宫颈癌表皮生长因子受体基因启动子甲基化的荧光偏振检测研究

发布时间：2018-06-25 03:55

本文选题：宫颈癌 + 表皮生长因子受体　；参考：《第四军医大学》2014年硕士论文

【摘要】：宫颈癌是一种严重威胁着女性生命和健康、由HPV感染导致的恶性肿瘤，发病率在全世界女性肿瘤中位居第二，死亡率位居第一，且目前发病率与死亡率仍呈上升趋势。多数患者在确诊时已达中晚期，因此大多要在手术治疗后接受放化疗。目前的化疗药物对晚期宫颈癌的治疗有一定效果，但仍存在严重的耐药性，导致预后差、生存期短。在大部分宫颈癌患者标本中，可检出表皮生长因子受体（epidermalgrowth factor receptor, EGFR）高水平表达。近年，以表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)为基础的分子靶向药物成为宫颈癌个体化用药治疗的研究热点，但临床试验却并未获得预期疗效。研究发现，在部分宫颈癌患者中，EGFR基因存在甲基化修饰，可能与EGFR靶向治疗药物效果较差有关。因此，对EGFR基因启动子甲基化状态的检测，有利于指导宫颈癌EGFR-TKI个体化治疗和去DNA甲基化临床用药的实施。荧光偏振技术（fluorescence polarization, FP）是一种通过检测荧光标记分子的荧光偏振值变化判断分子量改变的技术。偏振荧光的强度与荧光标记分子分子量的大小呈正相关，因此通过检测荧光偏振强度值的变化即可判断待测荧光标记分子是否参与了反应。本研究利用此技术在液相环境中均相检测的能力和效率的优势，对宫颈癌患者EGFR启动子甲基化状态进行检测研究。目的：建立基于荧光偏振技术的EGFR启动子甲基化标准检测方法与体系，检测并分析宫颈癌患者组织标本中EGFR基因启动子甲基化状态，并初步探讨其与肺癌相关EGFR-TKI敏感突变、宫颈癌病因HPV感染之间的联系，为指导宫颈癌的个体化治疗提供新思路和分子诊断技术方法。方法：首先建立荧光偏振检测标准体系。提取健康志愿者血液淋巴细胞DNA，对DNA进行亚硫酸氢盐修饰，一半经SssI甲基转移酶处理。设计EGFR启动子序列通用引物，扩增序列后测序，同时构建甲基化与非甲基化序列标准质粒载体。设计EGFR启动子甲基化特异性探针，利用标准品作为模板建立荧光偏振检测标准体系，对体系各项反应条件与敏感性、稳定性、特异性等进行研究和优化。随后利用已建立的检测方法对宫颈癌组织标本进行EGFR启动子甲基化状态检测，并与测序法结果进行比较。同时，检测标本中肺癌相关EGFR-TKI敏感突变以及HPV感染型别。利用SPSS13.0软件进行统计学分析，分析EGFR启动子甲基化与肺癌相关EGFR-TKI敏感突变、HPV感染等因素之间的关系。结果：本研究成功构建了基于荧光偏振技术的EGFR启动子甲基化状态的标准检测体系，对此标准检测体系的反应条件、灵敏度、准确度和稳定性均进行了研究和优化。本方法最低检测浓度为50拷贝/μl，，最低检测含量可达10%，敏感度高。利用该体系成功检测了宫颈癌标本组织中EGFR启动子甲基化状态，本方法检测EGFR启动子甲基化状态结果与直接测序法结果无统计学差异。99例宫颈癌标本中EGFR启动子甲基化阳性37例，阳性率为37.4%。未检测出EGFR基因18、19、21外显子EGFR-TKI敏感突变。HPV感染型别中HPV16阳性29例，HPV18阳性12例，HPV58阳性4例，HPV52阳性4例。HPV16感染阳性中EGFR启动子甲基化18例，占62.1%。HPV16阳性样本中EGFR启动子甲基化阳性比率最高。结论：本研究方法检测EGFR启动子甲基化操作简单高效，灵敏度与准确度较高，为临床宫颈癌个体化治疗相关检测提供了新技术。本研究中，宫颈癌标本中EGFR启动子甲基化阳性率为37.4%，未发现肺癌相关EGFR-TKI敏感基因突变， HPV16感染的宫颈癌标本中EGFR启动子甲基化阳性率较其他HPV型显著增高，提示HPV16感染与EGFR启动子甲基化可能有一定相关性。
[Abstract]:Cervical cancer is a malignant tumor that seriously threatens the life and health of women and is caused by HPV infection. The incidence of the disease is second in the world's female tumor. The mortality rate is the first, and the current incidence and mortality are still on the rise. Most patients have reached the middle late stage at the time of diagnosis, so most of them should receive chemotherapy after surgical treatment. The current chemotherapy drugs have some effect on the treatment of advanced cervical cancer, but there are still serious drug resistance, which results in poor prognosis and short survival time. In most of the cervical cancer patients, the epidermalgrowth factor receptor (EGFR) Gao Shui flat expression can be detected. In recent years, the epidermal growth factor receptor tyrosine kinase is used. Inhibitor (EGFR-TKI) based molecular targeting drugs have become a hot spot in the study of individualized drug therapy for cervical cancer, but clinical trials have not been expected. The study found that in some patients with cervical cancer, the EGFR gene methylation modification may be related to the poor effect of EGFR targeting therapy. Therefore, the promoter of the EGFR gene can be used as a promoter. The detection of basal status is helpful to guide the individualized treatment of cervical cancer EGFR-TKI and the implementation of clinical medication for removing DNA methylation.
Fluorescence polarization (FP) is a technique to determine the change of molecular weight by detecting the change of fluorescence polarization value of the fluorescent labeling molecule. The intensity of polarization fluorescence is positively correlated with the molecular weight of the fluorescent labeling molecule, so the fluorescence labeling can be judged by detecting the change of the fluorescence polarization intensity value. This study used this technique to detect the methylation status of EGFR promoter in cervical cancer patients by using the advantage of this technique to detect the ability and efficiency of homogeneous detection in the liquid environment.
Objective: to establish a standard detection method and system of EGFR promoter methylation based on fluorescence polarization technology, to detect and analyze the methylation status of EGFR gene promoter in the tissue specimens of cervical cancer patients, and to explore the relationship between the EGFR-TKI sensitive mutation associated with lung cancer and the HPV infection of the cause of cervical cancer in order to guide the individualized treatment of cervical cancer. The treatment provides new ideas and molecular diagnostic techniques.
Methods: first, a standard system of fluorescence polarization detection was established. DNA of blood lymphocyte in healthy volunteers was extracted, DNA was modified by hydrogen sulfite and half of SssI methyltransferase was treated. The universal primers for EGFR promoter sequence were designed, and the sequence was sequenced, and the standard plasmid vector of methylation and non methylation sequence was constructed. The EGFR initiation was designed. The methylation specific probe was used to establish a standard system for fluorescence polarization detection using standard products as a template. The reaction conditions and sensitivity, stability and specificity of the system were studied and optimized. Then the methylation status of EGFR promoter in cervical cancer tissues was detected by the established detection method, and the sequencing method was combined with the sequencing method. The results were compared. At the same time, the lung cancer related EGFR-TKI sensitive mutations and HPV infection types were detected. The SPSS13.0 software was used for statistical analysis to analyze the relationship between EGFR promoter methylation and EGFR-TKI sensitive mutations in lung cancer, HPV infection and other factors.
Results: the standard detection system for the methylation status of EGFR promoter based on fluorescence polarization technology was successfully constructed. The reaction conditions, sensitivity, accuracy and stability of the standard detection system were studied and optimized. The minimum detection concentration of this method was 50 copies / mu L, the minimum detection content was up to 10%, and the sensitivity was high. The system successfully detected the methylation status of EGFR promoter in cervical carcinoma tissue. This method was used to detect the methylation status of EGFR promoter and the results of direct sequencing, 37 cases of EGFR promoter methylation positive in.99 cases of cervical cancer, the positive rate was 37.4%. not detected the EGFR-TKI sensitivity of EGFR gene 18,19,21 exon There were 29 cases of HPV16 positive in the mutant.HPV infection, 12 cases of HPV18 positive, 4 cases of HPV58 positive, and 18 cases of EGFR promoter methylation in 4 HPV52 positive positive cases, which accounted for the highest percentage of EGFR promoter methylation positive in 62.1%.HPV16 positive samples.
Conclusion: the method of EGFR promoter methylation is simple and efficient, with high sensitivity and accuracy, which provides a new technique for the detection of clinical cervical cancer. In this study, the positive rate of EGFR promoter Zi Jiaji was 37.4% in cervical cancer specimens, and no EGFR-TKI sensitive gene mutation was found in lung cancer, and HPV16 infection was not found in this study. The positive rate of methylation of EGFR promoter in cervical cancer samples was significantly higher than that in other HPV types, suggesting that HPV16 infection may be related to methylation of EGFR promoter.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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