乳腺癌切缘判断新方法探索：拉曼光谱与荧光纳米材料

发布时间：2018-03-12 18:11

本文选题：乳腺癌　切入点：拉曼光谱　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：自从NSABP B-06研究证实了保乳术与改良根治术具有相似的长期生存,乳腺癌保乳手术开始广泛应用于临床,现在美国保乳手术比例接近60%,在国内保乳手术也逐年增加,目前在三甲医院也接近20%。而保乳手术最重要的是确保完整切除病灶,即保证手术切缘阴性,没有病灶残留,并尽可能保证剩余乳房的美观。目前尚没有一种快速、准确的理想的诊断方法。拉曼光谱是一种简便、快速、灵敏的光谱技术,可直接对组织进行检测,而无需任何前处理,具备术中即时快速检测的可能,是一种极其具有临床应用价值的技术。然而对于不进行处理的新鲜组织而言,组织本身的不均一性和检测位置的盲目性导致了检测结果差异较大,即使应用数学方法仍难以对结果进行有效分析及构建诊断模型。本研究应用拉曼光谱的mapping技术,通过微小面积的逐点扫描检测,排除组织自身特点的干扰,提高诊断准确率;并应用不同成像技术进行检测位置的成像,为直观定位阳性病灶位置提供研究基础。此外,荧光显微成像技术也是一类重要的乳腺癌早期诊断方法,荧光标记材料和它的理化特征在成像质量中起重要作用。但是具有AIE特性的荧光材料非常有限,荧光纳米材料的制备和性能也需要深入的优化,同时它对于肿瘤细胞的识别和靶向能力也需要改进。本研究中制备了具有高固态荧光量子效率的红色荧光的聚合物纳米粒子DPBA@PEG-PDLLA,并在乳腺癌MCF-7细胞中进行了荧光成像实验,以期为将来切缘成像奠定研究基础。本论文的主要研究内容如下:(1)乳腺正常组织及癌组织的mapping特征分析及成像采集乳腺癌及正常乳腺组织,冰冻固定,连续切片,一张用于染色诊断,一张用于拉曼光谱检测。应用共聚焦拉曼光谱仪对目标区域20μm×20μm范围进行逐点扫描,共获得mapping数据53组,2597条拉曼光谱数据;单点检测共获得1280条拉曼光谱数据。首先对mapping数据及单点测量数据求平均光谱,并进行特征峰归属及分析。发现脂类特征峰在正常乳腺组织中主要出现在1125、1307、1245、1445、1735cm-1波数位置,而在乳腺癌组织中则仅出现在1124、1302、1446cm-1波数位置,癌组织中脂类少于正常组织。正常乳腺组织中的核酸主要特征峰在1157cm-1,恶性组织中核酸的主要特征峰在967、1156cm-1处,而在879、1582cm-1处有强度相对较弱但是是恶性组织特有的核酸特征峰。在恶性组织的拉曼光谱中可以看到核物质所表现出的特征峰明显多于正常乳腺组织,这个结果显示出癌组织中核物质含量丰富,增殖旺盛。之后对mapping与单点数据稳健性对比分析发现,恶性组织Mapping离群数据占全部训练集数据的12.7%,而正常乳腺组织数为6.6%。常规单点检测恶性组离群数据占全部数据的24.5%;正常乳腺组织为26.0%。恶性数据mapping组与单点检测组离群值数量有显著差异(t=-6.169,P0.001);正常乳腺组织也有显著差异(t=-8.873,P0.001)。证实了逐点扫描所获得的数据具有更好的稳定性,更有利于诊断模型的构建。而基于mapping数据,采用KNN法构建的诊断模型,乳腺癌预测的准确率为99.56%,敏感性为96.6%,特异性98.48%,阳性预测值99.56%,阴性预测值89.04%。最后应用模型判断方法和特征峰强度方法分别对扫描区域进行了成像。图像显示根据模型判断后的结果进行成像与组织有很好的对应,可能成为切缘实时检测的良好方法;选择癌组织特有的特征峰位,根据强度的成像,在1088、1215与1582 cm-1波数位置具有很高的相似度,与936cm-1也有一定的相似度,显示了癌组织中高蛋白表达可能与细胞分裂、转移和细胞增殖有关,同时这种成像方式可能为进一步的分子诊断提供基础。(2)荧光纳米粒子的制备及其在乳腺癌MCF-7细胞系的成像利用两亲性聚合物聚乙二醇-聚乳酸(PEG-PDLLA)包覆具有聚集诱导发光特性的红光分子DBPA,制备了具有高效红光发射的荧光纳米粒子DPBA@PEG-PDLLA,其发光峰位在654 nm。通过调控不同的负载浓度,荧光纳米粒子的荧光量子效率可达26%。这类荧光纳米粒子具有很好的生物相容性和小的细胞毒性,并且这些纳米粒子具有均一的尺寸,其粒径为88 nm。这类纳米粒子作为荧光探针可以被乳腺癌细胞MCF-7通过内吞作用进入细胞质内,发出明亮的红光,表明了具有非常优异的荧光性能和显微成像潜力,为实现乳腺癌肿瘤组织的切缘检测奠定了良好的基础。此外,我们还利用共包覆的手段将药物紫杉醇负载在荧光纳米粒子,制备了多功能纳米粒子,实现了高效荧光发射和药物释放功能。因此,这类纳米粒子将可能在乳腺癌诊断中表现出了良好的应用前景。
[Abstract]:Since the NSABP B-06 study confirmed the long-term survival similar radical resection of breast conserving surgery and modified, is widely used in clinical breast conserving surgery, breast conserving surgery in America now account for nearly 60% of breast conserving surgery in China also increased year by year, is currently in the hospital is close to 20%. and breast conserving surgery is most important to ensure that complete excision of the lesion, which is to ensure a negative surgical margin, no residual lesions, and as far as possible to ensure that the residual breast appearance. There is not a quick and accurate diagnosis ideal. Raman spectroscopy is a simple, rapid and sensitive spectroscopy, can directly detect the organization, without any prior treatment may have immediate intraoperative rapid detection, is an extremely valuable technology. But for the fresh tissue is not treated, the organization itself heterogeneity and position of the blind detection The large difference between the results, even if the application of mathematical method is still difficult for the effective analysis of the results of this study and establish the diagnostic model. The application of mapping technology by Raman spectroscopy, scanning point detection of small area, to eliminate the interference of organization characteristics and improve the diagnosis accuracy; the imaging and detection position using different imaging techniques the study will provide the basis for visual positioning positive lesions location. In addition, fluorescence microscopic imaging technique is an important method for early diagnosis of breast cancer, fluorescent material and its physicochemical characteristics play an important role in image quality. But the fluorescent material with the characteristics of AIE are very limited, synthesis and properties of fluorescent nanomaterials the system also needs further optimization at the same time, it can identify and target tumor cells to ability need to be improved. This study was prepared with high solid fluorescence quantum efficiency The red fluorescence rate of polymer DPBA@PEG-PDLLA nanoparticles, and the fluorescence imaging experiments were carried out in breast cancer MCF-7 cells, in order to lay a foundation for the research on future margin imaging. The main research contents of this thesis are as follows: (1) normal breast tissue and cancer tissue mapping characteristic analysis and imaging of breast cancer and normal breast tissue frozen, fixed, continuous sections, one for dyeing diagnosis, one for Raman detection. Application of confocal Raman spectrometer on the target area of 20 m * 20 m range by point by point scanning, mapping received a total of 53 data groups, 2597 Raman spectrum data; single point detection were obtained from 1280 Raman spectrum data. The data of mapping and single point measurement data for the average spectrum and peaks and characteristic analysis. Found that lipid characteristic peaks occurred in 11251307124514451735cm-1 wave in normal breast tissues. The number of positions, and in breast cancer only appeared in 112413021446cm-1 wave position, less than normal tissue lipids in cancer tissue. Normal breast tissue nucleic acids in main characteristic peaks in 1157cm-1, nucleic acid and malignant lesions of the main characteristic peak at 9671156cm-1, and at 8791582cm-1 with the strength of the relatively weak but the characteristic peaks of the malignant tissue is a nucleic acid specific. Raman spectroscopy in malignant tissue can be seen in the characteristic peaks of nuclear matter showed significantly more than the normal breast tissue, the results showed that the cancer tissue rich nuclear matter content of proliferation. In the mapping and single point data comparative analysis of robustness, malignant tissue Mapping outlier data of total training set data 12.7%, while the number of normal breast tissue 6.6%. conventional single point detection of malignant group accounted for 24.5% of the outlier data; normal breast tissue for malignant 26.0%. The data of mapping group and single point detection of outliers group had significant difference (t=-6.169, P0.001); the number of normal breast tissue were significantly different (t=-8.873, P0.001). Confirmed the scanning point data obtained has better stability, more conducive to establishing a diagnosis model. Based on the mapping data, the diagnosis model constructed by KNN the accuracy of prediction of breast cancer was 99.56%, the sensitivity was 96.6%, specificity 98.48%, positive predictive value and negative predictive value of 99.56%, 89.04%. finally application model judgment method and the characteristic peak intensity method respectively on the scanning area of the image. The image display model according to the judgment of the results of the imaging correspond well with the the organization, may be a good method of cutting edge detection; feature selection for cancer specific peak, according to the strength of the imaging, has high similarity in the 10881215 and 1582 cm-1 wave position, and 9 36cm-1 also has a certain degree of similarity, and shows the possible cell division protein expression in cancer tissue, metastasis and cell proliferation, and this imaging method may provide a basis for further molecular diagnosis. (2) fluorescent nanoparticles preparation and imaging in breast cancer MCF-7 cell lines using two amphipathic polymer polyethylene glycol polylactic acid (PEG-PDLLA) coated with molecular aggregation induced by DBPA red luminescence characteristics, preparation of a highly efficient red light emission of DPBA@PEG-PDLLA fluorescent nanoparticles, the emission peak at 654 nm. load through the regulation of different concentration, fluorescence quantum efficiency of 26%. fluorescent nanoparticles this kind of fluorescent nanoparticles with biocompatibility and cell toxicity is very small good biological, and these nanoparticles have uniform size, the particle size of 88 nm. this kind of nanoparticles as fluorescent probes can be breast cancer cell M CF-7 by endocytosis into the cytoplasm, bright red, showed excellent fluorescence properties and microscopic imaging potential, laid a good foundation for the detection of tumor margin of breast cancer. In addition, we also use the co coated means drug paclitaxel loaded in fluorescent nanoparticles, multifunctional nanoparticles prepared to achieve an efficient fluorescence emission and drug release function. Therefore, this kind of nanoparticles may in the diagnosis of breast cancer showed a good application prospect.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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