表面增强拉曼光谱在肺癌EGFR突变检测中的应用研究

发布时间：2018-07-06 13:40

  本文选题：肺癌 + EGFR突变　； 参考：《第四军医大学》2017年博士论文

【摘要】：背景:针对EGFR突变基因的靶向治疗是晚期肺癌患者最有效的治疗方式之一。在治疗前准确了解EGFR基因的突变状态,在治疗中持续监测耐药基因突变情况,对肺癌的个体化靶向治疗有着重要的意义。目前常用的EGFR突变检测技术(如sARMS、ddPCR以及NGS等)在微量检测中(如胸腔积液或液态活检标本)的敏感度和特异度难以满足实际的临床需求,同时这些技术对实验平台要求高、耗时久、费用大等问题也长期制约着临床中肺癌EGFR突变基因的检测水准。因此开发一种高效的肺癌驱动基因检测技术对于提高肺癌的精准化诊治水平具有非常重要的意义。材料与方法:1.我们直接测取了肺癌组织的拉曼信号,并结合HE和IHC染色技术识别出肿瘤组织中EGFR突变位点出的拉曼信号,利用拉曼信号的原始数据构建了PCA/SVM判别模型,最终利用该判别模型诊断识别了30例肺癌组织标本的EGFR突变类型。2.我们制备了“海胆样”金颗粒,通过在颗粒表面包被CV分子、聚乙二醇和EGFR突变特异性抗体合成了金纳米细胞探针,利用该探针进行肺癌细胞EGFR突变检测,并构建出PCA/SVM判别模型,最终识别诊断了35例IV期肺癌患者胸腔积液的EGFR突变类型。3.我们制备了“海胆样”金颗粒,通过在颗粒表面包被EGFR突变特异性分子信标合成了金纳米核酸探针,并利用该核酸探针检测了肺癌细胞的DNA的EGFR突变状态。最终利用该探针诊断识别了28例肺癌患者外周血ctDNA的EGFR突变类型。结果:1.本研究首先使用sARMS法检测了153例肺腺癌患者的手术切除标本,其中75例(49.0%)检出EGFR突变,包括29例E19del,33例L858R,7例T790M,6例多重突变。按照入组标准,从上述标本中选择了30例肿瘤组织,野生型、E19del和L858R突变型各10例。IHC法检测EGFR突变的敏感度分别为90%(E19del)、80%(L858R),特异度100%(野生型)。通过IHC染色确定了肿瘤组织中EGFR突变区域,并选取了上述区域肿瘤组织的拉曼光谱,包括野生型149条,E19del和L858R突变型分别157条和135条。野生型组织在675,1107,1127,1307-1324,1582and 1660 cm~(-1)等峰位处强度明显升高,上述峰位可以归属为碱基对和DNA。L858R突变组织在1085,1175 and 1632 cm~(-1)等位置处升高的拉曼信号归属于精氨酸,而E19del组织也因为746-750碱基的缺失导致拉曼强度减弱。使用PCA和SVM模型诊断L858R或E19del突变的准确度为87.8%。2.本研究合成的“海胆样”金颗粒平均直径92.4 nm,颗粒表面的金刺长度15nm。由于颗粒表面尖锐的刺状结构,该颗粒具有良好的SERS性能,单颗粒单分子SERS信号增强因子达到了1.94×107。我们通过在颗粒表面包被CV分子、聚乙二醇和EGFR突变特异性抗体合成了金纳米细胞探针,该探针具有良好的SERS信号增强能力和肺癌EGFR突变识别能力。由于1617cm~(-1)处的SERS信号稳定,因而本研究中使用其作为标记峰位。通过对1617 cm~(-1)处的SERS信号强度持续监测,我们计算出该探针进行EGFR突变检测至少每毫升液体中需要25个肿瘤细胞。通过ICP-MS检测得到每个肺癌H1650细胞2小时内可以吞噬56-62枚金纳米细胞探针。进一步,我们应用该探针检测了35例IV期肺腺癌患者的胸腔积液EGFR突变情况,并构建了PCA/SVM的判别模型,该模型诊断EGFR突变的准确率达到了90.7%。3.本研究通过在“海胆样”金颗粒表面包被EGFR突变特异性分子信标合成了金纳米核酸探针,该探针具有良好的SERS信号增强能力和肺癌EGFR突变ssDNA识别能力。对DNA靶序列的最低检测浓度达到了1×10-8 mol/L,肺癌细胞的最低检测数目达到了100个。结合不对称PCR技术,我们能够获得大量的靶基因ssDNA,从而显著提升了待测靶基因的含量。进一步,在28例肺癌患者外周血ct DNA标本中,该探针检测了EGFR突变的敏感度和特异度分别达到了61.1%和100%。结论:本研究通过贵金属纳米颗粒介导的SERS光谱开展了肺癌EGFR突变基因的检测工作,建立了一种检测方法,制备了两种检测探针,分别针对肺癌组织、胸腔积液和外周血等三类临床标本开展了研究,具体包括:1.本研究建立了非标记型SERS技术检测肺癌EGFR突变的一整套技术方法,尤其在肺癌组织标本的实时、快速、精准检测方面展现了显著的优势;2.本研究合成的金纳米细胞探针和核酸探针,具有很高的检测灵敏度和特异度,检测水准高于目前临床常用的检测技术,在胸腔积液和液态活检标本的EGFR突变检测中展现了非常良好的临床应用前景。
[Abstract]:Background: targeted therapy for EGFR mutation is one of the most effective therapies for patients with advanced lung cancer. It is important to monitor the mutation status of EGFR gene accurately before treatment and monitor the mutation of resistant genes in the treatment. It is of great significance for the individualized targeting therapy of lung cancer. The current EGFR mutation detection technique (such as sARMS, DD) The sensitivity and specificity of PCR and NGS) in microdetection (such as pleural effusion or liquid biopsy specimen) are difficult to meet the actual clinical needs. At the same time, these techniques have long restricted the detection level of EGFR mutants in clinical lung cancer. The technique of driving gene detection is of great significance to improve the level of diagnosis and treatment of lung cancer. Materials and methods: 1. we directly measured the Raman signals of lung cancer tissues, and identified the Raman signals of the EGFR mutations in the tumor tissues by HE and IHC staining techniques, and constructed the PCA/SVM using the original data of the Raman signal. The discriminant model was used to diagnose the EGFR mutant.2. of 30 cases of lung cancer. We prepared the "sea urchin like" gold particles. The gold nanoscale probe was synthesized by the specific antibody of CV molecule, polyethylene glycol and EGFR mutation on the grain surface bread. The probe was used to detect the EGFR mutation of lung cancer cells. The PCA/SVM discriminant model was constructed and the EGFR mutation of the pleural effusion in 35 patients with IV lung cancer was identified. We prepared the "sea urchin like" gold particles. The gold nanoparticles were synthesized by the EGFR mutation specific molecular beacon on the grain surface bread. The nucleic acid probe was used to detect the EGFR process of the DNA in the lung cancer cells. The EGFR mutation of ctDNA in peripheral blood of 28 patients with lung cancer was identified by this probe. Results: 1. this study first used sARMS to detect the surgical excision specimens of 153 cases of lung adenocarcinoma. 75 cases (49%) detected EGFR mutations, including 29 cases of E19del, 33 cases of L858R, 7 T790M, 6 multiple mutations. According to the standard of entry group, From the above specimens, 30 cases of tumor tissue, wild type, E19del and L858R mutagenesis were detected by.IHC method, the sensitivity of EGFR mutation was 90% (E19del), 80% (L858R), and specificity 100% (wild type). The EGFR mutation area in tumor tissues was determined by IHC staining, and the Raman spectra of the tumor tissue in the above region were selected, including the wild. Type 149, E19del and L858R mutagenesis are 157 and 135 respectively. The intensity of wild type tissues at the peak of 675110711271307-13241582and 1660 cm~ (-1) is obviously increased. The above peak can belong to the arginine, and E19del of the DNA.L858R mutant tissues at the 10851175 and 1632 cm~ (-1), and E19del. The PCA and SVM models were used to diagnose the L858R or E19del mutation by using PCA and SVM models. The average diameter of the "sea urchin like" gold particles was 92.4 nm, and the gold thorn length 15nm. on the particle surface has a good SERS property due to the sharp spiny structure of the particle surface. Yes, single particle single molecule SERS signal enhancement factor reached 1.94 * 107.. We synthesized gold nanoscale probe by CV molecule, polyethylene glycol and EGFR mutant specific antibody on particle surface bread. The probe has good enhancement ability of SERS signal and identification ability of EGFR mutation in lung cancer. Because SERS signal at 1617cm~ (-1) is stable, because of the stability of SERS signal at -1 (-1) In this study, it was used as a marker peak. By continuous monitoring of the SERS signal intensity at 1617 cm~ (-1), we calculated that the probe for EGFR mutation detection required at least 25 tumor cells per milliliter of liquid. By ICP-MS detection, each lung cancer H1650 cell could phagocyt 56-62 gold nanoscale probe within 2 hours. Step, we used this probe to detect the EGFR mutation in the pleural effusion of 35 patients with IV lung adenocarcinoma and construct a discriminant model of PCA/SVM. The accuracy of the model for the diagnosis of EGFR mutation reached the 90.7%.3. study by synthesizing gold nanoscale nucleic acid probes by the EGFR mutation specific molecular beacon in the "sea urchin like" gold particle surface bread. The probe has good SERS signal enhancement ability and EGFR mutation ssDNA recognition ability of lung cancer. The minimum detection concentration of DNA target sequence reached 1 * 10-8 mol/L, and the minimum detection number of lung cancer cells reached 100. Combined asymmetric PCR technology, we can obtain a large number of target gene ssDNA, thus significantly enhancing the content of the target gene. Further, in the peripheral blood CT DNA specimens of 28 patients with lung cancer, the probe detected the sensitivity and specificity of EGFR mutation to 61.1% and 100%. respectively. This study carried out the detection of EGFR mutant gene in lung cancer by SERS spectrum mediated by noble metal nanoparticles. A detection method was established, and two kinds of detection were prepared. Three types of clinical specimens, including lung cancer tissue, pleural effusion and peripheral blood, were studied, including: 1. studies have established a complete set of technical methods for detecting EGFR mutation of lung cancer by unmarked SERS technology, especially in the real-time, rapid and accurate detection of lung cancer tissue specimens; 2. studies have been synthesized. The gold nanoscale probe and nucleic acid probe have high detection sensitivity and specificity, and the detection level is higher than the current clinical detection techniques. The EGFR mutation detection in the pleural effusion and liquid biopsy specimens shows a very good clinical application prospect.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2
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本文编号：2102955