非小细胞肺癌中EGFR表达及其启动子甲基化的临床意义研究

发布时间：2019-06-21 05:30

【摘要】：EGFR基因位于第7号染色体的p1 1.2,编码横跨细胞膜表面的糖蛋白受体。EGFR为酪氨酸激酶受体,是蛋白激酶家族重要一员。EGFR与其配体结合成二聚体,随后酪氨酸自身磷酸化启动下游各通路,从而调节细胞的增殖,分化,凋亡等。在多种癌症中EGFR信号通路的改变导致了癌细胞更难以被清除。大量文献报道EGFR蛋白的高表达与多种肿瘤的分期和预后相关。近年来表观遗传学的调控机制已经成为研究的热点,表观遗传学信息是指基因的DNA序列未发生变化而表型发生了改变,并且这种改变在发育和细胞增殖过程中能稳定遗传。DNA甲基化是表观遗传学调控机制之一。本研究旨在研究非小细胞肺癌中EGFR表达与临床各指标间的联系,并探讨EGFR启动子甲基化调控的改变与EGFR表达间的相关性。[目的]1.应用免疫印迹法检测肺癌组织和正常组织样本中EGFR蛋白表达,并研究样本中EGFR表达水平与组织分类,临床分期,样本病理等临床资料的相关性。2.应用甲基化特异性PCR检测肺癌组织和正常组织样本中EGFR基因启动子区CpG岛甲基化情况,并研究样品中EGFR启动子甲基化水平与EGFR的表达差异间的相关性。[方法]1.收集2015年12月至2016年5月间云南省肿瘤医院收治的手术切除的50例非小细胞肺癌患者的癌组织与正常组织样本。入选标准为:①手术切除标本经病理诊断为非小细胞肺癌②术前未经过任何抗肿瘤治疗③肿瘤经手术切除,切缘为阴性。④年龄≥30岁,≤75岁。2.将病人标本收回后,用液氮冰冻后至-80℃冰箱中保存。组织研磨后用试剂盒提取DNA,用Ripa提取蛋白,并测定其吸光度和浓度。提取后放回-80℃冰箱中保存待后续处理。3.BCA测定蛋白浓度,并使用Western-blot测定EGFR的蛋白表达。4.提取的DNA进行急速重亚硫酸盐处理,并用甲基化引物进行扩增,运用琼脂糖凝胶电泳,观测其表达差异。5.Western-blot 结果采用图像分析处理软件(ImageJ,National Institutes of Health)进行半定量分析。6.应用SPSS20.0软件包的分析和统计,各组间实验数据进行正态分布,方差齐性检验,非正态分布数据之间的差异用卡方(chi-square test)和秩和(Wilcoxon signed ranks test)检验法,应用非线性相关分析进行相关性分析。[结果]1.在非小细胞肺癌中癌和正常组织中EGFR的表达有统计学差异(p=0.009),EGFR在癌中高表达。2.EGFR的表达与淋巴结转移有统计学意义(p=0.008) , EGFR表达越高,淋巴结转移的可能性增加,两者呈正相关。3.EGFR的表达与EGFR启动子区甲基化具有统计学意义(p=0.025),甲基化程度降低时EGFR的表达上调,两者呈负相关。[结论]1.EGFR在肺癌组织中相对高表达,且EGFR在肺癌的癌组织中相对表达与淋巴结转移相关,说明EGFR的表达可能成为临床上判断疾病进展的指标和治疗的靶点。2.EGFR的表达与甲基化的改变具有相关性,肺癌的癌组织中EGFR启动子相对低甲基化时EGFR表达有增高趋势,EGFR启动子相对高甲基化时EGFR表达有降低趋势,所以EGFR的表达可能与启动子甲基化状态相关,为以EGFR为靶点的肿瘤靶向治疗提供了新的思路。
[Abstract]:The EGFR gene is located at p1 1.2 of chromosome 7 and encodes a glycoprotein receptor across the surface of the cell membrane. EGFR is a tyrosine kinase receptor and is an important member of the protein kinase family. EGFR binds to its ligand to form a dimer, followed by tyrosine autophosphorylation to initiate downstream pathways, thereby modulating cell proliferation, differentiation, apoptosis, and the like. The change in the EGFR signaling pathway in a variety of cancers leads to a more difficult removal of cancer cells. A large number of literature reports that the high expression of the EGFR protein is associated with the staging and prognosis of a variety of tumors. In recent years, the control mechanism of epigenetics has become the hot spot of the research, and the epigenetic information is that the DNA sequence of the gene has not changed and the phenotype has changed, and the change can be stably inherited in the course of development and cell proliferation. DNA methylation is one of the mechanisms of epigenetic control. The purpose of this study was to study the relationship between EGFR expression in non-small cell lung cancer and the clinical indicators, and to explore the relationship between the change of EGFR promoter methylation and the expression of EGFR. [Objective] 1. The expression of EGFR in lung cancer tissues and normal tissue samples was detected by immunoblotting method, and the correlation of the expression level of EGFR in the samples with the clinical data such as tissue classification, clinical stage and sample pathology was studied. Methylation-specific PCR was used to detect the methylation of the CpG island in the promoter region of the EGFR gene in the lung cancer tissue and the normal tissue sample, and the relationship between the methylation level of the EGFR promoter and the expression of EGFR in the sample was studied. [Method] 1. To collect 50 non-small cell lung cancer patients with non-small cell lung cancer and normal tissue samples from December 2015 to May 2016. The inclusion criteria were as follows: the surgical resection of the specimens was performed by the pathologic diagnosis of non-small cell lung cancer without any anti-tumor treatment, and the tumor was removed by operation and the cut edge was negative. She is 30 years old and 75 years of age. After the patient's specimen was recovered, the patient was frozen with liquid nitrogen and stored in a-80.degree. C. refrigerator. After the tissue was ground, the DNA was extracted with the kit, and the protein was extracted with the Ripa and its absorbance and concentration were determined. And the protein concentration of the EGFR was measured by Western-blot, and the protein expression of the EGFR was determined by Western-blot. The extracted DNA was subjected to rapid bisulfite treatment and amplified by a methylation primer, and the expression difference was observed by agarose gel electrophoresis. Based on the analysis and statistics of the SPSS10.0 software package, the normal distribution, the homogeneity test and the non-normal distribution data between the groups were analyzed by the chi-square test and the Wilcoxon signed test, and the correlation analysis was carried out with the non-linear correlation analysis. [Results] 1. The expression of EGFR in non-small cell lung cancer and normal tissue was statistically different (p = 0.009), and EGFR was highly expressed in cancer.2. The expression of EGFR and lymph node metastasis were of statistical significance (p = 0.008), and the higher the expression of EGFR, the possibility of lymph node metastasis was increased. There was a positive correlation between the expression of EGFR and the methylation of EGFR promoter region (p = 0.025). [Conclusion] 1. EGFR is relatively highly expressed in lung cancer tissues, and the relative expression of EGFR in the cancer tissues of lung cancer is related to lymph node metastasis, and it is indicated that the expression of EGFR may be a target for clinical judgment of disease progression and the target of treatment.2. The expression of EGFR is related to the change of methylation. In the case of lung cancer, the expression of EGFR is increased in the case of relatively low methylation of the EGFR promoter, and the expression of EGFR in the case of relatively high methylation of the EGFR promoter tends to decrease, so the expression of EGFR may be related to the methylation state of the promoter, and a new idea is provided for the target therapy of the tumor targeting EGFR.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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