乳腺癌细胞及其外泌体的拉曼表型快速鉴定与关系研究

Rapid Identification of the Raman Phenotypes of Breast Cancer Cell Derived Exosomes and the Relationship With Maternal Cells

外泌体(Exosome)是直径大小为30~150 nm的膜性囊泡,包裹DNA/RNA, miRNA,蛋白质和脂质等多种物质并参与微环境中的生物信息传递,是理想的癌症生物标志物,在液体活检领域具有重要的应用潜力,有望成为癌症快速检测的手段之一。表面增强拉曼光谱(SERS)是分子振动光谱,可从分子水平上探测物质的精细结构和信息变化,具有“指纹图谱”的特征。采用差速离心结合超速离心的方法获得乳腺癌细胞来源的外泌体,以金溶胶为增强基底,收集外泌体及其母细胞的SERS图谱,结合多元统计分析,进行乳腺癌细胞的快速鉴别与区分。研究结果表明,乳腺癌细胞及其外泌体在500~1 600 cm^(-1)波段范围内有特征拉曼信号,采用非标记检测所获得的图谱信息是样品“whole-organism fingerprint”整体信号的呈现。根据外泌体的拉曼表型并结合OPLS-DA分析,能够100%分辨3种不同类型的乳腺癌细胞。单细胞SERS检测联合PCA-LDA分析,区分乳腺癌细胞的准确率为83.7%。通过比较乳腺癌细胞及其外泌体的拉曼特征图谱发现,二者在拉曼谱图的波数高度表现一致,但是外泌体在特征波数上显著增强。具体体现为在506~569、 1 010~1 070 cm^(-1)等波段二者存在相似性,但外泌体在735、 963和1 318 cm^(-1)等处的特征信号显著高于细胞。分析认为外泌体结构比细胞更为简单,核酸、蛋白质等生物大分子信息更容易被表征。同时也提示了通过SERS检测外泌体实现快速鉴定乳腺癌的可行性。采用非标记、直接检测,建立了快速检测单细胞及外泌体的SERS分析技术,结合多元统计分析能够快速鉴别不同类型的乳腺癌细胞,并从拉曼组学角度探究了外泌体与母源细胞的关系。该方法具有非标记、快速、灵敏、准确、简便的优势,为乳腺癌的体外快速诊断与筛查提供有效的技术手段,为临床应用奠定基础。

Exosomes are nano-sized phospholipid bilayer-enclosed vesicles secreted by all cells into the extracellular milieu.Released exosomes contain cell-specific proteins,membrane lipids,mRNA,DNA and microRNA that can perform versatile roles in normal or diseased processes.Exosomes are ideal biomarkers of cancer,which have important application potential in liquid biopsy and are expected to become one of the means for rapid cancer detection.Surface enhanced Raman spectroscopy(SERS)is a molecular vibration spectrum,which can detect the fine structure and information changes of substances at the molecular level,and has the characteristics of a"fingerprint spectrum".In this study,the exosomes were isolated via differential centrifugation combined with ultracentrifugation of the supernatants of breast cells.The SERS profiles of breast cancer cells and their exosomes were collected with colloidal Au nanoparticles as the enhanced substrate,and multivariate statistical analysis was used to identify and distinguish breast cancer cells.The results showed that breast cancer cells and their exosomes had characteristic Raman signals in the range of 500~1600 cm^(-1).Their Raman phenotypes obtained by non-labeled detection were the presentation of all the signals of the"whole-organism fingerprint"of the sample.The accuracy rate reached 100%by using exosome-SERS detection and OPLS-DA analysis.Single-cellular SERS detection combined with PCA-LDA analysis showed that the accuracy of differentiating breast cancer cells was 83.7%.Breast cancer cells and their exosomes showed similarity at the bands of 506~569 and 1010~1070 cm^(-1),but the characteristic Raman peaks of exosomes at 735,963 and 1318 cm^(-1) were significantly higher than those of cells.It may be because the structure of exosomes is simpler than that of cells,and the information of biological macromolecules such as nucleic acids and proteins can be characterized more easily,indicating the feasibility of rapid identification of breast cancer by detecting exosomes by SERS technology.In summary,this study established a non-labeling and direct detection method for rapidly detecting single cells and their exosomes by SERS analysis.Combined with multivariate statistical analysis,different types of breast cancer cells could be quickly identified,and the relationship between exosomes and maternal cells was explored from the perspective of Raman omics.This method has the advantages of non-labeling,rapidness,sensitivity,accuracy and simplicity,which provides an effective technical means for rapid diagnosis and screening of breast cancer in vitro,and lays a foundation for clinical application.