技术领域technical field
本发明属于生物医药技术领域，具体涉及一种乳腺癌的生物标志物及其应用。The invention belongs to the technical field of biomedicine, and in particular relates to a biomarker of breast cancer and its application.
背景技术Background technique
神经节苷脂(Gangliosides，GAs)是一类酸性鞘糖脂，主要由带N-乙酰神经氨酸的糖链和神经酰胺链接而成，在生物体内，神经节苷脂通常通过疏水性的神经酰胺，插于细胞膜脂质双层中，作为膜组分的一部分，亲水的寡糖链伸展于细胞表面的外环境中。神经节苷脂不仅是细胞表面抗原的重要组成部分，而且是许多胞外生理活性物质的受体，并参与细胞识别和信息传递过程。神经节苷脂在体内的生物学功能主要有以下几个方面：(1)作为细胞表面标记及抗原，具有免疫学功能；(2)介导细胞间的相互识别，并可作为细胞识别受体；(3)诱导神经细胞分化抑制其生长，维持神经细胞的功能和形态；(4)调节细胞跨膜信号的传导等；(5)稳定细胞膜双分子层结构。神经节苷脂能促进神经系统的发育和并能修复保护损伤神经，因此引起人们极大关注。研究表明单唾液酸神经节昔脂GM1治疗帕金森病有良好疗效。Gangliosides (Gangliosides, GAs) are a class of acidic glycosphingolipids, which are mainly composed of sugar chains with N-acetylneuraminic acid and ceramide links. In organisms, gangliosides usually pass through hydrophobic neurons. Amides, inserted in the lipid bilayer of the cell membrane, as part of the membrane components, the hydrophilic oligosaccharide chains are extended in the external environment of the cell surface. Gangliosides are not only an important part of cell surface antigens, but also receptors for many extracellular physiologically active substances, and participate in the process of cell recognition and information transmission. The biological functions of gangliosides in the body mainly include the following aspects: (1) As a cell surface marker and antigen, it has immunological functions; (2) It mediates mutual recognition between cells and can be used as a cell recognition receptor ; (3) Induce nerve cell differentiation and inhibit its growth, maintain the function and shape of nerve cells; (4) Regulate cell transmembrane signal conduction, etc.; (5) Stabilize the cell membrane bilayer structure. Gangliosides can promote the development of the nervous system and can repair and protect damaged nerves, so it has attracted great attention. Studies have shown that monosialoganglioside GM1 has good curative effect in the treatment of Parkinson's disease.
在肿瘤的发生发展过程中，神经节苷脂的表达与结构会发生变化，其可能的机理包括：(1)细胞表面的神经节苷脂抗原或从细胞脱落的神经节苷脂抗原作为免疫抑制剂，如通常观察到抑制细胞毒性T细胞和树突细胞；(2)神经节苷脂如GD3或GM2促进肿瘤相关的血管生成；(3)神经节苷脂强烈调节细胞粘附/运动并由此启动肿瘤转移；(4)神经节苷脂抗原直接与微区中的转导分子连接以引发粘着；(5)神经节苷脂抗原及其分解代谢物通过与生长因子受体或其他蛋白激酶相关的酪氨酸激酶相互作用而进行信号转导。双唾液酸神经节苷脂GD2是在各种癌症中高表达的神经外胚层抗原，其对于三阴性乳腺癌具有较好的诊断作用。GM3在各种疾病中的重要作用已被广泛报道。在先天性帕金森病人血浆中发现了GM3浓度显著升高。早期复发-缓解型多发性硬化症病人血清中观测到GM3显着降低，可作为血脑屏障破坏的生物标志物。此外，GM3在黑色素瘤转移晚期表现出较高水平。GM3合酶基因是非小细胞肺癌组织学分级的新型生物标志物。中国专利(公开号：CN 104698059 B)公开了一种双唾液酸化的神经节苷脂作为脑胶质瘤肿瘤标志物及其应用；中国专利(公开号：CN105407916 A)公开了由生长因子受体HER-1和HER-2的胞外结构域或其片段、衍生自脑膜炎奈瑟氏球菌(Neisseria meningitidis)的外膜蛋白质的脂蛋白体及神经节苷脂GM3(vSSP-GM3)构成的二价疫苗组合物可用于恶性肿瘤的治疗；中国专利(公开号：CN 103998098 B)公开了一种用于治疗高表达EGFR和N-羟乙酰神经节苷脂(NeuGcGM3)的肿瘤的药物组合物；国际专利(WO2002/081661)公开了一种用于治疗过度表达神经节苷脂的肿瘤的免疫治疗组合物。During the development of tumors, the expression and structure of gangliosides will change, and the possible mechanisms include: (1) Ganglioside antigens on the cell surface or ganglioside antigens shed from cells act as immunosuppressive agents (2) gangliosides such as GD3 or GM2 promote tumor-associated angiogenesis; (3) gangliosides strongly regulate cell adhesion/motility and are driven by This initiates tumor metastasis; (4) ganglioside antigens directly connect to transduction molecules in microdomains to initiate adhesion; (5) ganglioside antigens and their catabolites interact with growth factor receptors or other protein kinases Associated tyrosine kinases interact for signal transduction. Disialoganglioside GD2 is a highly expressed neuroectodermal antigen in various cancers, and it has a good diagnostic effect on triple-negative breast cancer. The important role of GM3 in various diseases has been widely reported. Significantly elevated GM3 concentrations were found in the plasma of congenital Parkinson's patients. A significant decrease in GM3 was observed in the serum of patients with early relapsing-remitting multiple sclerosis, which can be used as a biomarker of blood-brain barrier disruption. In addition, GM3 showed higher levels in the advanced stage of melanoma metastasis. GM3 synthase gene is a novel biomarker for histological grade of non-small cell lung cancer. Chinese patent (publication number: CN 104698059 B) discloses a disialylated ganglioside as a glioma tumor marker and its application; Chinese patent (publication number: CN105407916 A) discloses a growth factor receptor The extracellular domains of HER-1 and HER-2 or fragments thereof, liposomes derived from the outer membrane protein of Neisseria meningitidis and ganglioside GM3 (vSSP-GM3) consist of two Vaccine compositions can be used for the treatment of malignant tumors; Chinese patent (publication number: CN 103998098 B) discloses a pharmaceutical composition for treating tumors that highly express EGFR and N-glycolyl ganglioside (NeuGcGM3); International patent (WO2002/081661) discloses an immunotherapeutic composition for the treatment of tumors overexpressing gangliosides.
乳腺癌是全球女性中最为常见的恶性肿瘤之一，根据最新统计数据，中国女性乳腺癌发病率与死亡率分别居于女性恶性肿瘤的第1位和第5位。根据临床免疫组化的结果，可以将乳腺癌分为四种分子分型：Luminal A(LA)型、Luminal B(LB)型、HER-2过表达(HER)型和基底样(BS)型乳腺癌。分子分型与乳腺癌的临床病理特征、疾病转归、患者预后和治疗反应密切相关。目前并未有专利或文献报道神经节苷脂作为乳腺癌血清标志物，并且尚未有关于不同分子分型癌症的区分。Breast cancer is one of the most common malignant tumors among women in the world. According to the latest statistics, the incidence and mortality of breast cancer in Chinese women rank first and fifth among female malignant tumors, respectively. According to the results of clinical immunohistochemistry, breast cancer can be divided into four molecular types: Luminal A (LA), Luminal B (LB), HER-2 overexpression (HER) and basal-like (BS) breast cancer. Molecular typing is closely related to clinicopathological features, disease outcome, patient prognosis and treatment response of breast cancer. At present, there are no patents or literature reports on gangliosides as serum markers of breast cancer, and there is no distinction between different molecular types of cancer.
发明内容Contents of the invention
针对目前技术对乳腺癌血清中神经节苷脂标志物及不同分子分型乳腺癌标志物研究的空白，本发明提供了一种乳腺癌的生物标志物及其应用。Aiming at the vacancy in current technology research on ganglioside markers in serum of breast cancer and markers of different molecular types of breast cancer, the present invention provides a biomarker of breast cancer and its application.
为实现上述发明目的，本发明采用以下技术方案予以实现：In order to achieve the above-mentioned purpose of the invention, the present invention adopts the following technical solutions to achieve:
本发明提供了一种乳腺癌筛查试剂盒，所述试剂盒包括任选的用于检测经节苷脂GM3表达水平的试剂。The present invention provides a breast cancer screening kit, which includes optional reagents for detecting the expression level of ganglioside GM3.
进一步的，所述检测神经节苷脂GM3表达水平的试剂为LC-MS/MS-MRM分析方法用试剂。Further, the reagent for detecting the expression level of ganglioside GM3 is a reagent for LC-MS/MS-MRM analysis method.
进一步的，所述分析方法采用的流动相为：Further, the mobile phase that described analysis method adopts is:
流动相A：10mM醋酸铵，含0.1％醋酸；Mobile phase A: 10mM ammonium acetate, containing 0.1% acetic acid;
流动相B：85％甲醇/15％异丙醇，含10mM醋酸铵、0.1％醋酸。Mobile phase B: 85% methanol/15% isopropanol, containing 10 mM ammonium acetate, 0.1% acetic acid.
进一步的，所述分析方法的洗脱程序为：0～3min保持30％流动相B，1min内升至100％流动相B，然后以100％流动相B保持10-15min。Further, the elution procedure of the analysis method is: maintain 30% mobile phase B for 0-3 minutes, increase to 100% mobile phase B within 1 min, and then maintain 10-15 min with 100% mobile phase B.
本发明还提供了用于检测生物标志物的表达水平的用品在制备用于指示对象中的乳腺癌的试剂中的应用，所述生物标志物为神经节苷脂GM3。The present invention also provides the application of the product for detecting the expression level of a biomarker in the preparation of a reagent for indicating breast cancer in a subject, and the biomarker is ganglioside GM3.
进一步的，所述试剂是用于检测神经节苷脂GM3的表达水平的试剂。Further, the reagent is a reagent for detecting the expression level of ganglioside GM3.
进一步的，所述试剂是用于检测所述对象血清中神经节苷脂GM3表达水平的试剂。Further, the reagent is a reagent for detecting the expression level of ganglioside GM3 in the serum of the subject.
进一步的，所述检测神经节苷脂GM3表达水平的试剂为LC-MS/MS-MRM分析方法用试剂，具体步骤为：Further, the reagent for detecting the expression level of ganglioside GM3 is a reagent for LC-MS/MS-MRM analysis method, and the specific steps are:
检测所述对象中神经节苷脂GM3的浓度；detecting the concentration of ganglioside GM3 in the subject;
将所述对象中神经节苷脂GM3的浓度与神经节苷脂GM3的参比浓度相比较，得到神经节苷脂GM3的表达水平。The expression level of ganglioside GM3 is obtained by comparing the concentration of ganglioside GM3 in the subject to a reference concentration of ganglioside GM3.
进一步的，所述分析方法采用的流动相为：Further, the mobile phase that described analysis method adopts is:
流动相A：10mM醋酸铵，含0.1％醋酸；Mobile phase A: 10mM ammonium acetate, containing 0.1% acetic acid;
流动相B：85％甲醇/15％异丙醇，含10mM醋酸铵、0.1％醋酸。Mobile phase B: 85% methanol/15% isopropanol, containing 10 mM ammonium acetate, 0.1% acetic acid.
进一步的，洗脱程序为：0～3min保持30％流动相B，1min内升至100％流动相B，然后以100％流动相B保持10-15min。Further, the elution procedure is: maintain 30% mobile phase B for 0-3 minutes, increase to 100% mobile phase B within 1 minute, and then maintain 10-15 minutes with 100% mobile phase B.
所述神经节苷脂GM3的表达水平大于1.5时，指示所述对象为乳腺癌。When the expression level of the ganglioside GM3 is greater than 1.5, it indicates that the subject has breast cancer.
所述神经节苷脂GM3的表达水平大于2.5时，指示所述对象的乳腺癌为Luminal-B型乳腺癌。When the expression level of ganglioside GM3 is greater than 2.5, it indicates that the subject's breast cancer is Luminal-B breast cancer.
所述神经节苷脂GM3的表达水平在1.3～1.5之间时，指示所述对象为良性肿瘤。When the expression level of the ganglioside GM3 is between 1.3 and 1.5, it indicates that the subject is a benign tumor.
与现有技术相比，本发明的优点和积极效果是：本发明提供的试剂盒操作步骤简单易学，容易普及，检测耗时短，检测成本低等特点。本发明提供的试剂盒样本用量少，血清血浆用量20～200μL，同时适用于组织(5-100mg)和细胞(105-107)的检测。本发明提供的试剂盒灵敏度高，检测限范围为0.15～0.52pg/μL，定量限范围为0.50～1.72pg/μL，且可以实现54种神经节苷脂的同时在线分离。本发明提供的试剂盒不仅可以区分正常人和乳腺癌病人，纤维限瘤病人及癌症病人，还可区分不同分子分型乳腺癌，用该方法有望找到血液癌症生物标志物。Compared with the prior art, the advantages and positive effects of the present invention are: the operation steps of the kit provided by the present invention are simple and easy to learn, easy to popularize, the detection time is short, and the detection cost is low. The kit provided by the invention has less sample consumption, 20-200 μL of serum and plasma, and is suitable for the detection of tissues (5-100 mg) and cells (105 -107 ). The kit provided by the invention has high sensitivity, the detection limit range is 0.15-0.52 pg/μL, the quantification limit range is 0.50-1.72 pg/μL, and simultaneous online separation of 54 gangliosides can be realized. The kit provided by the invention can not only distinguish between normal people and breast cancer patients, fibrous tumor patients and cancer patients, but also distinguish different molecular types of breast cancer, and the method is expected to find blood cancer biomarkers.
结合附图阅读本发明的具体实施方式后，本发明的其它优点和特点将变得更加清晰。Other advantages and features of the present invention will become clearer after reading the specific implementation manner of the present invention in conjunction with the accompanying drawings.
附图说明Description of drawings
图1为本发明所述基于RP-HPLC-FTMS方法的正常人与乳腺癌患者血清GAs差异。Figure 1 is the difference of serum GAs between normal people and breast cancer patients based on the RP-HPLC-FTMS method of the present invention.
图2为本发明所述正常人、乳腺良性瘤病人与乳腺癌病人血清中不同类型神经节苷脂的含量差异柱形图。Fig. 2 is a bar graph showing the content difference of different types of gangliosides in the serum of normal people, breast benign tumor patients and breast cancer patients according to the present invention.
图3为本发明所述健康人与乳腺癌患者血清中GM3系列的PCA分析图。Fig. 3 is a PCA analysis chart of GM3 series in the serum of healthy people and breast cancer patients according to the present invention.
图4为本发明所述乳腺癌患者相较于健康人血清中有极高诊断价值的GAs(AUC＞0.9)。Fig. 4 shows the GAs (AUC > 0.9) with extremely high diagnostic value in serum of breast cancer patients compared with healthy people according to the present invention.
图5为本发明所述不同分子分型乳腺癌病人血清GM3含量差异柱形图。Fig. 5 is a histogram showing the difference in serum GM3 content of breast cancer patients with different molecular types according to the present invention.
图6为本发明所述不同分子分型乳腺癌病人血清GAs的ROC曲线分析图。Fig. 6 is a graph of ROC curve analysis of serum GAs of breast cancer patients with different molecular types according to the present invention.
具体实施方式Detailed ways
下面结合附图和具体实施方式对本发明的技术方案进一步的详细说明，但本发明要求保护的范围并不局限于实例表述的范围。The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the scope of protection claimed by the present invention is not limited to the scope expressed by the examples.
实施例1：检测血清中神经节苷脂Example 1: Detection of gangliosides in serum
1、血清中神经节苷脂的提取方法1. Extraction method of gangliosides in serum
向20-200uL血清中加入1mL甲醇、2mL氯仿后，超声浸提20min，8000rmp离心10min，上清液用于糖脂提取。向上清液中加入1mL水，涡旋，室温静置10min，使其分层，8000rmp离心10min，上层转出。下层继续加入1mL甲醇、0.5mL水重复萃取两次，8000rmp离心10min，合并上层，即为神经节苷脂。将上层用氮气吹至干，复溶于80％甲醇中，用于LC-MS分析。Add 1mL of methanol and 2mL of chloroform to 20-200uL serum, ultrasonically extract for 20min, centrifuge at 8000rmp for 10min, and the supernatant is used for glycolipid extraction. Add 1mL of water to the supernatant, vortex, let stand at room temperature for 10min to make layers, centrifuge at 8000rmp for 10min, and transfer the upper layer out. Continue adding 1mL methanol and 0.5mL water to the lower layer to repeat the extraction twice, centrifuge at 8000rmp for 10min, and combine the upper layer to obtain gangliosides. The upper layer was blown to dryness with nitrogen, redissolved in 80% methanol, and used for LC-MS analysis.
2、通过LC-FTMS非靶向分析方法确定血清GA种类2. Determination of serum GA species by LC-FTMS non-targeted analysis method
质谱分析仪：Agilent 1290 LC UPLC液相色谱仪，LTQ ORBITRAP XL质谱仪，配有电喷雾离子源(ESI)；Mass spectrometer: Agilent 1290 LC UPLC liquid chromatograph, LTQ ORBITRAP XL mass spectrometer, equipped with electrospray ionization source (ESI);
液相色谱系统：色谱柱为Agilent Poroshell 120 EC-C18色谱柱(50 x 3.0mm，2.7μm)，色谱洗脱采用二元泵高压混合的梯度洗脱方式。流动相A为10mM醋酸铵(含0.1％醋酸)，流动相B为85％甲醇/15％异丙醇(含10mM醋酸铵，0.1％醋酸)。流速为0.2mL/min。梯度洗脱程序：0～3min，30％流动相B；3～3.1min，30～100％流动相B；3.1～14min，100％流动相B；14～14.1min，100～30％流动相B；14.1～17min，30％流动相B。检测器为负离子模式，喷雾电压4.2kV，毛细管温度275℃，氮气流速40L/min。Liquid chromatography system: the chromatographic column is Agilent Poroshell 120 EC-C18 chromatographic column (50 x 3.0mm, 2.7μm), and the chromatographic elution adopts the gradient elution method of binary pump high-pressure mixing. Mobile phase A was 10 mM ammonium acetate (containing 0.1% acetic acid), and mobile phase B was 85% methanol/15% isopropanol (containing 10 mM ammonium acetate, 0.1% acetic acid). The flow rate was 0.2 mL/min. Gradient elution program: 0～3min, 30% mobile phase B; 3～3.1min, 30～100% mobile phase B; 3.1～14min, 100% mobile phase B; 14～14.1min, 100～30% mobile phase B ; 14.1 ~ 17min, 30% mobile phase B. The detector is in negative ion mode, the spray voltage is 4.2kV, the capillary temperature is 275°C, and the nitrogen flow rate is 40L/min.
在本发明中，作为非靶向方法，首先采用LC-FTMS方法鉴定血清中所有的GA种类。在负离子模式下，通过RP-HPLC-FTMS方法分析了20名乳腺癌患者和10名健康志愿者的血清。根据准确的m/z，保留时间，同类GA的相对保留时间以及二级质谱解析，最终确定了49种GA种类。表1中列出了FTMS所鉴定到的所有GA种类、保留时间及其m/z。其中作为含量最为丰富的神经节苷脂，GM3共占有23种，并在癌症病人中显著增加，具有进一步研究的价值。如图1所示，提取的GAs的峰面积显示乳腺癌患者和健康对照之间的显着差异(p＜0.05)。但是，复杂的数据处理限制了该方法的应用。为了简化数据处理并实现定量分析，我们转向了一种有针对性的LC-MS/MS-MRM方法。In the present invention, as a non-targeting method, LC-FTMS method was first used to identify all GA species in serum. Sera from 20 breast cancer patients and 10 healthy volunteers were analyzed by RP-HPLC-FTMS method in negative ion mode. According to the accurate m/z, retention time, relative retention time of similar GAs and analysis of MS/MS, 49 kinds of GAs were finally determined. Table 1 lists all GA species identified by FTMS, their retention times and their m/z. Among them, as the most abundant ganglioside, GM3 has a total of 23 kinds, and it increases significantly in cancer patients, which has the value of further research. As shown in Figure 1, the peak areas of the extracted GAs showed significant differences (p<0.05) between breast cancer patients and healthy controls. However, complex data processing limits the application of this method. To simplify data processing and enable quantitative analysis, we turned to a targeted LC-MS/MS-MRM approach.
3：神经节苷脂LC-MS/MS-MRM分析方法的建立3: Establishment of LC-MS/MS-MRM analysis method for gangliosides
(1)质谱方法的建立(1) Establishment of mass spectrometry method
6种神经节苷脂标准品，经80％甲醇水溶液稀释至1ng/μL后，在50％甲醇流动相中，通过分别流动注射单一标准品，进行优化响应。检测器为负离子模式，喷雾电压2.8-3kV，离子传输管温度300-350℃，极化温度250-300℃，鞘气25-35Arb，辅助气2-8Arb。确定合适的子离子及最佳的Collisional energy和Tube lens。对于糖链相同而脂肪链长度不同的神经节苷脂，其优化参数相同。根据前期预实验结果可知血清中存在以下49种神经节苷脂。因此，得到MRM优化结果如表1所示。Six ganglioside standards were diluted to 1 ng/μL with 80% methanol in water, and the response was optimized by flow injection of a single standard in 50% methanol mobile phase. The detector is in negative ion mode, the spray voltage is 2.8-3kV, the ion transfer tube temperature is 300-350°C, the polarization temperature is 250-300°C, the sheath gas is 25-35Arb, and the auxiliary gas is 2-8Arb. Determine the appropriate product ion and the best Collisional energy and Tube lens. For gangliosides with the same sugar chain but different aliphatic chain lengths, the optimization parameters are the same. According to the preliminary experimental results, the following 49 gangliosides exist in serum. Therefore, the MRM optimization results are shown in Table 1.
表1糖脂标准品的质谱条件优化参数Table 1. Mass Spectrometry Condition Optimization Parameters of Glycolipid Standards
(2)色谱条件的建立(2) Establishment of chromatographic conditions
质谱分析仪：TSQ Quantiva质谱仪，配有电喷雾离子源(ESI)；Mass spectrometer: TSQ Quantiva mass spectrometer, equipped with electrospray ionization source (ESI);
检测所用液相：液相色谱系统；色谱柱为Agilent Poroshell 120 EC-C18色谱柱(50 x 3.0mm，2.7μm)，色谱洗脱采用二元泵高压混合的梯度洗脱方式。流动相A为10mM醋酸铵(含0.1％醋酸)，流动相B为85％甲醇/15％异丙醇(含10mM醋酸铵，0.1％醋酸)。流速为0.1-0.2mL/min。扫描方式为选择反应检测(SRM)。在优化好的质谱条件下，通过改变流动相比例、梯度、流速等，对色谱分离条件进行优化。最终确定如下洗脱程序：0～3min保持30％流动相B，1min内升至100％流动相B，然后以100％流动相B保持10-15min。基于MRM和保留时间，实现了54种神经节苷脂的全分离。The liquid phase used for detection: liquid chromatography system; the chromatographic column is Agilent Poroshell 120 EC-C18 chromatographic column (50 x 3.0mm, 2.7μm), and the chromatographic elution adopts the gradient elution method of binary pump high-pressure mixing. Mobile phase A was 10 mM ammonium acetate (containing 0.1% acetic acid), and mobile phase B was 85% methanol/15% isopropanol (containing 10 mM ammonium acetate, 0.1% acetic acid). The flow rate is 0.1-0.2 mL/min. The scanning method was selected reaction monitoring (SRM). Under the optimized mass spectrometry conditions, the chromatographic separation conditions were optimized by changing the mobile phase ratio, gradient, flow rate, etc. Finally, the following elution procedure was determined: keep 30% mobile phase B for 0-3 minutes, rise to 100% mobile phase B within 1 minute, and then keep 10-15 minutes with 100% mobile phase B. Based on MRM and retention time, a complete separation of 54 gangliosides was achieved.
(3)方法学评价(3) Methodological evaluation
6种标准品分别以GM1-36∶1、GM2-36∶1、GM3-41∶1、GD1-38∶1、GD3-41∶1、GT1b-38∶1为定量成分。将6种标准品混合后，用80％甲醇溶液稀释至系列不同浓度：5pg/μL，10pg/μL，50pg/μL，100pg/μL，500pg/μL，1ng/μL，5ng/μL，10ng/μL，20ng/μL，然后用于LC-MS/MS分析，进样量为5μL。基于标准二糖的进样量和多反应监测MRM的峰面积绘制标准曲线。信号强度(S)和背景噪声(N)的比值为信噪比(S/N)，不同浓度标准品分析数据可通过Xcalibur2.0.7计算S/N，将S/N为3时的浓度为检测限(LOD)，S/N为10时的浓度为定量限(LOQ)。基于标准品的进样浓度和MRM的峰面积绘制标准曲线，如表2所示，所有神经节苷脂标准曲线线性良好，R2均大于0.9994。根据Xcalibur2.0.7计算S/N，将S/N为3时的浓度为LOD，S/N为10时的浓度为LOQ。结果如表2所列，本方法对于所有二糖的LOD范围为0.15～0.52pg/μL，LOQ范围为0.50～1.72pg/μL。The six kinds of standard products respectively use GM1-36:1, GM2-36:1, GM3-41:1, GD1-38:1, GD3-41:1, GT1b-38:1 as quantitative components. After mixing the 6 standards, dilute with 80% methanol solution to a series of different concentrations: 5pg/μL, 10pg/μL, 50pg/μL, 100pg/μL, 500pg/μL, 1ng/μL, 5ng/μL, 10ng/μL , 20ng/μL, and then used for LC-MS/MS analysis with an injection volume of 5μL. A standard curve was drawn based on the injection volume of standard disaccharides and the peak area of multiple reaction monitoring MRM. The ratio of signal intensity (S) to background noise (N) is the signal-to-noise ratio (S/N), and the analysis data of different concentrations of standard products can be calculated by Xcalibur2.0.7 S/N, and the concentration when S/N is 3 is the detection The limit of quantification (LOD), the concentration at which the S/N was 10 was the limit of quantitation (LOQ). A standard curve was drawn based on the injection concentration of the standard and the peak area of the MRM, as shown in Table2 , all ganglioside standard curves had good linearity, and R2 was greater than 0.9994. Calculate S/N according to Xcalibur2.0.7, the concentration when S/N is 3 is LOD, and the concentration when S/N is 10 is LOQ. The results are listed in Table 2. The LOD range of this method for all disaccharides is 0.15-0.52 pg/μL, and the LOQ range is 0.50-1.72 pg/μL.
表2分析方法的线性与检测限、定量限Table 2 Linearity, detection limit and limit of quantification of analytical method
实施例2：乳腺癌患者血清中GAs的变化及GM3对于乳腺癌的诊断能力Example 2: Changes of GAs in the serum of breast cancer patients and the diagnostic ability of GM3 for breast cancer
所得数据通过Xcalibur软件进行处理，根据标准曲线计算每微升血清中神经节苷脂的绝对含量。通过差异分析比较正常人和乳腺癌患者血清中神经节苷脂的含量差异，以及不同分型乳腺癌患者之间神经节苷脂的含量差异，以发现癌症标志物。基于上述LC-MS/MS-MRM方法，对来自30名健康女性成人，20名乳腺良性瘤患者和70名乳腺癌患者血清样品中的GAs进行分析。结果显示，癌症组血清中GAs水平显着高于正常组(图2)。并且在非靶向和靶向方法下，GAs的变化趋势和相对丰度是一致的。乳腺癌患者血清中的GAs水平是健康血清中的2～3倍(表3)。根据ROC曲线分析，与正常血清相比，GM1，GM2，GM3，GD1和GD3的丰度在癌症血清中显著增加(AUC＞0.7，p值小于0.01)，这与已报导的通过2D-HPTLC和GC-MS方法测定到的结果一致。此外，作为主要的鞘糖脂成分，GM3在癌症血清中显示出极高的诊断准确性(AUC＞0.9)，具有进一步开发为标志物的价值。对具体GAs种类进行ROC分析，在癌症血清中共发现7种GAs具有极好的诊断准确性(图3)，并且它们都是GM3类型。目前已有报道证明GM3与致癌作用相关，例如通过整合素和CD9依赖的机制，在细胞表面表达的GM3与恶性肿瘤的逆转相关。The obtained data were processed by Xcalibur software, and the absolute content of gangliosides per microliter of serum was calculated according to the standard curve. Through differential analysis, the differences in the levels of gangliosides in the serum of normal people and breast cancer patients, as well as the differences in the levels of gangliosides in patients with different types of breast cancer, are used to discover cancer markers. Based on the LC-MS/MS-MRM method described above, GAs in serum samples from 30 healthy female adults, 20 breast benign tumor patients and 70 breast cancer patients were analyzed. The results showed that the level of GAs in the serum of the cancer group was significantly higher than that of the normal group (Fig. 2). And the changing trends and relative abundances of GAs were consistent under both non-targeted and targeted methods. The level of GAs in serum of breast cancer patients is 2-3 times that of healthy serum (Table 3). According to ROC curve analysis, compared with normal serum, the abundance of GM1, GM2, GM3, GD1 and GD3 was significantly increased in cancer serum (AUC>0.7, p value less than 0.01), which was consistent with the reported results by 2D-HPTLC and The results determined by GC-MS method are consistent. In addition, as the main glycosphingolipid component, GM3 shows extremely high diagnostic accuracy (AUC>0.9) in cancer serum, and has the value of being further developed as a marker. ROC analysis was performed on specific GAs types, and a total of 7 GAs were found in cancer serum with excellent diagnostic accuracy (Figure 3), and they were all GM3 types. It has been reported that GM3 is related to carcinogenesis, for example, GM3 expressed on the cell surface is related to the reversal of malignant tumors through integrin and CD9-dependent mechanisms.
为了进一步判断GM3对乳腺癌和正常人的区分能力，进一步进行了主成分分析(principal component analysis，PCA)，这是一种利用降维的思想，在损失很少信息的前提下把多个指标转化为几个综合指标的多元统计方法。对正常和癌症患者血清中获得的23种GM3进行PCA分析，结果如图4。尽管未实现癌症与正常血清之间的完全分离，但观察到明显的区分界线，表明GM3对乳腺癌的鉴别能力，也预示了GM3在乳腺癌进展中发挥着作用。In order to further judge the ability of GM3 to distinguish between breast cancer and normal people, principal component analysis (PCA) was further carried out. Transformed into a multivariate statistical approach for several composite indicators. The PCA analysis was performed on the 23 GM3s obtained from the sera of normal and cancer patients, and the results are shown in Fig. 4 . Although complete separation between cancer and normal serum was not achieved, a clear distinction line was observed, indicating the ability of GM3 to discriminate breast cancer and also predicting the role of GM3 in breast cancer progression.
此外，乳腺癌患者血清的GM3水平也与良性腺瘤患者显著不同。ROC曲线显示，与良性腺瘤患者相比，GM3在癌症患者血清中显示出较高的诊断价值(AUC＞0.8)(图2和表3)。然而，PCA分析无法有效区分良性组与其他两组(数据未给出)。此外，与健康人相比，GM3在良性腺瘤血清中显示出显着增加(AUC＞0.7)(图2和表3)。最终，基于AUC值和变化倍数，GM3作为一种高敏感性的新型生物标志物，可用于区分乳腺癌患者、健康志愿者和乳腺良性癌症患者。In addition, serum GM3 levels in patients with breast cancer were also significantly different from those in patients with benign adenomas. The ROC curve showed that GM3 showed a higher diagnostic value (AUC>0.8) in the serum of cancer patients compared with benign adenoma patients (Figure 2 and Table 3). However, PCA analysis could not effectively distinguish the benign group from the other two groups (data not shown). Furthermore, GM3 showed a significant increase (AUC > 0.7) in benign adenoma serum compared to healthy subjects (Fig. 2 and Table 3). Finally, based on AUC value and fold change, GM3, as a highly sensitive novel biomarker, can be used to distinguish breast cancer patients, healthy volunteers, and breast benign cancer patients.
表3正常、良性与癌症患者血清中GAs含量差异表。Table 3 Difference table of GAs content in serum of normal, benign and cancer patients.
实施例3：血清增加的GM3作为Luminal-B乳腺癌亚型的诊断标志物Example 3: Serum Increased GM3 as a Diagnostic Marker for Luminal-B Breast Cancer Subtype
在本发明的研究中，血清GM3升高被认为是乳腺癌诊断的潜在生物标志物。与正常组和良性组相比，总GM3，GM3-38∶1，GM3-40∶1和GM3-40∶2在乳腺癌血清中均表现出较高的诊断准确性(AUC＞0.8)。根据上述结果已经证实了GM3对乳腺癌的诊断能力，接下来本发明进一步探究了GM3在区分乳腺癌不同亚型的能力。In the present study, elevated serum GM3 was considered as a potential biomarker for breast cancer diagnosis. Compared with normal group and benign group, total GM3, GM3-38:1, GM3-40:1 and GM3-40:2 all showed higher diagnostic accuracy in breast cancer serum (AUC>0.8). According to the above results, the ability of GM3 to diagnose breast cancer has been confirmed. Next, the present invention further explores the ability of GM3 to distinguish different subtypes of breast cancer.
如图5所示，与LB分型相比，LA、HER和BS分型癌症病人血清GM3浓度显著降低。其中，各个分型病人血清中GM3含量如下：LB分型病人在12.58～13.83μg/mL之间，LA分型病人在6.34～7.64μg/mL之间，HER分型病人在6.64～8.34μg/mL之间，BS分型病人在7.19～9.22μg/mL之间。ROC曲线分析也证明GM3可以将LB分型与其他分型显著区分(ROC＞0.7，图6)。Luminal-B乳腺癌是ER阳性的一种增殖增加的乳腺癌亚型，与其他高增殖乳腺癌相比，显示出对化疗的耐药性以及内分泌治疗的不良预后。本发明的研究表明了GM3对Luminal-B乳腺癌的鉴别能力，对于临床个体化治疗具有指导意义。As shown in Figure 5, serum GM3 concentrations were significantly lower in LA, HER, and BS type cancer patients compared with LB type. Among them, the content of GM3 in the serum of patients with each type is as follows: LB type patients are between 12.58-13.83 μg/mL, LA type patients are between 6.34-7.64 μg/mL, HER type patients are between 6.64-8.34 μg/mL mL, the BS type patients were between 7.19 and 9.22 μg/mL. ROC curve analysis also proved that GM3 can significantly distinguish LB type from other types (ROC>0.7, Figure 6). Luminal-B breast cancer, an ER-positive, increased proliferation subtype of breast cancer, displays resistance to chemotherapy and a poor prognosis with endocrine therapy compared with other hyperproliferative breast cancers. The research of the present invention shows the ability of GM3 to distinguish Luminal-B breast cancer, which has guiding significance for clinical individualized treatment.
基于以上研究，首次发现血清中升高的神经节苷脂GM3可以作为生物标志物，用于乳腺癌的检测，并将Luminal B型乳腺癌与其他分型区分，有望为乳腺癌的临床诊断提供一种更为灵敏的标志物，并促进个体化治疗的发展。Based on the above studies, it was found for the first time that elevated ganglioside GM3 in serum can be used as a biomarker for the detection of breast cancer, and it can distinguish Luminal B breast cancer from other types, which is expected to provide a basis for the clinical diagnosis of breast cancer. A more sensitive marker and facilitates the development of individualized treatments.
以上实施例仅说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。The above embodiments only illustrate the technical solutions of the present invention, but are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still describe the contents of the foregoing embodiments. Modifications to the technical solutions, or equivalent replacement of some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.
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