WO2021213315A1 - Kit de détection de l'expression de mutation du gène braf v600e d'un patient atteint d'un cancer colorectal au moyen de cellules tumorales circulantes du sang périphérique - Google Patents

Kit de détection de l'expression de mutation du gène braf v600e d'un patient atteint d'un cancer colorectal au moyen de cellules tumorales circulantes du sang périphérique Download PDF

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WO2021213315A1
WO2021213315A1 PCT/CN2021/088093 CN2021088093W WO2021213315A1 WO 2021213315 A1 WO2021213315 A1 WO 2021213315A1 CN 2021088093 W CN2021088093 W CN 2021088093W WO 2021213315 A1 WO2021213315 A1 WO 2021213315A1
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peripheral blood
braf gene
tumor cells
filter
labeled goat
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PCT/CN2021/088093
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English (en)
Chinese (zh)
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黄宁
夏梅
李胜
李�浩
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山东第一医科大学(山东省医学科学院)
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Publication of WO2021213315A1 publication Critical patent/WO2021213315A1/fr

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    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N2333/82—Translation products from oncogenes

Definitions

  • the invention provides an immunofluorescence kit and a detection method for the expression of BRAF gene V600E mutation in peripheral blood circulating tumor cells of patients with colorectal cancer, and belongs to the technical field of molecular biology.
  • Colorectal cancer is one of the common malignant tumors of the digestive tract in China. According to statistics, the incidence and mortality of CRC are among the forefront of all malignant tumors and are on the rise. However, its pathogenic factors and pathogenesis are still not fully understood. It is currently considered to be a complex process involving multiple genes in the regulation, involving abnormal activation of oncogenes (such as RAF, etc.) and inactivation of tumor suppressor genes (such as APC, P53, etc.). Studies have shown that BRAF gene plays an important role in the pathogenesis and treatment of colorectal cancer. However, the relationship between BRAF gene mutation characteristics in colorectal cancer and the relationship between the two and the clinical pathology has not been reported in many studies.
  • the BRAF gene is located in the proto-oncogene of human chromosome 7q34, which encodes a serine/threonine specific kinase. It is an activator of the RAF gene and MAPK signal transduction cascade that regulates the serine/threonine kinase. After activating mutations, it can develop in cancer. Plays a carcinogenic effect in. Studies have found that BRAF mutations can inhibit colonic epithelial cell apoptosis and promote cell proliferation, and promote the immortal proliferation, survival and migration of cancer cells. Even in the absence of cytokines and other stimuli, the V600E mutation in BRAF can activate this signaling pathway, leading to immortal cell proliferation and eventually cancer. Based on the mutation of the tumor BRAF gene, the current clinical specimens are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to achieve multiple or real-time detection.
  • Circulating tumor cells are tumor cells that fall off from solid tumors and enter the peripheral blood circulation. Since they were discovered in 1989, there have been a variety of methods for detecting circulating tumor cells in the peripheral blood. Recent studies have shown that its detection has important clinical significance for evaluating the prognosis of cancer patients, especially advanced cancer patients, and selecting appropriate individualized treatments. Because CTC detection has the characteristics of minimally invasive and real-time detection, it is called "liquid biopsy" of tumors.
  • Immunofluorescence analysis technology combines immunological methods (antigen-antibody specific binding) and fluorescent labeling technology to study the method of specific protein antigen distribution in cells. Because the fluorescence emitted by fluorescein can be detected under a fluorescence microscope, fluorescein emits bright fluorescence (yellow-green or orange-red) when irradiated by the excitation light, and the cells or tissues where the fluorescence is located can be seen. Quantitative techniques are used to determine the content. In order to carry out cell qualitative and localization analysis of the antigen.
  • the specimens for BRAF gene V600E mutation detection in colorectal cancer patients are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to achieve multiple or real-time detection. Therefore, the detection of circulating tumor cells (CTC) BRAF gene V600E mutation expression is of great value for the prognosis of colorectal cancer and the evaluation of the efficacy of immunotherapy.
  • CTC circulating tumor cells
  • Shandong First Medical University, Shandong Pharmaceutical Research Institute and Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd. and other units have Research on the industrialization of key technologies for tumor cell detection and identification.
  • This project is a major scientific and technological innovation project in Shandong Province. This project will take the Shandong Provincial Pharmaceutical Research Institute on the Jinan Campus of Shandong First Medical University as the core and implement the registrant system.
  • Circulating tumor cell detection and identification of core diagnostic technology, and further registration and identification of diagnostic kits to include PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, TKI-EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catenin, E-Cadherin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, Cytokeratin, AE1/AE3, estrogen receptor, pregnancy Hormone receptors, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A, etc.
  • the identification and diagnosis kits are industrialized and promoted through cooperation with Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., and Shandong Discovery Biotechnology Co., Ltd., which are registered in Jinan.
  • the present invention provides an immunofluorescence kit for the expression of the BRAF gene V600E mutation in circulating tumor cells in the peripheral blood of colorectal cancer patients and a detection method.
  • a membrane filtration device is used to separate and obtain circulating tumor cells (CTC) in the peripheral blood of patients with advanced colorectal cancer.
  • CTC circulating tumor cells
  • An immunofluorescence kit for detecting the BRAF gene V600E mutation expression of circulating tumor cells in the peripheral blood of patients with colorectal cancer including 45mL dilution solution, 1mL decolorization solution, staining solution A 0.5mL, staining solution B 1mL, 200 ⁇ l methanol, 200 ⁇ l 2% PFA, 100 ⁇ l of 10% goat serum, 100 ⁇ l of primary antibody suspension composed of mouse anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutation, fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, fluorescently labeled goat 100 ⁇ L of anti-rabbit secondary antibody suspension, DAPI mounting plate;
  • mice anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutations in the primary antibody suspension were diluted 1:100, 1:400, and 1:500 respectively, and the total volume was 100 ⁇ L;
  • Fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit in the secondary antibody suspension were diluted 1:500.
  • the diluent is composed of 1 mmol/L EDTA + 1 mmol moon silicate + 0.1% BSA + 0.2% poloxamer.
  • the decolorizing liquid is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
  • the staining solution A is DAB staining solution
  • the staining solution B is hematoxylin staining solution.
  • the method for detecting the BRAF gene V600E mutation expression in peripheral blood circulating tumor cells of patients with colorectal cancer for non-diagnostic purposes of the above kit includes the following steps:
  • step (7) using immunofluorescence to detect the BRAF gene V600E mutation expression of peripheral blood CTC is as follows:
  • the membrane filtration device used in the present invention to separate circulating tumor cells includes a filter, a blood sample container, a waste liquid tank and an iron stand.
  • the iron stand is provided with a base, a stand and a bracket.
  • the blood sample container is set on the upper part of the iron stand through the bracket.
  • Below the blood sample container is a filter, the filter is connected to the waste liquid tank through the infusion set, and the waste liquid tank is arranged on the base.
  • the filter includes a filter upper port, a filter membrane, a filter membrane platform and a filter lower port.
  • the filter membrane is placed on the filter membrane platform; the upper port of the filter is connected to the blood sample container, and the lower port of the filter is connected to the waste liquid tank through the infusion device.
  • the filter membrane is made of hydrophobic material, and the filter holes with a diameter of 8 microns are uniformly spread on it; the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns, so when it contains CTC After the peripheral blood is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole, and the CTC is trapped on the filter membrane because the diameter is larger than the filter hole.
  • the detection method provided by the present invention can detect the BRAF gene V600E mutation expression of patients with advanced or recurrent colorectal cancer without puncture biopsy to obtain tissue samples, and can realize real-time dynamic detection by using minimally invasive technology.
  • the method provided by the present invention has good separation of circulating tumor cells, can avoid the interference of blood cells, can avoid false positive results caused by edge effects that may occur in the staining process, has good stability, reduces cell loss, and improves detection accuracy sex.
  • Figure 1 is a schematic diagram of the structure of the membrane filtration device of the present invention.
  • FIG. 2 is a schematic cross-sectional view of the structure of the filter of the membrane filtration device of the present invention
  • FIG. 3 is a schematic diagram of the structure of the filter membrane of the membrane filtration device of the present invention.
  • Figure 4 is an image diagram of circulating tumor cells obtained from peripheral blood of patients with colorectal cancer
  • Figure 5 is an immunofluorescence staining image of the BRAF gene V600E mutation of circulating tumor cells in the peripheral blood of patients with advanced colorectal cancer, where A is merge, B is the target gene expression (red), C is CK (green), and D is CD45 (blue).
  • Component content Diluent 45 mL Decolorizing liquid (volume ratio of 95% alcohol and 100% xylene 1:1) 1mL Staining solution A (DAB staining solution) 0.5 mL Staining solution B (hematoxylin staining solution) 1mL 10% goat serum (diluted in PBS) 100 ⁇ L A suspension of anti-aliasing 100 ⁇ L Secondary antibody suspensions 100 ⁇ L
  • the primary antibody suspension is composed of mouse anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutations.
  • Mouse anti-CK, rat anti-CD45 and rabbit anti-BRAF gene V600E mutations are respectively used with BD wash buffer at 1:100 , 1:500 and 1:400 dilution, after dilution, take 10 ⁇ L mouse anti-CK, 50 ⁇ L rat anti-CD45 and 40 ⁇ L rabbit anti-BRAF gene V600E mutation to form the primary antibody suspension;
  • the secondary antibody suspension is composed of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit. They are commercially available Alexa Fluor 546 goat Anti-mouse, Alexa Fluor 488 goat Anti -rat and Alexa Fluor 647 goat Anti-rabbit, take the same amount of the above three fluorescently labeled secondary antibodies, dilute them with BD wash buffer at 1:500 and mix to obtain the secondary antibody suspension.
  • the membrane filtration device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;
  • the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. Therefore, after the peripheral blood containing CTC is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, and the CTC is larger than the diameter.
  • the filter hole 10 is trapped on the filter membrane 7. What needs to be explained here is that the diluent plays the role of de-adhesion and dispersion. The laurate and poloxamer are used together to ensure that blood cells and CTC do not adhere and are fully dispersed in the diluent to effectively pass through the filter membrane. 7 was detained.
  • Figure 5 is an immunofluorescence staining image of circulating tumor cells in the peripheral blood of a patient with advanced colorectal cancer. According to immunological and morphological findings, it is found that the tumor cells are large in size, have abnormal nucleus-to-cytoplasm ratio, and immunologically show typical CTCs.
  • the detected circulating tumor cells were confirmed by immunofluorescence to confirm the BRAF gene V600E mutation and compared with the results of the BRAF gene V600E mutation gene of colorectal cancer specimens, to observe the differences, mainly for patients with negative BRAF gene V600E mutation in the gross specimens and positive expression of circulating tumor cells , To guide the targeted therapy of colorectal cancer and provide new ideas for targeted therapy of colorectal cancer.
  • the detected circulating tumor cells were confirmed by immunofluorescence to confirm the expression of BRAF gene V600E mutation and compared with the results of BRAF gene V600E mutation in colorectal cancer specimens. Observe the differences, mainly for the negative expression of BRAF gene V600E mutation in gross specimens and positive expression of circulating tumor cells Of patients, guide the targeted therapy of colorectal cancer, and provide new ideas for targeted therapy of colorectal cancer.

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Abstract

La présente invention concerne le domaine de la biologie moléculaire et concerne un kit permettant de détecter l'expression de mutation d'un gène BRAF V600E d'un patient atteint d'un cancer colorectal au moyen de cellules tumorales circulantes du sang périphérique. En particulier, le kit comprend principalement : une suspension d'anticorps primaire composée de sérum de chèvre, d'un anti-CK de souris, d'un anti-CD45 de rat et d'un anti-mutation de gène BRAF V600E de lapin, et une suspension d'anticorps secondaire composée d'un anti-souris de chèvre marqué par fluorescence, d'un anti-rat de chèvre marqué par fluorescence, et d'un anti-lapin de chèvre marquée par fluorescence. Une méthode de détection fait principalement appel à : la collecte de sang périphérique, le traitement du sang périphérique, le filtrage et l'enrichissement des cellules tumorales circulantes, et la détection de l'état d'expression de mutation du gène BRAF V600E des cellules tumorales circulantes au moyen d'un procédé d'immunofluorescence.
PCT/CN2021/088093 2020-04-22 2021-04-19 Kit de détection de l'expression de mutation du gène braf v600e d'un patient atteint d'un cancer colorectal au moyen de cellules tumorales circulantes du sang périphérique WO2021213315A1 (fr)

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CN202010319553.9 2020-04-22
CN202010319553.9A CN111521801A (zh) 2020-04-22 2020-04-22 通过外周血循环肿瘤细胞检测结直肠癌患者braf基因v600e突变表达的试剂盒

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Publication number Priority date Publication date Assignee Title
CN111521797A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 通过外周血循环肿瘤细胞检测结直肠癌患者braf基因v600e突变的非诊断目的方法
CN111521801A (zh) * 2020-04-22 2020-08-11 山东第一医科大学(山东省医学科学院) 通过外周血循环肿瘤细胞检测结直肠癌患者braf基因v600e突变表达的试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105588943A (zh) * 2016-01-28 2016-05-18 山东省肿瘤防治研究院 一种胃癌患者外周血循环肿瘤细胞Her-2基因的检测方法
WO2019232485A1 (fr) * 2018-05-31 2019-12-05 Nvigen, Inc. Analyse de sang précise permettant la prédiction de l'incidence et de la récurrence du cancer, et le guidage et la surveillance d'une intervention de traitement
CN110716041A (zh) * 2019-10-23 2020-01-21 郑州大学 一种用于胃癌早期筛查和诊断的血清蛋白标志物、试剂盒及检测方法
CN111521801A (zh) * 2020-04-22 2020-08-11 山东第一医科大学(山东省医学科学院) 通过外周血循环肿瘤细胞检测结直肠癌患者braf基因v600e突变表达的试剂盒

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105588943A (zh) * 2016-01-28 2016-05-18 山东省肿瘤防治研究院 一种胃癌患者外周血循环肿瘤细胞Her-2基因的检测方法
WO2019232485A1 (fr) * 2018-05-31 2019-12-05 Nvigen, Inc. Analyse de sang précise permettant la prédiction de l'incidence et de la récurrence du cancer, et le guidage et la surveillance d'une intervention de traitement
CN110716041A (zh) * 2019-10-23 2020-01-21 郑州大学 一种用于胃癌早期筛查和诊断的血清蛋白标志物、试剂盒及检测方法
CN111521801A (zh) * 2020-04-22 2020-08-11 山东第一医科大学(山东省医学科学院) 通过外周血循环肿瘤细胞检测结直肠癌患者braf基因v600e突变表达的试剂盒

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