Smac在食管鳞状细胞癌侵袭中的作用及临床意义

发布时间：2018-06-07 08:38

本文选题：食管鳞癌 + Smac　；参考：《山东大学》2017年硕士论文

【摘要】：[目的与背景]食管癌是最常见的恶性肿瘤之一,其发病率在全世界恶性肿瘤中居第9位,在我国居第5位,其死亡率在全球范围内居第8位,在我国居第4位。我国食管癌以食管鳞状细胞癌为主(简称食管鳞癌,esophageal squamous cell carcinoma,ESCC),占食管癌的90%以上。早期食管癌手术切除后5年生存率可达85%-90%,但因其缺乏典型的临床症状而难以早期诊断治疗。大部分患者在出现吞咽困难或转移症状时才去就诊,此时多已经处于中晚期,5年生存率仅为60%-15%。为了改善食管癌的预后,在评估及诊疗方面的分子技术尚有待改进。由于癌细胞通常具有逃避凋亡的进化机制,因此与细胞凋亡途径相接合的诊疗手段充满前景。凋亡抑制因子(inhibitor of apoptosis proteins,IAPs)阻碍了细胞凋亡,这与肿瘤侵袭、进展、治疗失败和预后不良有关。肿瘤细胞高表达IAPs是导致肿瘤细胞抵抗凋亡的关键。人类第二线粒体源性胱氨酸酶激活因子(Second mitochondria-derived activator of caspase,Smac)是凋亡途径中的一种重要蛋白,通过消除IPAs对胱天蛋白酶(cysteine aspartate-specific proteases,caspase)的抑制发挥促凋亡作用,因此Smac被认为是食管癌治疗的潜在靶点之一。阐明Smac在食管鳞状细胞癌侵袭中的表达情况、生物学功能及临床意义可能具有重要的价值。本课题通过免疫组织化学方法检测Smac在食管鳞癌组织、癌旁组织及正常食管组织中的表达情况,探讨其与临床病理特点之间的关系,并通过转染上调Smac在食管鳞癌细胞ECA109中的表达,验证Smac对食管鳞癌细胞体外侵袭行为的调控作用。[材料与方法]收集山大二院2015年1月至2016年8月期间手术切除的食管鳞癌组织、癌旁组织和正常食管组织共68组,常规石蜡包埋切片,通过免疫组织化学方法检测Smac食管鳞癌组织及对应癌旁组织和正常组织中的表达情况,运用X2检验分析Smac的表达与食管鳞癌患者临床病理参数的关系。通过qRT-PCR获取Smac蛋白的cDNA,从琼脂糖凝胶中回收靶片段,构建含Smac基因片段的真核表达载体pcDNA3.1-Smac,通过瞬时转染技术获得稳定高表达Smac的食管鳞癌细胞株 ECA109/pcDNA3.1-Smac(ECA109/Smac 组)和对照组 ECA109/pcDNA3.1(ECA109/对照组),并设立空白对照(ECA109组)。采用Western blotting技术验证Smac蛋白水平的过表达率。采用transwell小室检测Smac对食管鳞癌细胞体外侵袭能力的调控作用。[实验结果]根据免疫组化结果,食管鳞癌组织中的Smac阳性率为54.41%,明显低于癌旁组织和正常食管组织Smac的表达率(分别为89.71%,92.65%;P0.05),Smac的表达水平与食管鳞癌的TNM分期、浸润深度、淋巴结转移情况显著相关(P0.05),与年龄、性别、分化程度无关(P0.05)。Western blotting显示,ECA109/Smac组的Smac蛋白表达升高。Transwell侵袭实验表明与ECA109/对照组和ECA109组相比,ECA109/Smac组的细胞侵袭能力降低(P＜0.05)。[实验结论]食管鳞癌组织中的Smac明显降低,上调Smac的表达后,食管鳞癌细胞的侵袭能力降低,表明Smmac在食管鳞癌的发生发展及转移中发挥重要的作用,进一步研究Smac的生物学特性可能有益于治疗食管癌。
[Abstract]:[Objective and background] cancer of the esophagus is one of the most common malignant tumors. Its incidence is the ninth most malignant tumor in the world and the fifth place in our country. The mortality rate is the eighth in the world and the fourth in China. Esophageal squamous cell carcinoma is mainly esophageal squamous cell carcinoma (esophageal squamous cell carcinoma, ESCC), More than 90% of the cancer of the esophagus is accounted for. The 5 year survival rate of early esophageal cancer is up to 85%-90%, but it is difficult to be diagnosed early because of its lack of typical clinical symptoms. Most patients go to the doctor when they have dysphagia or metastases. At this time, most of them are in the middle and late stages, and the 5 year survival rate is only 60%-15%. in order to improve the precondition of esophageal cancer. Molecular techniques in evaluation and diagnosis have yet to be improved. Because cancer cells usually have an evolutionary mechanism to escape apoptosis, the diagnostic and therapeutic methods that join the apoptotic pathway are full of prospects. Apoptosis inhibitor (inhibitor of apoptosis proteins, IAPs) hinders apoptosis, which is associated with tumor invasion, progress, treatment failure and The high expression of IAPs in tumor cells is the key to the resistance to apoptosis of tumor cells. Human second mitochondrial Second mitochondria-derived activator of caspase (Smac) is an important protein in the apoptotic pathway, by eliminating IPAs to cystine (cysteine aspartate-specific Pro). The inhibition of teases, caspase) plays a role in promoting apoptosis, so Smac is considered to be one of the potential targets for the treatment of esophageal cancer. It is possible to clarify the expression, biological function and clinical significance of Smac in the invasion of squamous cell carcinoma of the esophagus. This topic is to detect Smac in the tissue of esophageal squamous cell carcinoma and the para cancer by immunohistochemical method. The relationship between the expression of tissue and normal esophageal tissue and its relationship with the clinicopathological characteristics, and the expression of Smac in ECA109 of esophageal squamous cell carcinoma cells by transfection, and to verify the regulation of Smac on the invasion of esophageal squamous cell carcinoma cells in vitro. [materials and methods] collect the operation cut from January 2015 to August 2016. 68 groups of esophageal squamous cell carcinoma, para cancerous tissue and normal esophageal tissue were divided into 68 groups. Routine paraffin embedded sections were used to detect the expression of Smac esophageal squamous cell carcinoma tissue and its para cancerous tissue and normal tissues by immunohistochemistry. The relationship between the expression of Smac and the clinicopathological parameters of the patients with esophageal squamous cell carcinoma was analyzed by X2 test. R obtained the cDNA of Smac protein, recovered the target fragment from agarose gel and constructed the eukaryotic expression vector containing the Smac gene fragment, pcDNA3.1-Smac, and obtained the ECA109/pcDNA3.1-Smac (ECA109/Smac group) and the control group ECA109/pcDNA3.1 (ECA109/ control group), which stabilized the high expression of Smac by transient transfection, and set up a blank pair. Western blotting technique was used to verify the overexpression of Smac protein level. The regulation of Smac on the invasion ability of esophageal squamous cell carcinoma cells was detected by Transwell chamber. [experimental results] the positive rate of Smac in esophageal squamous cell carcinoma was 54.41% according to the immunohistochemical results, which was significantly lower than that of the Para cancerous tissue and the normal esophageal tissue S. The expression rate of MAC (89.71%, 92.65%; P0.05). The expression level of Smac was significantly correlated with the TNM staging, infiltration depth and lymph node metastasis of esophageal squamous cell carcinoma (P0.05). It was not related to age, sex, differentiation degree (P0.05).Western blotting, the increase of Smac protein expression in the ECA109/Smac group showed that.Transwell invasion experiment showed that the ECA109/ control group was in the ECA109/ control group. Compared with the ECA109 group, the cell invasion ability of ECA109/Smac group decreased (P < 0.05). [experimental conclusions] the Smac in esophageal squamous cell carcinoma decreased obviously. After the expression of Smac, the invasion ability of esophageal squamous cell carcinoma cells decreased, indicating that Smmac plays an important role in the development and metastasis of esophageal squamous cell carcinoma, and further studies the biological characteristics of Smac. Sex may be beneficial to the treatment of cancer of the esophagus.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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