为食管鳞癌的预后判定指标之一。Twist-1蛋白在食管鳞癌中的过表达机制与Twist-1基因启动子区甲基化密切相关,并有望成为食管鳞癌治疗的新靶点。更多还原

【Abstract】 Objectives: To investigate the clinicopathological significance of Twist-1 gene expression in esophageal squamous cell carcinoma (ESCC) by RNAi, Western blot, Real-time PCR, MS-PCR, immunohistochemical staining and other techniques.Materials and Methods: Total 201 cases of specimen, including 167 of ESCC and 34 of normal esophagus, were selected from Department of Pathology, Samsung Medical Center (South Korea) in the period of 1995-2008, and the tissue microarray (TMA) was made by using all above tissues. To select the best antibodies of Twist-1 protein, four kinds of different Twist antibodies were tested in 10 gastric cell lines by RNAi, Western blot analysis and cell block immunocytochemistry (ICC), and the best one was used to investigate the protein expression of Twist-1 in ESCC and normal esophagus by immunohistochemical staining. The correlation between Twist-1 over-expression and clinicolpathological parameters of ESCC was also analyzed. Then the Twist-1 mRNA level and DNA methylation status of Twist-1 gene promoter were detected by Real-time PCR and Methylation Specific PCR (MS-PCR) for exploring the mechanism of Twist-1 protein over-expression in ESCC.Results: Monoclonal antibody, Twist2Cla (ab50887), was the best antibody for detection of Twist-1 antigen specificity with low level of background. Twist-1 protein expression level was significantly increased in ESCC than in normal esophagus (86/167, 51.5% versus 9/34, 26.5%, p =0.008<0.01). The over-expression of Twist-1 in ESCC indicated the poor-prognosis (low 5 years survival) (p<0.001), however, there was no significant difference between Twist-1 protein expression and the clinicopathological parameters, including age, sex, lymph node metastasis, tumor size,stage, differentiation, chemical therapy and radiation therapy (p>0.05). Also the correlation of Twist-1 over-expression with overall survival rate of the ESCC patients was analyzed, and it showed the statistical significance in univariate analysis (RR=3.019, p=0.000) and multivariate analysis (RR=3.131, p=0.000) by the Cox-proportional hazards regression analysis (Figure 3). According to the IHC result, 7 ESCC cases with Twist-1 over-expression, 7 ESCC cases with Twist-1 lower expression, and 5 cases of normal esophagus were selected; the mRNA expression level of the blocks was detected by RT-PCR. It showed that Twist-1 mRNA expression level is also significant high in the cases with Twist-1 strong positivity by IHC. The result of MS-PCR showed that the hypermethylation of Twist-1 might be corrrelated with the low expression of Twist-1 protein in esophageal lesions, whereas the unmethylation of Twist-1 might be the cause of Twist-1 high expression.Conclusions: The monoclonal antibody Twist2Cla (ab50887) was the best one for detection of Twist-1 protein. Twist-1 was highly expressed in ESCC, and the positive expression was strongly associated with patients’ survival with a statistical significance. Meanwhile, the mechanism of Twist-1 high expression in ESCC was correlated with the methylation status of Twist-1 gene promoter, and Twist-1 was involved in the progression of ESCC and could be the molecular target for ESCC therapy.更多还原