WO2020147252A1 - 外泌体TβRII蛋白作为标志物在制备乳腺癌检测试剂盒中的应用 - Google Patents

外泌体TβRII蛋白作为标志物在制备乳腺癌检测试剂盒中的应用 Download PDF

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WO2020147252A1
WO2020147252A1 PCT/CN2019/090046 CN2019090046W WO2020147252A1 WO 2020147252 A1 WO2020147252 A1 WO 2020147252A1 CN 2019090046 W CN2019090046 W CN 2019090046W WO 2020147252 A1 WO2020147252 A1 WO 2020147252A1
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breast cancer
exosomes
tβrii
detection kit
cancer detection
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张龙
谢枫
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Zhejiang University
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    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57515—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the breast
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/57585—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds identifiable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/365—Breast disorders, e.g. mastalgia, mastitits, Paget's disease

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  • the invention relates to the technical field of breast cancer detection kits, in particular to the application of exosomal T ⁇ RII protein as a marker in preparing a breast cancer diagnostic kit.
  • Breast cancer is the most common malignant tumor in women. It ranks second among the most common cancers in the world. A prominent feature of breast cancer is that it is prone to metastasis. The most common metastatic organ is bone. 80% of patients who die of breast cancer have bone metastases. Tumor metastasis is a very important cause of death in cancer patients. As many as 90% of solid tumor patients die of tumor metastasis.
  • Exosomes play an important role in the occurrence, development and metastasis of cancer.
  • Exosomes are a type of special extracellular vesicles with a size of 30-100nm secreted by most cells. They are microvesicles released from different cells into body fluids such as plasma, cerebrospinal fluid, urine or saliva. . These transmission substances are also closely related to the pathogenesis of most human malignant tumors. Tumor cell exosomes released into the blood can provide cancer-related information and have a good prospect for non-invasive tumor diagnosis.
  • Glypican-1 a protein contained in the exosomes of pancreatic cancer cancer cells, may be suitable as a non-invasive diagnosis and screening Early pancreatic cancer in the surgical treatment stage.
  • the purpose of the present invention is to provide a kit for detecting breast cancer, which evaluates the occurrence and development of tumors by detecting peripheral blood, so as to overcome the unsuitability of tissue specimens for breast cancer patients, but cannot evaluate the malignancy and metastasis of the patients And other issues.
  • exosomal T ⁇ RII protein as a marker in the preparation of breast cancer detection kits.
  • Tumor exosomes are extracellular vesicles secreted from tumor cells into the peripheral blood circulation.
  • the research of the present invention found for the first time that the content of T ⁇ RII-positive exosomes in the serum of breast cancer patients increased significantly.
  • the metastatic growth factor ⁇ (TGF- ⁇ ) signaling pathway plays a very important role in the occurrence and development of tumors.
  • TGF- ⁇ inhibits tumor growth in the early stage of carcinogenesis, and transforms into a tumor promoting factor in the later stage of tumor development. In the late stage of breast cancer, the TGF- ⁇ pathway will be abnormally activated, leading to tumor metastasis.
  • T ⁇ RII protein transforming growth factor beta receptor 2, TGFBR2
  • TGFBR2 transforming growth factor beta receptor 2
  • the present invention also provides a breast cancer detection kit based on detecting T ⁇ RII protein in peripheral blood exosomes, which includes a direct-labeled primary antibody against T ⁇ RII protein.
  • the kit provided by the invention utilizes the specific binding of the primary antibody to the T ⁇ RII protein on the exosomes, and realizes the qualitative or quantitative determination of the breast cancer marker T ⁇ RII protein by detecting the label on the direct-labeled primary antibody.
  • the direct-labeled primary antibody is labeled with APC.
  • the exosomes were incubated with the APC-T ⁇ RII directly labeled primary antibody, and then the number of labeled exosomes was detected by flow cytometry.
  • FSC adopts Line linear form
  • SSC and FL1 adopt Log logarithmic form
  • FSC-H/SSC- The exosomes are circled on the H scatter chart, the excitation light is 633nm, the fluorescence value of the exosomes is obtained in the FSC/APC scatter chart, more than 10,000 exosomes are read, and the scatter chart is analyzed.
  • the detection kit provided by the present invention also includes reagents for pretreatment of peripheral blood to separate and enrich exosomes.
  • the breast cancer detection kit further includes a sodium citrate anticoagulant, wherein the mass percentage concentration of sodium citrate is 2.5%.
  • the breast cancer detection kit further includes PBS buffer.
  • the breast cancer detection kit also includes T ⁇ RII-positive exosomes standards with known concentrations.
  • the standard T ⁇ RII-positive exosomes are obtained from the culture supernatant of the breast cancer cell line MDA-MB-231.
  • the T ⁇ RII expression level on the surface of exosomes secreted by MDA-MB-231 breast cancer cells is high, which can be used as a positive control for detecting the number of T ⁇ RII-positive exosomes in the serum.
  • the present invention also provides a method for separating exosomes from an isolated blood sample, which includes the following steps:
  • the present invention finds that the content of T ⁇ RII-positive exosomes in the serum of breast cancer patients is significantly higher than that of healthy people, and that the content of T ⁇ RII exosomes in the serum of breast cancer patients is significantly reduced after surgery, suggesting its role in breast cancer diagnosis and prognostic diagnosis Therefore, the content of T ⁇ RII protein in peripheral blood exosomes can be used as an indicator for judging breast cancer metastasis, and it can also be used as a new target for tumor treatment.
  • the detection of exosomes has the characteristics of minimally invasive and real-time monitoring. It can be used as a "liquid biopsy" for tumor development and metastasis.
  • a breast cancer diagnostic kit based on the detection of T ⁇ RII content of exosomes in serum has a good application prospect in breast cancer diagnosis.
  • the detection kit provided by the present invention is simple to operate, only a small amount of peripheral blood of the population to be tested can be drawn to detect whether the population to be tested has breast cancer, and compared with the existing detection technology, it is convenient and quick, and has little impact on the population to be tested ; It does not use organic solvents such as phenol and chloroform, which is safe for operators and has no toxic side effects. It is suitable for breast cancer prevention and detection in a large number of people.
  • Figure 1 shows the Western blot detection results of the T ⁇ RII protein content of human breast cancer cells MDA-MB-231 exosomes.
  • Figure 2 shows the result of electron microscopy detection of the T ⁇ RII protein content of human breast cancer cells MDA-MB-231 exosomes.
  • Figure 3 shows the results of flow cytometric detection of the T ⁇ RII protein content of human breast cancer cells MDA-MB-231 exosomes.
  • Figure 4 shows the diameter (A) and density (B) of the isolated exosomes detected by nanosight and the shape of the exosomes (C) photographed by an electron microscope.
  • Figure 5 shows the exosomal particles with exosomal marker proteins photographed with an electron microscope.
  • Figure 6 shows the results of flow cytometry detection of T ⁇ RII protein content in peripheral blood of breast cancer patients and normal people.
  • Fig. 7 is a graph showing the comparison result of T ⁇ RII protein content in peripheral blood of breast cancer patients and normal persons.
  • Figure 8 is a graph showing the sensitivity and specificity analysis results of T ⁇ RII protein in peripheral blood exosomes of breast cancer patients and normal people.
  • Figure 9 is a schematic diagram of the extraction process of peripheral blood exosomes before and after surgery for breast cancer patients (A) and the comparison result diagram of the T ⁇ RII protein content of peripheral blood exosomes (B).
  • Figure 10 shows the results of electron microscopy detection of T ⁇ RII protein content in peripheral blood of breast cancer patients before and after surgery.
  • the supernatant of MDA-MB-231 cell culture was centrifuged at 4000rpm/min for 10min; the supernatant was collected and centrifuged at 100,000g for 1h; the pellet was collected and resuspended in 28ml PBS buffer, centrifuged at 100,000g for 1h, and the pellet was exosomes. Resuspend the exocrine body in 0.2ml PBS buffer, and store in aliquots at -80°C for later use.
  • exosomal lysate composition: 2.5% SDS and 8M urea, the balance is water
  • BCA protein concentration is determined to determine the concentration of exosomes
  • Western blotting is used to determine that the isolated product is extra-tumor Exosomes.
  • the isolated exosomes in step 1 also have a high protein content of T ⁇ RII.
  • FSC adopts Line linear form
  • SSC and FL1 adopt Log logarithmic form, in FSC-H/SSC-H scatter point
  • the exosomes are circled on the graph, the excitation light is 633nm, the fluorescence value of the exosomes is obtained in the FSC/APC scatter chart, more than 10,000 exosomes are read, and the scatter chart is analyzed.
  • Example 1 The technique of Example 1 was used to isolate, obtain and identify 36 breast cancer patients (20 normal samples were tested at the same time as a negative control) peripheral blood exosomes, and the expression of T ⁇ RII was detected.
  • Test results The particle diameter, density and other parameters detected by nanosight are consistent with the characteristics of exosomes, and the particle size observed by electron microscopy is also consistent with the size of exosomes.
  • the isolated exosomes are obtained ( Figure 4). ).
  • the immunogold electron microscopy method was used to detect the exosomal-specific markers CD63 and TSG101, and the presence of these markers on the surface of the particles was indeed observed, which once again showed that the exosomal particles were separated and extracted ( Figure 5).
  • APC-T ⁇ RII direct-labeled primary antibody was added to the isolated exosomes at a ratio of 1:100, incubated at room temperature in the dark for 30 minutes, and washed three times with PBS.
  • the processed samples were tested for the content of T ⁇ RII in the exosomes by flow cytometry.
  • Test results The content of T ⁇ RII in the exosomes of breast cancer patients is higher than that of normal people ( Figure 6, 7).
  • peripheral blood exosomal T ⁇ RII protein itself has a greater advantage as a diagnostic marker for breast cancer, and its sensitivity and specificity have a high level (Figure 8).
  • T ⁇ RII protein content in peripheral blood exosomes of breast cancer patients before and after surgery showed that the T ⁇ RII protein content in peripheral blood exosomes of breast cancer patients after surgery decreased significantly ( Figure 9), and the method was detected by immunogold electron microscope
  • the T ⁇ RII protein content of exosomes in breast cancer patients before and after surgery showed that the T ⁇ RII protein content of peripheral blood exosomes in breast cancer patients also decreased significantly after surgery ( Figure 10). It shows that the T ⁇ RII protein content of peripheral blood exosomes can also be used as the detection of postoperative recovery level of breast cancer patients.
  • the present invention collects serum of healthy people and breast cancer patients, and detects the content of T ⁇ RII-positive exosomes in them by flow cytometry. Compare the content of T ⁇ RII-positive exosomes in the serum of healthy people and breast cancer patients to obtain the best cut-off value, and evaluate the sensitivity and specificity of the serum T ⁇ RII-positive exosomes in the diagnosis of breast cancer;
  • T ⁇ RII-positive exosomes in the serum of breast cancer patients was significantly higher than that of healthy people. It shows the application prospect of detecting the content of T ⁇ RII positive exosomes in human serum in the diagnosis of breast cancer.

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Abstract

外泌体TβRII蛋白作为标志物在制备乳腺癌检测试剂盒中的应用。乳腺癌病人血清中TβRII阳性的外泌体含量要明显高于健康人，且乳腺癌病人术后血清中TβRII外泌体的含量明显减少，提示其在乳腺癌诊断及预后诊断中的价值。一种基于检测外周血外泌体中TβRII蛋白的乳腺癌检测试剂盒，操作简单，只需抽取待测人群少量外周血，即可检测待测人群是否患有乳腺癌及评估患者肿瘤恶性程度及转移状况。

Description

外泌体TβRII蛋白作为标志物在制备乳腺癌检测试剂盒中的应用 技术领域
本发明涉及乳腺癌检测试剂盒技术领域，具体涉及外泌体TβRII蛋白作为标志物在制备乳腺癌诊断试剂盒中的应用。
背景技术
乳腺癌是女性最常见的恶性肿瘤。在全球最常见的癌症中发病率排名第二。乳腺癌的一个突出的特点是易发生转移，其最常见的转移器官为骨，死于乳腺癌的患者中有80％都发生了骨转移。肿瘤转移是导致癌症病人死亡的一个非常重要的原因，多达90％的实体瘤患者死于肿瘤转移。
目前通常使用的乳腺癌转移检测方法有两种，一种是免疫组织化学方法，通过采集病理组织制作病理学切片，并通过免疫组织化学检测相关蛋白质的方法来检测肿瘤转移；另外一种是蛋白质免疫印迹法(Westernblot)，特异性更高。但这种方法都需要以肿瘤组织为检测对象，对于部分缺乏手术指征或存在手术禁忌症的癌症患者，很难切除其组织进行检测。且肿瘤转移具有随机性，这就增加了样本的数量，使得检测患者是否转移变得更加困难。
近年的研究发现，外泌体在癌症的发生、发展以及转移的过程中发挥重要的作用。外泌体是一类大多数细胞会分泌的大小30-100nm的特殊的细胞外囊泡，它是从不同细胞中释放到血浆，脑脊液，尿液或唾液等体液中的微小囊泡(microvesicle)。这些传输物质也与大多数人类恶性肿瘤的发病机制密切相关，释放到血液中的肿瘤细胞外泌体能够提供与癌症相关的信息，具有良好的肿瘤无创诊断应用前景。
Kalluri于2015年6月24日在《自然》上刊文称胰腺癌癌细胞外泌体中包含的一种蛋白Glypican-1，这种蛋白有可能作为一种非侵入性诊断和筛查处于适合手术治疗阶段的早期胰腺癌。
乳腺癌转移是乳腺癌致死率高发的一大主因，外泌体蛋白可先于细胞达到转移部分，可对细胞转移进行深度介入，若能在此基础上发现特异表 面抗原，将指导研究人员对病人进行精确诊断及治疗。如获得表征乳腺癌转移所分泌外泌体的特异性表面抗原，便可通过对病人血液中外泌体进行提取检测，精确判断病人的疾病状况，对其进行治疗。
因此，研究乳腺癌外泌体中携带的有效成分及其捕获靶细胞并影响其功能的分子机制及生理意义已成为未来癌症研究的热点，为开发新的防治乳腺癌等肿瘤治疗药物奠定理论基础。
发明内容
本发明的目的在于提供一种用于检测乳腺癌的试剂盒，通过检测外周血来评估肿瘤发生、发展情况，以克服乳腺癌患者不适宜组织标本取材，而无法评估患者肿瘤恶性程度及转移状况等问题。
为实现上述目的，本发明采用如下技术方案：
外泌体TβRII蛋白作为标志物在制备乳腺癌检测试剂盒中的应用。
肿瘤外泌体(exosome)是从肿瘤细胞分泌进入外周血液循环的一种细胞外囊泡。本发明研究首次发现乳腺癌病人的血清中TβRII阳性的外泌体含量显著提升。转移生长因子β(TGF-β)信号通路在肿瘤发生和发展中有着非常重要的作用，TGF-β在早期癌变中抑制肿瘤生长，而在后期肿瘤发展过程中则转变成一个肿瘤促进因子。在乳腺癌发生后期，TGF-β通路会异常激活从而导致肿瘤转移，且在转移初期可以发现乳腺癌细胞分泌的外泌体中大量含有TβRII蛋白(transforming growth factor beta receptor 2，TGFBR2)。因此，该指标能够作为乳腺癌诊断的标志物，特别是对于诊断难度较大的早期肿瘤具有良好的诊断效果。
本发明还提供了一种基于检测外周血外泌体中TβRII蛋白的乳腺癌检测试剂盒，包括抗TβRII蛋白的直标一抗。
本发明提供的试剂盒利用一抗与外泌体上TβRII蛋白的特异性结合，通过对直标一抗上的标记物的检测，实现对乳腺癌标志物TβRII蛋白的定性或定量测定。
作为优选，所述直标一抗上标记有APC。通过APC-TβRII直标一抗孵育外泌体，然后通过流式细胞仪检测具有标记的外泌体数量。
具体操作：机型上样，调整并确定仪器前向角散射光、侧向角散射光和FL1基本参数，FSC采用Line线性形式，SSC和FL1采用Log对数形 式，在FSC-H/SSC-H散点图上圈定外泌体，激发光选用633nm，在FSC/APC散点图中获取外泌体的荧光值，读取外泌体10000个以上，分析散点图。
本发明提供的检测试剂盒中还包括对外周血进行预处理的试剂，对外泌体进行分离、富集。
作为优选，所述乳腺癌检测试剂盒还包括柠檬酸钠抗凝剂，其中柠檬酸钠的质量百分比浓度为2.5％。
作为优选，所述乳腺癌检测试剂盒还包括PBS缓冲液。
作为优选，所述乳腺癌检测试剂盒还包括浓度已知的TβRII阳性外泌体标准品。
更为优选，所述TβRII阳性外泌体标准品由乳腺癌细胞株MDA-MB-231细胞培养上清液中分离获得。MDA-MB-231乳腺癌细胞分泌的外泌体表面TβRII表达水平较高，可以作为检测血清中TβRII阳性外泌体数量时的阳性对照。
本发明还提供了一种分离离体血液样本中外泌体的方法，包括以下步骤：
(1)将采集的外周血样本中加入柠檬酸钠抗凝剂，混合均匀，2000-4000rpm，离心10-20min，收集上层血浆；
(2)利用PBS缓冲液稀释血浆，100000-120000g超速离心1-1.5h，弃上清，再加入PBS缓冲液重悬沉淀，100000-120000g超速离心1-1.5h，收集沉淀即为所述外泌体。
利用nanosight和电镜以及通过免疫印迹检测外泌体的已知Marker(TSG101,CD63,Alix等)等检测手段皆验证了由上述方法对乳腺癌病人的外周血样本分离获得的沉淀颗粒即含有乳腺癌外泌体。
本发明具备的有益效果：
本发明发现乳腺癌病人血清中TβRII阳性的外泌体含量要明显高于健康人，且乳腺癌病人术后血清中TβRII外泌体的含量明显减少，提示其在乳腺癌诊断及预后诊断中的价值，因此外周血外泌体中TβRII蛋白含量可以作为乳腺癌转移判断的指标，也可作为肿瘤治疗的新靶点。外泌体检测具有微创、实时监测等特点，可作为肿瘤发展转移的“液体活检”，基于血清中外泌体TβRII含量检测的乳腺癌诊断试剂盒，在乳腺癌诊断中具有良 好的应用前景。
本发明提供的检测试剂盒操作简单，只需抽取待测人群少量外周血，即可检测待测人群是否患有乳腺癌，且相对于现有的检测技术方便又快捷，对待测人群影响较小；不使用苯酚、氯仿等有机溶剂，对操作人员安全无毒副作用，适用于大量人群乳腺癌预防检测。
附图说明
图1为人乳腺癌细胞MDA-MB-231外泌体TβRII蛋白含量的免疫印迹检测结果。
图2为人乳腺癌细胞MDA-MB-231外泌体TβRII蛋白含量的电镜检测结果。
图3为人乳腺癌细胞MDA-MB-231外泌体TβRII蛋白含量流式细胞仪检测结果。
图4为用nanosight检测到的分离的外泌体的直径(A)，密度(B)及电镜拍摄的外泌体形状(C)。
图5为用电子显微镜拍摄的带有外泌体标志物蛋白的外泌体颗粒。
图6为乳腺癌病人和正常人的外周血外泌体TβRII蛋白含量流式细胞仪检测结果。
图7为乳腺癌病人和正常人的外周血外泌体TβRII蛋白含量的对比结果图。
图8为乳腺癌病人和正常人的外周血外泌体TβRII蛋白的敏感性和特异性分析结果图。
图9为乳腺癌病人术前术后外周血外泌体提取的流程示意图(A)及外周血外泌体TβRII蛋白含量的对比结果图(B)。
图10为乳腺癌病人术前术后外周血外泌体TβRII蛋白含量的电镜检测结果。
具体实施方式
为了更充分的理解本发明的技术内容，下面结合实验，进一步说明本发明的技术方案。
实施例1
1、人乳腺癌细胞株MDA-MB-231细胞来源外泌体的提取和鉴定。提取方法如下：
MDA-MB-231细胞培养上清，4000rpm/min，离心10min；收集上清，100000g离心1h；收集沉淀，沉淀用28ml PBS缓冲液重悬，100000g离心1h，沉淀即为外泌体。将外泌体重悬于0.2ml PBS缓冲液，分装保存于-80℃备用。
2、外泌体裂解液裂解(组成：2.5％SDS和8M尿素，余量为水)外泌体30min，经BCA蛋白浓度测定，确定外泌体浓度；利用免疫印迹检测确定分离产物为肿瘤外泌体。
如图1所示，步骤1的分离的外泌体中除了含有已知的标志物TSG101,ALIX,CD63外，TβRII的蛋白含量也很高。
3、利用流式细胞检测方法检测外泌体TβRII：
(1)按照1:100的比例在外泌体中加入APC-TβRII直标一抗(R&D Systems：Catalog#FAB241A)，室温黑暗孵育30min，用PBS洗三遍；
(2)利用流式细胞仪测定外泌体中TβRII平均荧光强度。
机型上样，调整并确定仪器前向角散射光、侧向角散射光和FL1基本参数，FSC采用Line线性形式，SSC和FL1采用Log对数形式，在FSC-H/SSC-H散点图上圈定外泌体，激发光选用633nm，在FSC/APC散点图中获取外泌体的荧光值，读取外泌体10000个以上，分析散点图。
4、电镜对MDA-MB-231细胞外泌体的鉴定结果显示，制备的外泌体均为直径在100nm左右的小囊泡，具有典型的脂质双分子层结构，且TβRII含量较高(图2)。流式细胞仪检测结果同样显示，MDA-MB-231细胞外泌体TβRII含量较高(图3)。
实施例2
运用实施例1的技术分离和获取并鉴定36例乳腺癌患者(同时检测20例正常样本做阴性对照)外周血外泌体，检测TβRII的表达情况。
一、利用超速离心分离获取待测人群外周血中的外泌体：
采集各样本静脉血1ml，加入0.5ml 2.5％柠檬酸钠抗凝剂，混合均匀，以4000rpm/min，离心10min，取上层血浆。
加入PBS稀释至28ml，倒入超速离心管中，100000g超速离心1h。 用移液器或吸液管弃去上清，留离心管管底1ml液体，再加入PBS至28ml重悬沉淀，100000g超速离心，1h。用移液器或吸液管弃去上清，把超速离心管倒扣在滤纸上5min彻底吸尽液体，加入0.5ml PBS重悬沉淀，转移至1.5ml离心管中，置于-80℃保存用于研究。
利用nanosight或用外泌体裂解液裂解外泌体30mim，然后用BCA试剂盒测定其浓度，以及用电镜观察其形态，确定分离肿瘤外泌体。
检测结果：利用nanosight检测出的颗粒直径，密度等参数均与外泌体的特征相一致，且电镜观察到的颗粒大小也与外泌体大小相一致，分离得到的为外泌体(图4)。
进一步利用免疫胶体金电镜的方法检测外泌体特异性标志物CD63和TSG101，确实观察到颗粒表面有这些标志物的存在，再一次说明分离提取的是外泌体颗粒(图5)。
二、利用流式细胞检测方法检测外泌体上TβRII的表达情况。
在分离得到的外泌体中按1:100的比例加入APC-TβRII直标一抗，室温黑暗孵育30min，用PBS洗三遍。
把处理过的样品通过流式细胞仪检测其外泌体中TβRII的含量。
检测结果：乳腺癌病人外泌体中TβRII的含量相对于正常人高表达(图6,7)。
进一步进行的受试者工作曲线分析显示，外周血外泌体TβRII蛋白本身作为乳腺癌诊断标志物具有较大优势，其敏感度和特异度均有较高水平(图8)。
进一步分析乳腺癌病人术前术后外周血外泌体中TβRII蛋白含量，分析显示术后乳腺癌病人外周血外泌体TβRII蛋白含量显著下降(图9)，以及利用免疫胶体金电镜的方法检测乳腺癌病人术前术后外泌体中TβRII蛋白含量，结果显示术后乳腺癌病人外周血外泌体TβRII蛋白含量同样显著下降(图10)。说明外周血外泌体TβRII蛋白含量还可作为乳腺癌病人术后康复水平的检测。
本发明搜集了健康人以及乳腺癌病人血清，并通过流式细胞仪对其中TβRII阳性外泌体含量进行检测。对比健康人与乳腺癌病人血清中TβRII阳性外泌体的含量，获得最佳的cut-off值，评价血清中TβRII阳性外泌体含量对乳腺癌诊断的灵敏度与特异性；
发现了乳腺癌病人血清中TβRII阳性外泌体的含量要明显高于健康人。表明检测人体血清中TβRII阳性外泌体的含量在乳腺癌诊断中的应用前景。

Claims (8)

  1. 外泌体TβRII蛋白作为标志物在制备乳腺癌检测试剂盒中的应用。
  2. 一种基于检测外周血外泌体中TβRII蛋白的乳腺癌检测试剂盒，其特征在于，包括抗TβRII蛋白的直标一抗。
  3. 如权利要求2所述的乳腺癌检测试剂盒，其特征在于，所述直标一抗上标记有APC。
  4. 如权利要求2所述的乳腺癌检测试剂盒，其特征在于，还包括柠檬酸钠抗凝剂，其中柠檬酸钠的质量百分比浓度为2.5％。
  5. 如权利要求2所述的乳腺癌检测试剂盒，其特征在于，还包括PBS缓冲液。
  6. 如权利要求2所述的乳腺癌检测试剂盒，其特征在于，还包括TβRII阳性外泌体标准品。
  7. 如权利要求6所述的乳腺癌检测试剂盒，其特征在于，所述TβRII阳性外泌体标准品由乳腺癌细胞株MDA-MB-231细胞培养上清液中分离获得。
  8. 一种分离离体血液样本中外泌体的方法，其特征在于，包括以下步骤：
    (1)将采集的外周血样本中加入柠檬酸钠抗凝剂，混合均匀，4000rpm，离心10min，收集上层血浆；
    (2)利用PBS缓冲液稀释血浆，100000-120000g超速离心1-1.5h，弃上清，再加入PBS缓冲液重悬沉淀，100000-120000g超速离心1-1.5h，收集沉淀即为所述外泌体。
PCT/CN2019/090046 2019-01-14 2019-06-05 外泌体TβRII蛋白作为标志物在制备乳腺癌检测试剂盒中的应用 Ceased WO2020147252A1 (zh)

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CN110373471A (zh) * 2019-09-05 2019-10-25 贵州医科大学附属医院 血浆外泌体tRFs标志物及其在乳腺癌诊断中的应用
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