三阴性乳腺癌中TOP2A、EGFR和Ⅳ型胶原的量子点探针影像技术检测及价值研究

发布时间：2018-09-09 13:52

【摘要】：乳腺癌是女性最常见的恶性肿瘤,发病率逐年上升,成为最主要的导致女性死亡的恶性肿瘤。目前乳腺癌的分类越来越细化,更加注重乳腺癌的分子分型。三阴性乳腺癌作为乳腺癌的一种特殊类型,缺乏有效的靶向治疗和内分泌治疗手段,导致该类肿瘤极易出现复发和转移。然而,目前对于三阴性乳腺癌侵袭和转移的机制尚不明确,尚无临床公认的有效的具有预后预测价值的分子靶向标记物。纳米量子点标记探针分子成像技术是本世纪最重要的生物医学技术之一,大量学者对其运用于癌症诊断、分子分型、治疗和预后评估方面的可行性进行了探索,发现其在癌症基础和临床研究中具有广泛前景。尤其是量子点免疫荧光成像和定量检测系统(quantum dot-based immuno fluorescent imaging and quantitative analytical system, QD-IIQAS)的发展,使得其广泛应用于恶性肿瘤分子标记物的检测。本课题借助量子点纳米技术的优势,对三阴性乳腺癌组织中拓扑异构酶2α(Topoisomerase 2 alpha, TOP2A)、表皮生长因子受体(epidermal growth factor receptor, EGFR)和Ⅳ型胶原的检测进行了可行性探索,并与传统免疫组织化学检测方法(immunohistochemistry, IHC)进行对比。拟通过对三阴性乳腺癌这三种分子标记物的检测,初步寻找三阴性乳腺癌化疗药物敏感性的分子靶标。拟通过对癌细胞表面分子标记物(如EGFR)和肿瘤微环境主要组分(如Ⅳ型胶原)的联合分析,探索三阴性乳腺癌的侵袭和转移是否为癌细胞的独立行为,是否需要肿瘤微环境的参与,为恶性肿瘤侵袭和转移机制的研究提供新理念。并且通过量子点荧光成像和定量分析系统,计算出EGFR/Ⅳ型胶原比值,积极寻找三阴性乳腺癌有效的独立预后因素指标,探索三阴性乳腺癌生物学行为,为推动三阴性乳腺癌的个体化医疗提供理论基础和临床启示。本课题从以下两部分开展了研究：第一部分量子点标记探针技术检测三阴性乳腺癌组织TOP2A的表达及价值分析乳腺癌是高异质性恶性肿瘤,相同分期和病理类型的患者预后往往不同。三阴性乳腺癌作为乳腺癌的一种特殊亚型,具有高侵袭性,极易发生复发和远处转移。目前,三阴性乳腺癌唯一有效的治疗手段就是化疗,主要以蒽环和紫衫类化疗药物为主,目前其治疗效果已经到达了瓶颈,新突破的取得显得尤为困难。TOP2A是DNA复制的重要酶,也是细胞毒性化疗药物,比如蒽环类的靶向作用分子,在乳腺癌领域研究广泛。然而,由于各种不同检测方法的存在和不同阳性阂值的差异,用来研究TOP2A在乳腺癌患者的疗效预测和预后价值的研究结论并不统一。我们以往的研究证实了量子点纳米技术在生物标记物定量检测中的显著优势,并且与传统的IHC进行了比较。本研究拟利用量子点标记探针技术在乳腺癌领域研究的优势,尤其是QD-IIQAS技术,对三阴性乳腺癌组织TOP2A进行原位荧光成像和定量检测,研究TOP2A的表达与主要的临床病理参数之间的关系。我们分别采用传统IHC和QD-IIQAS方法对145例三阴性乳腺癌石蜡切片中的TOP2A的表达进行了检测,并且对这两种检测方法进行了比较,还评估了TOP2A的表达与主要的临床病理参数之间的关系。我们发现,这两种方法具有相同的抗原定位,高的染色率相关性(r=0.79),和高的检测一致性(λ=0.763)(在选取45%作为临界值时)。并且,TOP2A的表达在直径偏大的肿瘤(p=0.002),高组织学分级肿瘤(p=0.005)和有淋巴结转移(p0.001)的患者中表达更高。QD-IIQAS是进行TOP2A检测的更简易和更精确的方法。我们的研究为以后研究TOP2A的表达与蒽环类药物的敏感性以及其在三阴性乳腺癌组织的预后价值打下了坚实的基础。第二部分三阴性乳腺癌组织EGFR和IV型胶原的量子点标记双色荧光成像和定量检测及预后价值三阴性乳腺癌是乳腺癌的一种特殊亚型,具有明显异质性和不良预后。癌症的侵袭和转移并不完全是癌细胞的自发过程,它要求癌细胞穿透其周围的细胞外基质屏障。为了研究三阴性乳腺癌侵袭和转移的机制,发展一种新的检测方法,该方法可以同时研究肿瘤微环境中的重要组分和癌细胞表面重要分子标记物,显得尤为重要。我们使用量子点多光谱荧光成像对三阴性乳腺癌组织进行研究,在第一部分单标量子点免疫荧光成像和定量检测系统的基础上,发展了一种计算机辅助的新型量子点多光谱荧光成像和定量检测技术。使用该技术,我们实现了表达于癌细胞膜的EGFR和表达于细胞外基质的Ⅳ型胶原的原位、同时共成像和定量检测,实现了量子点的双标多光谱成像。我们计算了EGFR/Ⅳ型胶原比值,并且研究了EGFR/Ⅳ型胶原比值在三阴性乳腺癌患者的独立预后价值。通过对三阴性乳腺癌组织EGFR和Ⅳ型胶原的同时荧光成像和定量检测,我们发现,EGFR和Ⅳ型胶原的表达呈显著负相关,差异具统计学意义。在高EGFR/Ⅳ型胶原比值组和低EGFR/Ⅳ型胶原比值组,两者的5年无病生存率有显著差别。EGFR/Ⅳ型胶原比值具有预后预测作用,并且是三阴性乳腺癌的独立预后指标。与单纯的EGFR表达比较,EGFR/Ⅳ型胶原比值更能反应三阴性乳腺癌患者的预后差异。我们的研究联合癌细胞和细胞外基质中的重要组分进行分析,为临床预测三阴性乳腺癌患者的预后和探索三阴性乳腺癌侵袭和转移机制的研究提供了新视野。
[Abstract]:Breast cancer is the most common malignant tumor in women. The incidence of breast cancer is increasing year by year, and it has become the most important malignant tumor leading to female death. However, the mechanism of invasion and metastasis of triple-negative breast cancer is still unclear, and there is no clinically recognized molecular targeted marker with predictive value of prognosis. A large number of scholars have explored its feasibility in cancer diagnosis, molecular typing, treatment and prognosis evaluation, and found that it has broad prospects in basic and clinical research of cancer, especially quantum dot-based immuno fluorescent imaging and quantitative analysis system. The development of system, QD-IIQAS, has made it widely used in the detection of molecular markers of malignant tumors. With the advantage of quantum dot nanotechnology, topoisomerase 2 alpha (TOP2A), epidermal growth factor receptor (EGFR) and collagen IV in triple-negative breast cancer tissues were detected. The feasibility of the assay was explored and compared with the traditional immunohistochemical assay (IHC). The molecular targets of chemosensitivity of triple-negative breast cancer were preliminarily found by the detection of three molecular markers, such as EGFR and swelling on the surface of cancer cells. Combined analysis of major components of tumor microenvironment (e.g. collagen type IV) to explore whether invasion and metastasis of triple-negative breast cancer are independent behaviors of cancer cells and whether the involvement of tumor microenvironment is necessary provides new ideas for the study of mechanisms of invasion and metastasis of malignant tumors. The ratio of collagen type IV to collagen type IV was used to explore the biological behavior of triple-negative breast cancer and provide theoretical basis and clinical inspiration for promoting the individual treatment of triple-negative breast cancer. The expression and value of TOP2A in sexual breast cancer tissues Chemotherapy is mainly anthracycline and purple shirt chemotherapeutics. At present, its therapeutic effect has reached a bottleneck, and new breakthroughs are particularly difficult. TOP2A is an important enzyme in DNA replication, and also a cytotoxic chemotherapeutics, such as anthracycline targeting molecules, has been widely studied in the field of breast cancer. The existence of these methods and the difference of different positive thresholds are inconsistent in predicting the efficacy and prognostic value of TOP2A in breast cancer patients. Our previous studies have confirmed the significant advantages of quantum dot nanotechnology in quantitative detection of biomarkers and compared it with traditional IHC. The advantages of sub-point labeling probe technique in breast cancer research, especially QD-IIQAS technique, were studied in situ fluorescence imaging and quantitative detection of TOP2A in triple-negative breast cancer tissues to study the relationship between the expression of TOP2A and the main clinicopathological parameters. The expression of TOP2A was detected and compared with the two methods. The relationship between the expression of TOP2A and the main clinicopathological parameters was also evaluated. QD-IIQAS is a simpler and more accurate method for the detection of TOP2A. Our study is to investigate the expression of TOP2A and the sensitivity of anthracycline drugs in the future. Part 2 Quantum dot-labeled double-color fluorescence imaging and quantitative detection and prognostic value of EGFR and collagen IV in triple-negative breast cancer tissue. Triple-negative breast cancer is a special subtype of breast cancer with obvious heterogeneity and poor prognosis. And metastasis is not entirely a spontaneous process of cancer cells, it requires cancer cells to penetrate the surrounding extracellular matrix barrier. In order to study the mechanism of invasion and metastasis of triple-negative breast cancer, a new detection method is developed, which can simultaneously study important components in the tumor microenvironment and important molecular markers on the surface of cancer cells. We used quantum dot multispectral fluorescence imaging to study triple-negative breast cancer tissues. Based on the first part of the single-label quantum dot immunofluorescence imaging and quantitative detection system, we developed a new computer-aided quantum dot multispectral fluorescence imaging and quantitative detection technology. We calculated the ratio of EGFR to collagen IV and studied the independent prognostic value of EGFR to collagen IV in triple-negative breast cancer patients. The expression of EGFR and collagen IV in breast cancer tissues was negatively correlated with the expression of collagen IV. The 5-year disease-free survival rate was significantly different between the high EGFR/IV collagen ratio group and the low EGFR/IV collagen ratio group. The ratio of EGFR to collagen type IV was more sensitive to the prognostic differences in patients with triple negative breast cancer than the expression of EGFR alone. To explore the mechanism of invasion and metastasis of three negative breast cancer provides a new perspective.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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