本文選題：肺癌 + EGFR突變��； 參考：《第四軍醫(yī)大學》2017年博士論文

【摘要】：背景:針對EGFR突變基因的靶向治療是晚期肺癌患者最有效的治療方式之一。在治療前準確了解EGFR基因的突變狀態(tài),在治療中持續(xù)監(jiān)測耐藥基因突變情況,對肺癌的個體化靶向治療有著重要的意義。目前常用的EGFR突變檢測技術(如sARMS、ddPCR以及NGS等)在微量檢測中(如胸腔積液或液態(tài)活檢標本)的敏感度和特異度難以滿足實際的臨床需求,同時這些技術對實驗平臺要求高、耗時久、費用大等問題也長期制約著臨床中肺癌EGFR突變基因的檢測水準。因此開發(fā)一種高效的肺癌驅(qū)動基因檢測技術對于提高肺癌的精準化診治水平具有非常重要的意義。材料與方法:1.我們直接測取了肺癌組織的拉曼信號,并結合HE和IHC染色技術識別出腫瘤組織中EGFR突變位點出的拉曼信號,利用拉曼信號的原始數(shù)據(jù)構建了PCA/SVM判別模型,最終利用該判別模型診斷識別了30例肺癌組織標本的EGFR突變類型。2.我們制備了“海膽樣”金顆粒,通過在顆粒表面包被CV分子、聚乙二醇和EGFR突變特異性抗體合成了金納米細胞探針,利用該探針進行肺癌細胞EGFR突變檢測,并構建出PCA/SVM判別模型,最終識別診斷了35例IV期肺癌患者胸腔積液的EGFR突變類型。3.我們制備了“海膽樣”金顆粒,通過在顆粒表面包被EGFR突變特異性分子信標合成了金納米核酸探針,并利用該核酸探針檢測了肺癌細胞的DNA的EGFR突變狀態(tài)。最終利用該探針診斷識別了28例肺癌患者外周血ctDNA的EGFR突變類型。結果:1.本研究首先使用sARMS法檢測了153例肺腺癌患者的手術切除標本,其中75例(49.0%)檢出EGFR突變,包括29例E19del,33例L858R,7例T790M,6例多重突變。按照入組標準,從上述標本中選擇了30例腫瘤組織,野生型、E19del和L858R突變型各10例。IHC法檢測EGFR突變的敏感度分別為90%(E19del)、80%(L858R),特異度100%(野生型)。通過IHC染色確定了腫瘤組織中EGFR突變區(qū)域,并選取了上述區(qū)域腫瘤組織的拉曼光譜,包括野生型149條,E19del和L858R突變型分別157條和135條。野生型組織在675,1107,1127,1307-1324,1582and 1660 cm~(-1)等峰位處強度明顯升高,上述峰位可以歸屬為堿基對和DNA。L858R突變組織在1085,1175 and 1632 cm~(-1)等位置處升高的拉曼信號歸屬于精氨酸,而E19del組織也因為746-750堿基的缺失導致拉曼強度減弱。使用PCA和SVM模型診斷L858R或E19del突變的準確度為87.8%。2.本研究合成的“海膽樣”金顆粒平均直徑92.4 nm,顆粒表面的金刺長度15nm。由于顆粒表面尖銳的刺狀結構,該顆粒具有良好的SERS性能,單顆粒單分子SERS信號增強因子達到了1.94×107。我們通過在顆粒表面包被CV分子、聚乙二醇和EGFR突變特異性抗體合成了金納米細胞探針,該探針具有良好的SERS信號增強能力和肺癌EGFR突變識別能力。由于1617cm~(-1)處的SERS信號穩(wěn)定,因而本研究中使用其作為標記峰位。通過對1617 cm~(-1)處的SERS信號強度持續(xù)監(jiān)測,我們計算出該探針進行EGFR突變檢測至少每毫升液體中需要25個腫瘤細胞。通過ICP-MS檢測得到每個肺癌H1650細胞2小時內(nèi)可以吞噬56-62枚金納米細胞探針。進一步,我們應用該探針檢測了35例IV期肺腺癌患者的胸腔積液EGFR突變情況,并構建了PCA/SVM的判別模型,該模型診斷EGFR突變的準確率達到了90.7%。3.本研究通過在“海膽樣”金顆粒表面包被EGFR突變特異性分子信標合成了金納米核酸探針,該探針具有良好的SERS信號增強能力和肺癌EGFR突變ssDNA識別能力。對DNA靶序列的最低檢測濃度達到了1×10-8 mol/L,肺癌細胞的最低檢測數(shù)目達到了100個。結合不對稱PCR技術,我們能夠獲得大量的靶基因ssDNA,從而顯著提升了待測靶基因的含量。進一步,在28例肺癌患者外周血ct DNA標本中,該探針檢測了EGFR突變的敏感度和特異度分別達到了61.1%和100%。結論:本研究通過貴金屬納米顆粒介導的SERS光譜開展了肺癌EGFR突變基因的檢測工作,建立了一種檢測方法,制備了兩種檢測探針,分別針對肺癌組織、胸腔積液和外周血等三類臨床標本開展了研究,具體包括:1.本研究建立了非標記型SERS技術檢測肺癌EGFR突變的一整套技術方法,尤其在肺癌組織標本的實時、快速、精準檢測方面展現(xiàn)了顯著的優(yōu)勢;2.本研究合成的金納米細胞探針和核酸探針,具有很高的檢測靈敏度和特異度,檢測水準高于目前臨床常用的檢測技術,在胸腔積液和液態(tài)活檢標本的EGFR突變檢測中展現(xiàn)了非常良好的臨床應用前景。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R734.2
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本文編號：2102955