WO2015165290A1 - Uses of blood evs and associated molecules in tumor drug resistance detection - Google Patents

Uses of blood evs and associated molecules in tumor drug resistance detection Download PDF

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WO2015165290A1
WO2015165290A1 PCT/CN2015/071054 CN2015071054W WO2015165290A1 WO 2015165290 A1 WO2015165290 A1 WO 2015165290A1 CN 2015071054 W CN2015071054 W CN 2015071054W WO 2015165290 A1 WO2015165290 A1 WO 2015165290A1
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evs
blood
drug resistance
expression
tumor
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French (fr)
Chinese (zh)
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马鑫
金坚
陈震
何冬旭
陈蕴
蔡燕飞
陆扬帆
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/02—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material
    • G01N23/04—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and forming images of the material
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to extracellular vesicles (EVs) and related molecules thereof, including cell membrane proteins, RNA and DNA, in particular, transient receptor potential channel (TRPC5), breast cancer resistance protein (BCRP), multidrug resistance
  • TRPC5 transient receptor potential channel
  • BCRP breast cancer resistance protein
  • MRP1 drug-related protein-1
  • Cancer also known as Malignant neoplasm, is a disease caused by a disorder that controls the growth and proliferation of cells. In addition to uncontrolled growth of cancer cells, cancer cells locally invade the surrounding normal tissues and even transfer to other parts of the body via the internal circulatory system or lymphatic system. Among various diseases, cancer is the "number one killer.” According to the World Health Organization report, there were approximately 12.7 million new cancer patients in the world in 2008, and 7.6 million died of cancer, especially in developing countries. The number of new cancer cases reached 56%. It is estimated that 2,020 years ago, the global annual 15 million cancer patients will be added.
  • drugs are: (i) anthracyclines, taxanes, antimetabolites, anti-microtubules, alkylating agents, miscellaneous, etc.; Antiestrogens, aromatase inhibitors, etc.; (iii) Trastuzumab (anti-Her- 2 ), bevacizumab (anti-VEGF).
  • P-glycoprotein is currently The most important and most important ABC drug transporter, encoded by the multidrug resistance gene mdrl; (2) reduce the drug activity by causing the drug to be catabolized, common enzymes include GST, CYP450, etc.; (3) drug target mutation or Changes in expression levels, etc.; (4) tumor heterogeneity; (5) cancer stem cell resistance.
  • Tumor markers refer to some intracellular substances that can respond to cancerous cells during tumorigenesis and development.
  • MUC1 is a mucin which is a transmembrane glycoprotein expressed in normal cells and is normally expressed at the apical end of epithelial cells of the breast, gastrointestinal tract and genitourinary tract, and glycosylation is complete. MUC1 normal epithelium provides lubrication and protection, mediates signal transduction and cell adhesion.
  • MUC1 By phosphorylation in breast cancer cell line MCF-7, MUC1 binds to Rrb/SOS and is involved in receptor tyrosine kinase-mediated signal transduction, whereas tyrosine phosphorylation is a membrane receptor involved in signal transduction A key step in the process.
  • the expression characteristics of breast cancer MUC1 include: high expression, low abnormal expression; glycosylation, high sialylation; apical unclear, polar confusion; overexpression of cytoplasm and cell surface, and these molecules will be from breast cancer The cells enter the serum.
  • TRPC5 is a subtype of the transient receptor potential channels (TRP channel) family, which is a non-selective channel on the cell membrane that is transparent to calcium ions, mainly distributed in the brain, lungs, testes and Placenta, and is mainly involved in the formation of growth cones and brain development.
  • TRP channel transient receptor potential channels
  • MCF-7/ADM adriamycin-resistant breast cancer cells
  • TRPC5 is up-regulated in adriamycin-resistant breast cancer cells (MCF-7/ADM) and can indirectly mediate tumors. Expression levels of multidrug-resistant P-gp proteins in multidrug resistant tumor cells (eg, MCF-7/ADM).
  • MCF-7/ADM multidrug-resistant P-gp proteins in multidrug resistant tumor cells
  • BCRP Breast cancer resistance protein
  • BCRP A transmembrane protein of 655 amino acid residues belonging to the ABC transporter superfamily. BCRP has only 6 transmembrane regions and 1 ATP binding site, so it is called incomplete transporter molecule. It is speculated that BCRP has the same composition. Dimers or heterodimers function as transmembrane channels. The expression of BCRP can be detected in placental syncytiotrophoblast cells, small intestine and colonic mucosal epithelial cells, bile duct membrane, mammary gland lobular and vascular endothelial cells and stem cells in human normal tissues. It is speculated that BCRP can inhibit certain exogenous absorption of digestive tract.
  • Substances include anticancer drugs and toxic substances participate in the formation of physiological functions such as the placental barrier.
  • BCRP is associated with clinical chemosensitivity in a variety of tumors including acute myeloid leukemia, non-small cell lung cancer, and breast cancer.
  • Multidrug resistance-associated protein-1 is mainly distributed in tissues such as lung, testis, and peripheral blood mononuclear cells, but can be detected in almost all tissues.
  • M RP1 has a molecular weight of 190 kD and consists of 1531 amino acid residues and is a glycosylated phosphoprotein integrated into the cell membrane.
  • the mRNA encoding MRP1 is approximately 6.5 kb, and a small portion of the sequence is homologous to P-glycoprotein mRNA, both of which are members of the superfamily of transporters of ATPase activity. Therefore, the tissue distribution and function of MRP is similar to that of P-glycoprotein. It is also used as a drug output pump.
  • ATP energy is used to remove drugs from cells.
  • Many natural drugs such as antibiotics, alkaloids and the like are substrates of MRP1, but MRP The effect does not appear to be inhibited by the MD R modulator.
  • Transfection of mrp gene into normal cells can confer resistance to many chemotherapeutic drugs, suggesting that overexpression of mrp gene can lead to MDR.
  • Extracellular vesicles refer to vesicles in which the extracellular environment contains a large number of mobile cell membrane sources. These EVs mainly include exosomes, microvesicles, and apoptotic bodies. These dynamic EVs play an important role in cell-to-cell communication and immune regulation.
  • the lipid raft marker protein flotillin-2 is specifically expressed on EVs and can be used as a specific marker in the research process. Tumor cells also produce EVs, and researchers have found large amounts of EVs in the blood of patients with malignant gliomas, pancreatic cancer, stomach cancer, and acute leukemia.
  • EVs contain cell membrane proteins, RNA and DNA, which transport vesicle contents from donor cells to recipient cells via EVs, thereby mediating cell-to-cell communication, which in turn leads to tumor migration and invasion.
  • RNA and DNA transport vesicle contents from donor cells to recipient cells via EVs, thereby mediating cell-to-cell communication, which in turn leads to tumor migration and invasion.
  • RNA and DNA transport vesicle contents from donor cells to recipient cells via EVs, thereby mediating cell-to-cell communication, which in turn leads to tumor migration and invasion.
  • RNA and DNA transport vesicle contents from donor cells to recipient cells via EVs, thereby mediating cell-to-cell communication, which in turn leads to tumor migration and invasion.
  • the formation of these EVs The reasons for its association with chemotherapy resistance in breast cancer remain to be further studied.
  • the Applicant has provided an application of EVS-TRPC5 in detecting the degree of tumor resistance.
  • the present invention can show the transformation of tumor cell resistance, and has the advantages of simple detection method, high speed, high sensitivity and low cost.
  • the tumor is a breast cancer or other type of tumor.
  • the related cell membrane protein refers to the transient receptor potential channel, TRPC5.
  • the related cell membrane protein refers to a breast cancer resistance protein, BCRP.
  • the related cell membrane protein refers to a multidrug resistance-associated protein-1, MRP1.
  • the present invention can initially obtain the drug resistance of breast tumors by detecting the expression level of related molecules in blood EVs as an intermediate result.
  • the detection method adopted by the invention is simple and rapid, and can be detected only by taking a blood sample, which greatly reduces the suffering of the tumor patient;
  • the patient's drug resistance at this stage can be initially detected, and the other existing tumor drug resistance diagnosis technology can help the doctor to propose a more accurate treatment plan to obtain the best. Efficacy.
  • FIG. 1 is a schematic diagram showing the detection of the expression of TRPC5 of Example 1 on drug-resistant breast cancer cells EVs;
  • FIG. 2 is a diagram showing the blood and the process of TRPC5 of Example 2 in xenograft nude mice with drug-resistant breast cancer. Chemotherapy of the breast Schematic diagram of the detection of expression in blood EVs of neoplastic patients;
  • FIG. 3 is a schematic diagram showing the detection of MRP1 expression in the drug-resistant breast cancer cell MVs of Example 4;
  • FIG. 4 is a schematic diagram showing the detection of the expression of BCRP in the drug-resistant breast cancer cell MVs of Example 4;
  • FIG. 5 is a schematic diagram showing the detection of MRP1 and Flotillin-2 in the drug-resistant breast cancer cell MVs of Example 4;
  • FIG. 6 is a diagram showing the detection of the expression of BCRP and Flotillin-2 in drug-resistant breast cancer cells MVs of Example 4;
  • FIG. 7 is a schematic diagram showing the expression of MRP1 of Example 5 in blood MVs of a chemotherapy-derived breast tumor patient;
  • Figure 8 shows the indication of the expression of BCRP in Example 5 in blood MVs of patients with chemotherapy-derived breast tumors.
  • Wild type human breast cancer cells MCF-7/WT was purchased from the American Type Culture Collection (ATCC).
  • Adriamycin-resistant human breast cancer cells MCF-7/ADM is prepared and preserved by the Department of Pharmaceutical Design and Molecular Pharmacology of Jiangnan University, and its preparation method is as follows:
  • the newly revived wild-type human breast cancer cell MCF-7/WT cells (purchased from ATCC) were cultured for 2 to 3 generations under normal cell culture conditions to stabilize cell growth, and the cells were confluent with trypsin. Digestion and passage, the medium was updated the next day, and doxorubicin was added at the same concentration as 1/10 of MCF-7/WT IC50. The drug was changed again after the second day of administration, and the concentration of doxorubicin was maintained. Conventional subculture, after the cells grow stably, increase the drug concentration and continue to culture until the cells can grow stably in the medium with the doxorubicin concentration of 5 g/ml. That is, the whole preparation process lasts for 8 months.
  • TRPC5 antibody (ab63151) was purchased from Abeam, USA; goat polyclonal to rabbit IgG 15 nm Gold (ab27236) was purchased from Abeam, USA; TRPC5-siRNA was purchased from Invitrogen, USA. [0040] 3, the patient
  • TEM Hitachi purchased from Japan; laser confocal microscope available from Zeiss, Germany; the PCR instrument was purchased from Bio-Rad Company; CO 2 cell incubator temperature purchased from Thermo Corporation; nucleic acid electrophoresis system available from U.S. Bio-Rad.
  • Example 1 TRPC5 is expressed on EVs of drug-resistant breast cancer cells and participates in the formation of EVs.
  • MCF-7/ADM cells were seeded in a confocal dish at 3000 cells/well, and immunostained after the cells were attached, and the samples were incubated with BSA, TRPC5-anti-green fluorescent secondary antibody, and The expression and distribution of TRPC5 were observed under a focused microscope and photographed. The result is shown in Fig. 1B.
  • MCF-7/ADM cells cultured in vitro were first treated with Scrambled siRNA and TrpC5 siRNA, and then prepared into transmission electron microscope samples. The structural differences of MCF-7/ADM cells before and after TrpC5 siRNA interference were observed under transmission electron microscope. Take a photo record. The result is shown in Figure 1C.
  • Example 2 RT-PCR was used to detect the expression of TRPC5 in blood samples.
  • MCF-7/WT and MCF-7/ADM cells (5 ⁇ 10 6 cells/nude mice) were injected into the flank of female nude mice, and cultured for 4-8 weeks.
  • ADM (3mg/kg) was injected every three days in the tumor site of drug-resistant tumor-bearing mice, and TRPC5-siRNA or control siRNA (40pmol) was injected every three days in the tumor site of all tumor-bearing mice, and passed after 30 days.
  • Cardiac puncture techniques collect blood stored in heparin sodium solution and collect EVs.
  • Plasma samples were collected in a polypropylene centrifuge tube containing EDTA, first centrifuged at 3000 xg for 10 minutes to remove intact cells, and centrifuged at 2500 xg for 20 minutes to remove platelets and cell debris, and repeated the operation twice. Then, it was centrifuged at 16000 x g for 1 hour at 16000 x g for 1 hour, and precipitated as EVs. After washing with PBS, it was resuspended in PBS solution for use.
  • RT-PCR Using Reverse Transcription Kit Reverse Transcriptase M-MLV (RNase H-) Kit
  • TrpC5 forward, 5'-GACCTGATAA CCACTGAGAACCTGCTGAGC-3';
  • flotillin2 forward, 5'-CCAGAGACACTGTCCTTCCC-3';
  • MUC1 forward, 5'-TTCTTCCTGCTGCTGCTCCTCAC-3';
  • mdrl forward, 5'-CTTTCGAACTGCAAATATGCCTCC-3';
  • GAPDH forward, 5'-CAACGTGTCAGTGGTGGACC-3';
  • Primers for detecting a blood sample of a patient include:
  • TrpC5 forward, 5'-AGACTTGCCATGGGCCACCTCTCATCAGAACC-3'; [0069] reverse, 5-GAGGCGAGTTGTAACTTGTTCTTCCTGTCCATC-3';
  • flotillin2 forward, 5'-AGATCCGGCAGGAAGAGATT-3';
  • MUC 1 forward, 5'-CGACTACTACCA AGAGCTGCAGAGACAT-3';
  • GAPDH forward, 5'-GGACTCATGACCACAGTCCATGCCATCACT-3';
  • RT-PCR was used to detect the mRNA level of each protein in EVs of nude mice.
  • the mRNA levels of TrpC5, flotillin2, mdrl and MUCl were positive, indicating drug resistance.
  • EVs expressing TrpC5 secreted by tumor-bearing mice were released into the peripheral blood of nude mice as shown in Fig. 2A.
  • the transcription levels of TRPC5, flotillin2 and mdrl were significantly decreased compared with the control group.
  • the statistical results are shown in Figure 2A. This result further suggests that TRPC5 is closely related to the formation and transport of EVs.
  • RT-PCR was used to detect the mRNA levels of various proteins in EVs of blood samples.
  • the mRNA levels of TRPC5, flotillin2, mdrl and MUCl were highly expressed, and TRPC5 expression was strongly positive, while 12 In patients without chemotherapy, the mRNA levels of these proteins are lower, as shown in Figure 2B.
  • the results further indicate that EVs carrying TRPC5 are closely related to tumor resistance, suggesting that the expression of TRPC5 in blood EVs of patients with breast cancer can be used as a detection index in the diagnosis and treatment of drug-resistant breast cancer.
  • Example 3 Elisa method was used to detect the expression of TRPC5 in EVs in blood samples.
  • the content of TRPC5 in blood sample EVs was quantitatively determined by the double antibody sandwich method (Elisa method).
  • the commercially available or manually coated Elisa assay kit for the detection of human TRPC5 protein is used to detect samples of blood EVs lysed with protein lysate, and the content of TRP C5 in the sample is determined according to the conventional Elisa assay. , use the corresponding detection instrument to detect the signal strength.
  • the more TRPC5 in the sample the higher the relative detection value, and the higher the TRPC5 expression, the more likely the person who provides the blood sample will become resistant. High.
  • MRP1 antibody (ab24102) and BCRP antibody (ab3380) were purchased from Abeam, USA; goat anti-MUCl monoclonal antibody (sc-6825) was purchased from Santa Cruz, USA; Alexa Fluor 488 labeled Donkey Anti-Mouse IgG (H+ L) Antibody and Alexa Fluor 546-labeled donkey anti-goat IgG (H+L) Antibody was purchased from Invitrogen, USA.
  • the tumor samples were selected before surgery for 2-4 chemotherapy courses or preoperative untreated tumors. Blood samples were taken from 55 breast cancer patients, including 34 patients receiving 3-6 cycles after surgery. Patients with taxoid-like adjuvant chemotherapy (extracted before the next cycle) and 21 patients who did not receive chemotherapy before surgery.
  • Laser confocal microscope was purchased from Zeiss, Germany; PCR instrument was purchased from Bio-Rad, USA; CO 2 thermostatic cell culture incubator was purchased from Thermo Corporation, USA; nucleic acid electrophoresis system was purchased from Bio-Rad, USA; flow cytometry The instrument was purchased from BD Corporation of the United States.
  • Example 4 FISH method was used to detect the expression of MRP1 and BCRP in MVs of blood samples.
  • Method The patient tumor cells were inoculated into a confocal dish at 3000 cells/well, and immunostained after the cells were attached, and the samples were incubated with BSA blocking, MRP1/BCRP-anti, red fluorescent secondary antibody, and the nuclei were incubated. After counterstaining with PADI, the expression and distribution of MRP1 and BCRP were observed under laser confocal microscopy and photographed. The results are shown in Figure 3 and Figure 4.
  • the patient tumor cells were seeded in a confocal dish at 3000 cells/well, and immunostained after the cells were attached, and the samples were incubated with BSA blocking, MRP1/BCRP and Flotillin-2 anti-green fluorescent secondary antibody, and After counterstaining the nucleus with PADI, the expression and distribution of MRP1, BCRP and Flotillin-2 were observed under laser confocal microscopy and photographed. The results are shown in Figure 5 and Figure 6.
  • Example 5 RT-PCR method, flow cytometry was used to detect the expression of MRP1 and BCRP in MVs of blood samples.
  • Plasma samples were collected in polypropylene tubes containing EDTA, first centrifuged at 300 ⁇ g for 10 minutes to remove intact cells, centrifuged at 2500 ⁇ g for 20 minutes to remove platelets and cell debris, and repeated operations twice. Then, after centrifugation at 16000 xg for 1 hour, centrifuge at 16000 xg for 1 hour, precipitate into MVs, wash with PBS, and resuspend in PBS solution for use.
  • RT-PCR Using Reverse Transcription Kit Reverse Transcriptase M-MLV (RNase H-) Kit
  • TaKaRa Reverse transcribes the genome of MVs into cDNAs, which are then detected by conventional RT-PCR methods.
  • Primers for detecting a blood sample of a patient MRP1 include:
  • GAPDH forward, 5'-CTCCTGCACCACCACCAACTGCTTAGC-3';
  • MRP1 forward, 5'-CTGCACGACCTCCGCTTCAAGAT-3';
  • Flotillin-2 forward, 5'-AGATCCGGCAGGAAGAGATT-3';
  • MUC1 forward, 5 ' -CGACTACTACCAAGAGCTGCAGAG AGACAT-3 ';
  • Primers for detecting a blood sample of a patient BCRP include:
  • BCRP forward, 5'-CAG CCG TGG AAC TCT TTG TGG TAG AGA AG-3';
  • Flotillin-2 forward, 5,-AGATCCGGCAGGAAGAGATT-3,; [0116] Reverse, 5 ' -GCTTCTGCCTTGAGCTTCAT-3 '
  • MUCl forward, 5 ' -CGACTACTACCAAGAGCTGCAGAG AGACAT-3 '; [0118] Reverse, 5 ' -TGTAAGAGAGGCTGCTGCCACCATTACCTG-3 ' .
  • GAPDH forward. 5,-CTC CTG CAC CAC CAA CTG CTT AGC-3';
  • MVs were incubated at 4 degrees and then incubated with BSA-blocked, MRP1/BCRP and M UC 1 primary antibodies, Alexa Fluor 488 and Alexa Fluor 546 labeled secondary antibodies, using flow cytometry Detection.
  • mRNA levels of MRP1, BCRP, Flotillin-2, MUCl and GAPDH showed high expression (A curve of Fig. 7 and Fig. 8), and MRP1 and BCRP expression were strongly positive (B curves of Fig. 7 and Fig. 8).
  • the mRNA levels of these proteins were lower in 21 patients without chemotherapy.
  • MRP1 or BCRP in blood MVs of breast cancer patients can be used as a detection index in the diagnosis and treatment of drug-resistant breast cancer.
  • breast cancer tumors were selected as samples, and cell membrane proteins, RNA and DNA (TRPC5, BCRP, MRP1, Flotillin-2) specifically expressed in EVs of breast cancer cells were selected, by RT-PCR, flow Cell technology, or Elisa method to detect their specific expression, according to the level of expression, indicating the possibility of multidrug resistance in breast cancer.
  • RNA and DNA the amount of expression of these substances was measured by the same method as described above, thereby demonstrating the possibility of multidrug resistance.
  • the expression molecules used in the examples are all molecules related to multidrug resistance of all tumors, therefore, The method provided by the application is universal.

Abstract

Disclosed are uses of blood EVs and associated molecules in tumor drug resistance detection. A specific detection method comprises: (1) collecting blood samples of patients or animals suffering from tumors, removing, by centrifugation, complete cells, platelets, and cell debris in the samples, and collecting, by centrifugation, EVs in the samples; and (2) detecting, by using a chemiluminescence method, an RT-PCR method, flow cytometry, or an ELISA method, an expression of related cellular membrane protein, RNA, or DNA in the collected EVs, wherein if the expression is positive, a suspicious patient or animal with drug resistance is found. The associated molecules of the EVs comprise TRPC5, BCRP, or MRP1.

Description

发明名称：血液 EVs及其关联分子在肿瘤耐药检测中的应用 技术领域  Title of Invention: Application of Blood EVs and Their Associated Molecules in Tumor Resistance Detection
[0001] 本发明涉及细胞外囊泡 （EVs) 及其关联分子， 包括细胞膜蛋白、 RNA及 DNA ， 尤其是瞬吋受体电位通道 （TRPC5) ， 乳腺癌耐药蛋白 （BCRP) ， 多药耐药 相关蛋白 -1 (MRP1) 在检测肿瘤耐药程度中的应用， 属于生物蛋白质检测及利 用技术领域。  [0001] The present invention relates to extracellular vesicles (EVs) and related molecules thereof, including cell membrane proteins, RNA and DNA, in particular, transient receptor potential channel (TRPC5), breast cancer resistance protein (BCRP), multidrug resistance The application of drug-related protein-1 (MRP1) in the detection of tumor resistance is a field of biological protein detection and utilization technology.
背景技术  Background technique
[0002] 癌症 （Cancer) ， 亦称恶性肿瘤 （Malignant neoplasm) ， 为由控制细胞生长增 殖机制失常而引起的疾病。 癌细胞除了生长失控外， 还会局部侵入周遭正常组 织甚至经由体内循环系统或淋巴系统转移到身体其他部分。 在各种疾病中， 癌 症可谓是"头号杀手"。 世界卫生组织报告显示， 2008年全世界约有 1270万癌症新 增患者， 760万死于癌症， 尤其在发展中国家， 癌症新增例数达 56%， 据推测到 2 020年前， 全球每年将新增 1500万癌症患者。  [0002] Cancer, also known as Malignant neoplasm, is a disease caused by a disorder that controls the growth and proliferation of cells. In addition to uncontrolled growth of cancer cells, cancer cells locally invade the surrounding normal tissues and even transfer to other parts of the body via the internal circulatory system or lymphatic system. Among various diseases, cancer is the "number one killer." According to the World Health Organization report, there were approximately 12.7 million new cancer patients in the world in 2008, and 7.6 million died of cancer, especially in developing countries. The number of new cancer cases reached 56%. It is estimated that 2,020 years ago, the global annual 15 million cancer patients will be added.
[0003] 乳腺癌是女性常见的癌症， 严重威胁女性身心健康。 中国抗癌协会公布的最新 统计数字显示， 从 20世纪 90年代以来， 乳腺癌发病率和死亡率均持续稳定上升 ， 发病率以每年 4.476%的速度增长， 死亡率以每年 3%的速度增长。 化学药物治 疗是乳腺癌的主要治疗手段之一， 目前临床常用药物有： （i) 蒽环类， 紫杉类 ， 抗代谢类， 抗微管类， 烷化剂， 杂类等； （ii) 抗雌激素， 芳香化酶抑制剂等 ； (iii) 曲妥珠单抗 （抗 Her-2) 、 贝伐单抗 （抗 VEGF) 。 [0003] Breast cancer is a common cancer in women, which is a serious threat to women's physical and mental health. According to the latest statistics released by the China Anti-Cancer Association, since the 1990s, breast cancer morbidity and mortality have continued to rise steadily. The incidence rate has increased by 4.476% per year, and the mortality rate has increased at a rate of 3% per year. Chemotherapy is one of the main treatments for breast cancer. Currently, commonly used drugs are: (i) anthracyclines, taxanes, antimetabolites, anti-microtubules, alkylating agents, miscellaneous, etc.; Antiestrogens, aromatase inhibitors, etc.; (iii) Trastuzumab (anti-Her- 2 ), bevacizumab (anti-VEGF).
[0004] 但是， 据美国癌症协会报道， 90%以上肿瘤患者死于不同程度的化疗耐药。 对 化疗药物产生耐药已经成为肿瘤治疗的一大难题。 经过近几十年的研究， 目前 发现的耐药机制有 （1) 细胞膜 ABC药物转运蛋白表达增强， 减少药物摄入及增 加药物排出， P-糖蛋白 （P-glycoprotein, P-gp) 是目前研究最多也是最为重要的 ABC药物转运蛋白， 由多药耐药基因 mdrl编码； （2) 通过使药物发生异化降低 药物活性， 常见的酶有 GST、 CYP450等； （3) 药物作用靶点突变或表达水平变 化等； （4) 肿瘤异质性； （5) 肿瘤干细胞耐药等。 [0005] 目前， 早期诊断肿瘤的最新、 最有效的方法是通过验血寻找肿瘤标记物。 肿瘤 标志物指的是在肿瘤发生、 发展的过程中， 能反应细胞癌变的一些细胞内物质 。 临床上通过对各种肿瘤标志物的检测， 可以对相应肿瘤做早期诊断、 检测疗 效和肿瘤复发状况。 因此， 在深入研究耐药机制的基础上， 幵发肿瘤耐药分子 诊断指标、 方法， 实现试剂化， 达到耐药早期、 动态监测， 在现阶段对于乳腺 癌耐药的诊疗至关重要， 也为优化药物结构设计、 研发耐药逆转药物提供有益 借鉴， 因而具有重大而现实的社会价值。 [0004] However, according to the American Cancer Society, more than 90% of cancer patients die from varying degrees of chemotherapy resistance. Resistance to chemotherapy drugs has become a major problem in cancer treatment. After several decades of research, the current drug resistance mechanisms are (1) enhanced expression of ABC drug transporter in cell membranes, reduced drug intake and increased drug excretion. P-glycoprotein (P-gp) is currently The most important and most important ABC drug transporter, encoded by the multidrug resistance gene mdrl; (2) reduce the drug activity by causing the drug to be catabolized, common enzymes include GST, CYP450, etc.; (3) drug target mutation or Changes in expression levels, etc.; (4) tumor heterogeneity; (5) cancer stem cell resistance. [0005] Currently, the most recent and most effective method for early diagnosis of tumors is to find tumor markers by blood test. Tumor markers refer to some intracellular substances that can respond to cancerous cells during tumorigenesis and development. Clinically, through the detection of various tumor markers, early diagnosis, detection of curative effect and tumor recurrence can be performed on the corresponding tumor. Therefore, on the basis of in-depth study of drug resistance mechanisms, it is necessary to diagnose the molecular markers and methods of drug resistance, and to achieve reagents, to achieve early drug resistance and dynamic monitoring. At this stage, it is essential for the diagnosis and treatment of breast cancer resistance. It provides a useful reference for optimizing drug structure design and developing drug resistance reversal drugs, and thus has significant and realistic social value.
[0006] MUC1是一种粘蛋白， 在正常细胞表达吋是一种跨膜糖蛋白， 正常情况下， 在 乳腺、 胃肠道及泌尿生殖道的上皮细胞顶端表达， 糖基化完全。 MUC1度正常上 皮起润滑和保护作用、 介导信号转导和细胞黏附。  [0006] MUC1 is a mucin which is a transmembrane glycoprotein expressed in normal cells and is normally expressed at the apical end of epithelial cells of the breast, gastrointestinal tract and genitourinary tract, and glycosylation is complete. MUC1 normal epithelium provides lubrication and protection, mediates signal transduction and cell adhesion.
[0007] 在乳腺癌细胞株 MCF-7中通过磷酸化， MUC1能结合 Rrb/SOS， 参与受体酪氨 酸激酶介导的信号转导， 而酪氨酸磷酸化是膜受体参与信号转导的一个关键步 骤。 乳腺癌 MUC1的表达特点包括： 高表达、 异常表达低； 糖基化、 高唾液酸化 ； 顶端定为不清， 极性混乱； 细胞浆和细胞表面都有过度表达， 并且这些分子 会从乳腺癌细胞进入血清。 有研究者检测基底样乳腺癌患者， 发现高表达 MUC1 ， 大约有 92%的患者能检测出过表达的 MUC1。  [0007] By phosphorylation in breast cancer cell line MCF-7, MUC1 binds to Rrb/SOS and is involved in receptor tyrosine kinase-mediated signal transduction, whereas tyrosine phosphorylation is a membrane receptor involved in signal transduction A key step in the process. The expression characteristics of breast cancer MUC1 include: high expression, low abnormal expression; glycosylation, high sialylation; apical unclear, polar confusion; overexpression of cytoplasm and cell surface, and these molecules will be from breast cancer The cells enter the serum. Some researchers have detected basal-like breast cancer patients and found that MUC1 is highly expressed, and about 92% of patients can detect overexpressed MUC1.
[0008] TRPC5是瞬吋受体电位通道 （transient receptor potential channels, TRP通道） 家族的一个亚型， 它是细胞膜上能通透钙离子的非选择性通道， 主要分布于脑 、 肺、 睾丸和胎盘， 并主要参与生长锥的形成和脑的发育。 本研究室研究发现 ， 瞬吋受体电位通道 TRPC5与肿瘤多药耐药密切相关， TRPC5在耐阿霉素的乳 腺癌细胞 （MCF-7/ADM) 中发生明显上调， 并能间接介导肿瘤产生多药耐药的 P-gp蛋白在多药耐药肿瘤细胞 （如 MCF-7/ADM) 中的表达水平。 本申请人针对 该性质申请了相关专利， 专利名称为 《TRPC5作为药物靶点在逆转肿瘤多药耐 药中的应用》 （专利号： 201210318389.5) 。  [0008] TRPC5 is a subtype of the transient receptor potential channels (TRP channel) family, which is a non-selective channel on the cell membrane that is transparent to calcium ions, mainly distributed in the brain, lungs, testes and Placenta, and is mainly involved in the formation of growth cones and brain development. This study found that the transient receptor potential channel TRPC5 is closely related to multidrug resistance of tumors. TRPC5 is up-regulated in adriamycin-resistant breast cancer cells (MCF-7/ADM) and can indirectly mediate tumors. Expression levels of multidrug-resistant P-gp proteins in multidrug resistant tumor cells (eg, MCF-7/ADM). The applicant applied for a patent for this property, and the patent name was “TRPC5 as a drug target in reversing tumor multidrug resistance” (Patent No.: 201210318389.5).
[0009] 乳腺癌耐药蛋白（ breast cancer resistance protein, BCRP)是近年发现的与肿瘤多 药耐药有关的新的药物排出泵。 BCRP是含  [0009] Breast cancer resistance protein (BCRP) is a new drug discharge pump found in recent years related to tumor multidrug resistance. BCRP is included
655个氨基酸残基的跨膜蛋白， 属于 ABC转运蛋白超家族的成员。 BCRP仅有 6个 跨膜区和 1个 ATP结合位点， 故被称为不完整转运分子， 推测 BCRP通过组成同 二聚体或异二聚体构成跨膜通道而发挥功能。 人体正常组织中胎盘合体滋养层 细胞、 小肠和结肠粘膜上皮细胞、 胆小管膜、 乳腺小叶及血管内皮细胞和干细 胞均能检测到 BCRP的表达； 推测 BCRP具有抑制消化道吸收某些外源性物质 (包 括抗癌药和有毒物质)， 参与形成胎盘屏障等生理功能。 BCRP与急性髓性白血病 、 非小细胞肺癌、 乳腺癌等多种肿瘤的临床化疗敏感性有关。 A transmembrane protein of 655 amino acid residues belonging to the ABC transporter superfamily. BCRP has only 6 transmembrane regions and 1 ATP binding site, so it is called incomplete transporter molecule. It is speculated that BCRP has the same composition. Dimers or heterodimers function as transmembrane channels. The expression of BCRP can be detected in placental syncytiotrophoblast cells, small intestine and colonic mucosal epithelial cells, bile duct membrane, mammary gland lobular and vascular endothelial cells and stem cells in human normal tissues. It is speculated that BCRP can inhibit certain exogenous absorption of digestive tract. Substances (including anticancer drugs and toxic substances) participate in the formation of physiological functions such as the placental barrier. BCRP is associated with clinical chemosensitivity in a variety of tumors including acute myeloid leukemia, non-small cell lung cancer, and breast cancer.
[0010] 多药耐药相关蛋白 -1 (Multidrug resistance-associated protein- 1 , MRPl) 主要分 布于肺、 睾丸、 外周血单个核细胞等组织， 但在几乎所有组织中均可检测到。 M RP1的分子量为 190kD， 由 1531个氨基酸残基组成， 是整合于细胞膜的糖基化磷 蛋白。 编码 MRP1的 mRNA约有 6.5kb， 有一小部分序列与 P-糖蛋白 mRNA同源， 都属于 ATP酶活性的转运蛋白的超家族成员。 因此 MRP的组织分布和作用与 P-糖 蛋白相似， 也是作为药物输出泵， 利用 ATP能量将药物排出细胞之外， 许多天然 药物如抗生素、 生物碱之类都是 MRP1的底物， 但是 MRP的作用似乎不能被 MD R调控剂抑制。 将 mrp基因转染正常细胞可使之对许多化疗药物产生耐药性， 提 示 mrp基因过度表达能导致产生 MDR。  [0010] Multidrug resistance-associated protein-1 (MRP1) is mainly distributed in tissues such as lung, testis, and peripheral blood mononuclear cells, but can be detected in almost all tissues. M RP1 has a molecular weight of 190 kD and consists of 1531 amino acid residues and is a glycosylated phosphoprotein integrated into the cell membrane. The mRNA encoding MRP1 is approximately 6.5 kb, and a small portion of the sequence is homologous to P-glycoprotein mRNA, both of which are members of the superfamily of transporters of ATPase activity. Therefore, the tissue distribution and function of MRP is similar to that of P-glycoprotein. It is also used as a drug output pump. ATP energy is used to remove drugs from cells. Many natural drugs such as antibiotics, alkaloids and the like are substrates of MRP1, but MRP The effect does not appear to be inhibited by the MD R modulator. Transfection of mrp gene into normal cells can confer resistance to many chemotherapeutic drugs, suggesting that overexpression of mrp gene can lead to MDR.
[0011] 细胞外囊泡 （extracellular vesicles , EVs) 是指细胞外环境含有大量可移动的细 胞膜来源的囊泡。 这些 EVs主要包括外来体 exosomes、 微泡 microvesicles以及凋 亡小体 apoptotic bodies, 这些动态的 EVs在细胞间交流和免疫调节过程中起着至 关重要的作用。 脂筏标记蛋白 flotillin-2在 EVs上特异表达， 可作为特异性标志物 应用于研究过程中。 肿瘤细胞也产生 EVs， 研究者在恶性胶质瘤、 胰腺癌、 胃癌 和急性白血病病人的血液中发现都有大量的 EVs存在。 这些 EVs中包含有细胞膜 蛋白、 RNA及 DNA， 通过 EVs将囊泡内容物由供体细胞向受体细胞转运， 从而 介导细胞间交流， 进而引发肿瘤的迁移和侵袭， 然而这些 EVs结构的形成原因及 其与乳腺癌化疗耐药的相关性还有待进一步研究。  [0011] Extracellular vesicles (EVs) refer to vesicles in which the extracellular environment contains a large number of mobile cell membrane sources. These EVs mainly include exosomes, microvesicles, and apoptotic bodies. These dynamic EVs play an important role in cell-to-cell communication and immune regulation. The lipid raft marker protein flotillin-2 is specifically expressed on EVs and can be used as a specific marker in the research process. Tumor cells also produce EVs, and researchers have found large amounts of EVs in the blood of patients with malignant gliomas, pancreatic cancer, stomach cancer, and acute leukemia. These EVs contain cell membrane proteins, RNA and DNA, which transport vesicle contents from donor cells to recipient cells via EVs, thereby mediating cell-to-cell communication, which in turn leads to tumor migration and invasion. However, the formation of these EVs The reasons for its association with chemotherapy resistance in breast cancer remain to be further studied.
技术问题  technical problem
[0012] 针对现有技术存在的上述问题， 本申请人提供了一种 EVS-TRPC5在检测肿瘤耐 药程度中的应用。 本发明根据血液 EVs中 TRPC5的表达的高低， 可显示出肿瘤细 胞耐药转变的情况， 具有检测方法简单、 速度快、 灵敏度高、 成本低的优点。  [0012] In view of the above problems in the prior art, the Applicant has provided an application of EVS-TRPC5 in detecting the degree of tumor resistance. According to the expression of TRPC5 in blood EVs, the present invention can show the transformation of tumor cell resistance, and has the advantages of simple detection method, high speed, high sensitivity and low cost.
[0013] 同吋， 发现 MVs-BCRP, MVs-MRPl在肿瘤耐药程度检测中也具有相同作用， 且检测方法也相同。 [0013] Peer, found that MVs-BCRP, MVs-MRPl also have the same role in the detection of tumor resistance. And the detection method is the same.
问题的解决方案  Problem solution
技术解决方案  Technical solution
[0014] 血液 EVs及其关联分子在肿瘤耐药检测中的应用， 具体检测方法为：  [0014] The application of blood EVs and their associated molecules in the detection of tumor resistance, the specific detection methods are:
[0015] (1) 收集患有肿瘤的病人或动物的血液样本， 分别通过离心去除样本中的完 整细胞、 血小板及细胞碎片， 最后离心收集样本中的 EVs;  [0015] (1) collecting a blood sample of a patient or animal having a tumor, respectively removing whole cells, platelets and cell debris in the sample by centrifugation, and finally collecting the EVs in the sample by centrifugation;
[0016] (2) 采用化学发光法， RT-PCR法、 流式细胞技术、 或者 Elisa法检测收集到的[0016] (2) using chemiluminescence, RT-PCR, flow cytometry, or Elisa detection
EVs中相关细胞膜蛋白、 RNA或 DNA的表达情况， 若表达阳性， 为出现耐药性 的可疑病人或动物。 The expression of related cell membrane proteins, RNA or DNA in EVs, if positive, is a suspicious patient or animal with drug resistance.
[0017] 所述肿瘤为乳腺癌或其他类型肿瘤。 [0017] The tumor is a breast cancer or other type of tumor.
[0018] 所述相关细胞膜蛋白是指瞬吋受体电位通道即 TRPC5。 [0018] The related cell membrane protein refers to the transient receptor potential channel, TRPC5.
[0019] 或者， 所述相关细胞膜蛋白是指乳腺癌耐药蛋白即 BCRP。 [0019] Alternatively, the related cell membrane protein refers to a breast cancer resistance protein, BCRP.
[0020] 或者， 所述相关细胞膜蛋白是指多药耐药相关蛋白 -1即 MRP1。 [0020] Alternatively, the related cell membrane protein refers to a multidrug resistance-associated protein-1, MRP1.
发明的有益效果  Advantageous effects of the invention
有益效果  Beneficial effect
[0021] 1、 本发明通过检测血液 EVs中关联分子的表达水平， 作为一个中间结果， 可初 步得到乳腺肿瘤的耐药情况。 关联分子的表达水平越高， 则提供血液样本者发 生耐药的可能性也就越高。  [0021] 1. The present invention can initially obtain the drug resistance of breast tumors by detecting the expression level of related molecules in blood EVs as an intermediate result. The higher the expression level of the associated molecule, the higher the likelihood that the blood sample will be resistant.
[0022] 2、 本发明采用的检测方法简单快速， 只需抽取血液样本便可检测， 极大程度 地减轻了肿瘤病人的痛苦； [0022] 2. The detection method adopted by the invention is simple and rapid, and can be detected only by taking a blood sample, which greatly reduces the suffering of the tumor patient;
[0023] 3、 通过本发明可以初步检测出病人在该阶段的耐药情况， 同吋联合其它已有 的肿瘤耐药诊断技术， 可有利于指导医生提出更加准确的治疗方案， 以获得最 佳疗效。 [0023] 3. Through the invention, the patient's drug resistance at this stage can be initially detected, and the other existing tumor drug resistance diagnosis technology can help the doctor to propose a more accurate treatment plan to obtain the best. Efficacy.
对附图的简要说明  Brief description of the drawing
附图说明  DRAWINGS
[0024] 图 1为实施例 1的 TRPC5在耐药乳腺癌细胞 EVs上的表达情况检测示意图； [0025] 图 2为实施例 2的 TRPC5在耐药乳腺癌的异种移植瘤裸鼠血液和经化疗的乳腺肿 瘤病人的血液 EVs中的表达情况检测示意图； 1 is a schematic diagram showing the detection of the expression of TRPC5 of Example 1 on drug-resistant breast cancer cells EVs; [0025] FIG. 2 is a diagram showing the blood and the process of TRPC5 of Example 2 in xenograft nude mice with drug-resistant breast cancer. Chemotherapy of the breast Schematic diagram of the detection of expression in blood EVs of neoplastic patients;
[0026] 图 3为实施例 4的 MRP1在耐药乳腺癌细胞 MVs上的表达情况检测示意图； 3 is a schematic diagram showing the detection of MRP1 expression in the drug-resistant breast cancer cell MVs of Example 4;
[0027] 图 4为实施例 4的 BCRP在耐药乳腺癌细胞 MVs上的表达情况检测示意图； 4 is a schematic diagram showing the detection of the expression of BCRP in the drug-resistant breast cancer cell MVs of Example 4;
[0028] 图 5为实施例 4的 MRP1和 Flotillin-2在耐药乳腺癌细胞 MVs上的表达情况检测示 意图； 5 is a schematic diagram showing the detection of MRP1 and Flotillin-2 in the drug-resistant breast cancer cell MVs of Example 4;
[0029] 图 6为实施例 4的 BCRP和 Flotillin-2在耐药乳腺癌细胞 MVs上的表达情况检测示 意图；  6 is a diagram showing the detection of the expression of BCRP and Flotillin-2 in drug-resistant breast cancer cells MVs of Example 4;
[0030] 图 7为实施例 5的 MRP1在经化疗的乳腺肿瘤病人的血液 MVs中的表达情况检测 示意图；  7 is a schematic diagram showing the expression of MRP1 of Example 5 in blood MVs of a chemotherapy-derived breast tumor patient;
[0031] 图 8实施例 5的 BCRP在经化疗的乳腺肿瘤病人的血液 MVs中的表达情况检测示 意图。  Figure 8 shows the indication of the expression of BCRP in Example 5 in blood MVs of patients with chemotherapy-derived breast tumors.
本发明的实施方式 Embodiments of the invention
[0032] 一、 TRPC5在检测乳腺癌耐药程度中的应用 [0032] First, the application of TRPC5 in detecting the degree of drug resistance of breast cancer
[0033] 实施例中所使用的材料来源或制备方法如下： [0033] The source or preparation method of the materials used in the examples are as follows:
[0034] 1、 细胞 [0034] 1, cells
[0035] (1) 野生型人乳腺癌细胞 MCF-7/WT购自美国模式培养物寄存库 （ATCC) 。  [0035] (1) Wild type human breast cancer cells MCF-7/WT was purchased from the American Type Culture Collection (ATCC).
[0036] (2) 耐阿霉素人乳腺癌细胞 MCF-7/ADM由江南大学药物设计与分子药理学研 究室制备并保存， 其制备方法如下：  [0036] (2) Adriamycin-resistant human breast cancer cells MCF-7/ADM is prepared and preserved by the Department of Pharmaceutical Design and Molecular Pharmacology of Jiangnan University, and its preparation method is as follows:
[0037] 将新复苏的野生型人乳腺癌细胞 MCF-7/WT细胞 （购自 ATCC) 于细胞培养常 规条件下培养 2代〜 3代， 使细胞生长稳定， 待细胞汇合吋用胰酶进行消化并传 代， 第二天更新培养基， 同吋以 MCF-7/WT IC50的 1/10为起始浓度加入阿霉素， 加药第二天后再次换液， 并维持阿霉素的浓度进行常规传代培养， 待细胞稳定 生长后提高药物浓度继续培养， 直到细胞可在阿霉素浓度为 5 g/ml的培养基中稳 定生长， 即得， 整个制备过程历吋 8个月。  [0037] The newly revived wild-type human breast cancer cell MCF-7/WT cells (purchased from ATCC) were cultured for 2 to 3 generations under normal cell culture conditions to stabilize cell growth, and the cells were confluent with trypsin. Digestion and passage, the medium was updated the next day, and doxorubicin was added at the same concentration as 1/10 of MCF-7/WT IC50. The drug was changed again after the second day of administration, and the concentration of doxorubicin was maintained. Conventional subculture, after the cells grow stably, increase the drug concentration and continue to culture until the cells can grow stably in the medium with the doxorubicin concentration of 5 g/ml. That is, the whole preparation process lasts for 8 months.
[0038] 2、 抗体及 siRNA  2, antibodies and siRNA
[0039] TRPC5 antibody (ab63151)购自美国 Abeam公司； goat polyclonal to rabbit IgG 15 nm Gold (ab27236)购自美国 Abeam公司； TRPC5-siRNA购自美国 Invitrogen公司 [0040] 3、 病人 [0039] TRPC5 antibody (ab63151) was purchased from Abeam, USA; goat polyclonal to rabbit IgG 15 nm Gold (ab27236) was purchased from Abeam, USA; TRPC5-siRNA was purchased from Invitrogen, USA. [0040] 3, the patient
[0041] 血液样本采自 29位乳腺癌患者， 其中 17位患者接受过蒽环类 /紫杉烷类药物化 疗， 12位患者未接受过任何化疗药物治疗。  [0041] Blood samples were collected from 29 breast cancer patients, 17 of whom underwent anthracycline/taxane chemotherapy, and 12 patients did not receive any chemotherapy.
[0042] 4、 实验仪器 [0042] 4. Experimental instrument
[0043] 透射电镜购自日本 Hitachi公司； 激光共聚焦显微镜购自德国 Zeiss公司； PCR仪 购自美国 Bio-Rad公司； CO 2恒温细胞培养箱购自美国 Thermo公司； 核酸电泳仪 系统购自美国 Bio-Rad公司。 [0043] TEM Hitachi, purchased from Japan; laser confocal microscope available from Zeiss, Germany; the PCR instrument was purchased from Bio-Rad Company; CO 2 cell incubator temperature purchased from Thermo Corporation; nucleic acid electrophoresis system available from U.S. Bio-Rad.
[0044]  [0044]
[0045] 实施例 1 : TRPC5在耐药乳腺癌细胞的 EVs上表达， 且参与 EVs的形成。  Example 1 : TRPC5 is expressed on EVs of drug-resistant breast cancer cells and participates in the formation of EVs.
[0046] 方法： 收集体外培养的 MCF-7/ADM和 MCF-7/WT细胞， 制备成透射电镜样本Method: Collecting MCF-7/ADM and MCF-7/WT cells cultured in vitro to prepare TEM samples
， 在透射电镜下观察两种细胞的结构差异， 并拍照记录。 结果如图 1A所示。 The structural differences between the two cells were observed under transmission electron microscopy and photographed. The result is shown in Fig. 1A.
[0047] MCF-7/ADM细胞的透射电镜样本经过 BSA封闭、 TRPC5—抗、 免疫金标记二 抗孵育后， 在透射电镜下观察 TRPC5的分布情况， 并拍照记录。 结果如图 1B所 示。 [0047] Transmission electron microscopy samples of MCF-7/ADM cells were incubated with BSA, TRPC5-antibody, and immunogold-labeled secondary antibodies, and the distribution of TRPC5 was observed under transmission electron microscopy. The result is shown in Figure 1B.
[0048] 将 MCF-7/ADM细胞以 3000个 /孔接种于共聚焦小皿中， 待细胞贴壁后进行免疫 染色， 样本经 BSA封闭、 TRPC5—抗、 绿色荧光二抗孵育后， 在激光共聚焦显微 镜下观察 TRPC5的表达及分布情况， 并拍照记录。 结果如图 1B所示。  [0048] MCF-7/ADM cells were seeded in a confocal dish at 3000 cells/well, and immunostained after the cells were attached, and the samples were incubated with BSA, TRPC5-anti-green fluorescent secondary antibody, and The expression and distribution of TRPC5 were observed under a focused microscope and photographed. The result is shown in Fig. 1B.
[0049] 体外培养的 MCF-7/ADM细胞先经 Scrambled siRNA和 TrpC5 siRNA处理， 然后 同样制备成透射电镜样本， 在透射电镜下观察 MCF-7/ADM细胞经 TrpC5 siRNA 干扰前后的结构差异， 并拍照记录。 结果如图 1C所示。  [0049] MCF-7/ADM cells cultured in vitro were first treated with Scrambled siRNA and TrpC5 siRNA, and then prepared into transmission electron microscope samples. The structural differences of MCF-7/ADM cells before and after TrpC5 siRNA interference were observed under transmission electron microscope. Take a photo record. The result is shown in Figure 1C.
[0050] 结果：  [0050] Results:
[0051] 如图 1A所示， 透射电镜结果显示， 与 MCF-7/WT细胞相比， MCF-7/ADM细胞 膜表面产生了大量的 EV结构。 如图 1B所示， 免疫荧光染色和免疫透射电镜结果 均显示， TrpC5在 EVs上高量表达。 如图 1C所示， 当 MCF-7/ADM细胞分别经 Sera mbled siRNA和 TRPC5 siRNA  As shown in FIG. 1A, transmission electron microscopy results showed that a large amount of EV structure was generated on the surface of MCF-7/ADM cells as compared with MCF-7/WT cells. As shown in Figure 1B, immunofluorescence staining and immuno-TEM results showed that TrpC5 was highly expressed on EVs. As shown in Figure 1C, when MCF-7/ADM cells were Sera mbled siRNA and TRPC5 siRNA, respectively
处理后， 透射电镜结果显示 TRPC5被抑制后， MCF-7/ADM细胞膜表面的 EVs明 显减少。 这些结果表明， 与野生型 MCF-7细胞相比， 耐阿霉素的 MCF-7细胞表面 产生大量的 EVs结构， TRPC5蛋白在该结构上大量表达， 并参与 EVs的形成。 因 为在耐药细胞的 EVs中才能检测到 TRPC5 , 说明 TRPC5和耐药是相对应的。 After treatment, the results of transmission electron microscopy showed that the EVs on the surface of MCF-7/ADM cells were significantly reduced after TRPC5 was inhibited. These results indicate that the doxorubicin-resistant MCF-7 cell surface is compared to wild-type MCF-7 cells. A large number of EVs are produced, and the TRPC5 protein is abundantly expressed on the structure and participates in the formation of EVs. Because TRPC5 can be detected in EVs of drug-resistant cells, it indicates that TRPC5 and resistance are corresponding.
[0052]  [0052]
[0053] 实施例 2: RT-PCR法检测血液样本 EVs中 TRPC5的表达情况。  Example 2: RT-PCR was used to detect the expression of TRPC5 in blood samples.
[0054] 方法： 在雌性裸鼠的侧腹分别注射 MCF-7/WT和 MCF-7/ADM细胞 （5x10 6个细 胞 /裸鼠） ， 养殖 4~8周， 当肿瘤长至约 200mm 3吋， 在耐药荷瘤小鼠肿瘤部位每 三天注射一次 ADM (3mg/kg) ， 同吋在所有荷瘤小鼠肿瘤部位每三天注射一次 TRPC5-siRNA或对照 siRNA (40pmol) ， 30天后通过心脏穿刺技术收集血液保 存在肝素钠溶液中， 并收集 EVs。 Methods: MCF-7/WT and MCF-7/ADM cells (5× 10 6 cells/nude mice) were injected into the flank of female nude mice, and cultured for 4-8 weeks. When the tumor grew to about 200 mm 3吋, ADM (3mg/kg) was injected every three days in the tumor site of drug-resistant tumor-bearing mice, and TRPC5-siRNA or control siRNA (40pmol) was injected every three days in the tumor site of all tumor-bearing mice, and passed after 30 days. Cardiac puncture techniques collect blood stored in heparin sodium solution and collect EVs.
[0055] 收集血液样本中的 EVs: 血浆样本收集在含 EDTA的聚丙烯离心管中， 首先以 3 OOxg离心 10分钟去除完整细胞， 以 2500xg离心 20分钟去除血小板和细胞碎片， 重复操作 2次， 然后在 4度条件下以 16000xg离心 1小吋、 lOOOOOxg离心 1小吋， 沉 淀为 EVs， 用 PBS洗涤后， 重悬在 PBS溶液中备用。  [0055] Collecting EVs in blood samples: Plasma samples were collected in a polypropylene centrifuge tube containing EDTA, first centrifuged at 3000 xg for 10 minutes to remove intact cells, and centrifuged at 2500 xg for 20 minutes to remove platelets and cell debris, and repeated the operation twice. Then, it was centrifuged at 16000 x g for 1 hour at 16000 x g for 1 hour, and precipitated as EVs. After washing with PBS, it was resuspended in PBS solution for use.
[0056] RT-PCR: 运用反转录试剂盒 Reverse Transcriptase M-MLV (RNase H-) Kit [0056] RT-PCR: Using Reverse Transcription Kit Reverse Transcriptase M-MLV (RNase H-) Kit
(TaKaRa)将 EVs的基因组反转录成 cDNAs, 然后用常规的 RT-PCR方法进行检测 。 用于检测裸鼠血液样本的引物包括： (TaKaRa) The EVs genome was reverse transcribed into cDNAs and then detected by conventional RT-PCR methods. Primers for detecting blood samples from nude mice include:
[0057] TrpC5: forward, 5'-GACCTGATAA CCACTGAGAACCTGCTGAGC-3';  [0057] TrpC5: forward, 5'-GACCTGATAA CCACTGAGAACCTGCTGAGC-3';
[0058] reverse, 5'-GACAACCTCT TGCCAAATGGGTCTTGATGC-3';  [0058] reverse, 5'-GACAACCTCT TGCCAAATGGGTCTTGATGC-3';
[0059] flotillin2: forward, 5'-CCAGAGACACTGTCCTTCCC-3';  [0059] flotillin2: forward, 5'-CCAGAGACACTGTCCTTCCC-3';
[0060] reverse, 5'-CATTGGAGCAAGGAGACAGAG-3';  [0060] reverse, 5'-CATTGGAGCAAGGAGACAGAG-3';
[0061] MUC1: forward, 5'-TTCTTCCTGCTGCTGCTCCTCAC-3';  [0061] MUC1: forward, 5'-TTCTTCCTGCTGCTGCTCCTCAC-3';
[0062] reverse, 5'-GCACATCACT CACGCTGACGTCTG-3';  [0062] reverse, 5'-GCACATCACT CACGCTGACGTCTG-3';
[0063] mdrl: forward, 5'-CTTTCGAACTGCAAATATGCCTCC-3';  [0063] mdrl: forward, 5'-CTTTCGAACTGCAAATATGCCTCC-3';
[0064] reverse, 5'-GAGTTAGGAATGTAGCCCAGG-3';  [0064] reverse, 5'-GAGTTAGGAATGTAGCCCAGG-3';
[0065] GAPDH: forward, 5'-CAACGTGTCAGTGGTGGACC-3';  [0065] GAPDH: forward, 5'-CAACGTGTCAGTGGTGGACC-3';
[0066] reverse,5'-AGCAGTGAGGGTCTCTCTCTTC-3'.  [0066] reverse, 5'-AGCAGTGAGGGTCTCTCTCTTC-3'.
[0067] 用于检测病人血液样本的引物包括：  [0067] Primers for detecting a blood sample of a patient include:
[0068] TrpC5: forward, 5'-AGACTTGCCATGGGCCACCTCTCATCAGAACC-3'; [0069] reverse, 5-GAGGCGAGTTGTAACTTGTTCTTCCTGTCCATC-3'; [0068] TrpC5: forward, 5'-AGACTTGCCATGGGCCACCTCTCATCAGAACC-3'; [0069] reverse, 5-GAGGCGAGTTGTAACTTGTTCTTCCTGTCCATC-3';
[0070] flotillin2: forward, 5'-AGATCCGGCAGGAAGAGATT-3'; [0070] flotillin2: forward, 5'-AGATCCGGCAGGAAGAGATT-3';
[0071] reverse, 5'-GCTTCTGCCTTGAGCTTCAT-3'; [0071] reverse, 5'-GCTTCTGCCTTGAGCTTCAT-3';
[0072] MUC 1： forward,5'-CGACTACTACCA AGAGCTGCAGAGAGACAT-3';  [0072] MUC 1: forward, 5'-CGACTACTACCA AGAGCTGCAGAGAGACAT-3';
[0073] reverse, 5'-TGTAAGAGAGGCTGCTGCCA CCATTACCTG-3';  [0073] reverse, 5'-TGTAAGAGAGGCTGCTGCCA CCATTACCTG-3';
[0074] mdrl： forward, 5'-CTGTTTGACTGCAGCATTGCTGAG AACAT-3';  [0074] mdrl: forward, 5'-CTGTTTGACTGCAGCATTGCTGAG AACAT-3';
[0075] reverse, 5'-CTGGCGCTTTGTTCCAGCCTGGACACTGAC-3';  [0075] reverse, 5'-CTGGCGCTTTGTTCCAGCCTGGACACTGAC-3';
[0076] GAPDH: forward, 5'-GGACTCATGACCACAGTCCATGCCATCACT-3';  [0076] GAPDH: forward, 5'-GGACTCATGACCACAGTCCATGCCATCACT-3';
[0077] reverse, 5'-CTTGGAGGCCATGTGGGCCATGAGGTCCAC-3'.  [0077] reverse, 5'-CTTGGAGGCCATGTGGGCCATGAGGTCCAC-3'.
[0078] 结果： RT-PCR检测裸鼠血液样本 EVs中各蛋白的 mRNA水平， 7只耐药荷瘤小 鼠中， TrpC5、 flotillin2、 mdrl和 MUCl的 mRNA水平均显示阳性表达， 表明耐药 荷瘤小鼠肿瘤分泌的表达有 TrpC5的 EVs被释放到了裸鼠的外周血中， 如图 2A所 示。 当耐药肿瘤经 TRPC5-siRNA处理后， 与对照组相比 TRPC5、 flotillin2和 mdrl 的转录水平明显下降， 统计结果见图 2A， 该结果进一步提示 TRPC5与 EVs的形成 和转运密切相关。  [0078] Results: RT-PCR was used to detect the mRNA level of each protein in EVs of nude mice. In 7 drug-resistant mice, the mRNA levels of TrpC5, flotillin2, mdrl and MUCl were positive, indicating drug resistance. EVs expressing TrpC5 secreted by tumor-bearing mice were released into the peripheral blood of nude mice as shown in Fig. 2A. When the drug-resistant tumors were treated with TRPC5-siRNA, the transcription levels of TRPC5, flotillin2 and mdrl were significantly decreased compared with the control group. The statistical results are shown in Figure 2A. This result further suggests that TRPC5 is closely related to the formation and transport of EVs.
[0079] RT-PCR检测病人血液样本 EVs中各蛋白的 mRNA水平， 17位经化疗的病人中， TRPC5、 flotillin2、 mdrl和 MUCl的 mRNA水平均显示高表达， 且 TRPC5表达为 强阳性， 而 12位未经化疗的病人中， 这些蛋白的 mRNA水平均较低， 如图 2B所 示。 该结果进一步表明了携带有 TRPC5的 EVs与肿瘤耐药有密切的联系， 提示乳 腺癌病人血液 EVs中 TRPC5的表达可作为检测指标应用于耐药乳腺癌的诊断治疗 中。  [0079] RT-PCR was used to detect the mRNA levels of various proteins in EVs of blood samples. In 17 patients with chemotherapy, the mRNA levels of TRPC5, flotillin2, mdrl and MUCl were highly expressed, and TRPC5 expression was strongly positive, while 12 In patients without chemotherapy, the mRNA levels of these proteins are lower, as shown in Figure 2B. The results further indicate that EVs carrying TRPC5 are closely related to tumor resistance, suggesting that the expression of TRPC5 in blood EVs of patients with breast cancer can be used as a detection index in the diagnosis and treatment of drug-resistant breast cancer.
[0080]  [0080]
[0081] 实施例 3: Elisa法检测血液样本 EVs中 TRPC5的表达情况。  Example 3: Elisa method was used to detect the expression of TRPC5 in EVs in blood samples.
[0082] 采用双抗体夹心法 （Elisa法） 定量测定血样本 EVs中 TRPC5的含量。 运用商业 化的或手动包被的用于检测人源性 TRPC5蛋白的 Elisa检测试剂盒， 检测样本为 用蛋白裂解液裂解好的血液 EVs样本， 根据常规的 Elisa检测方法测定样本中 TRP C5的含量， 用相应检测仪器检测信号强度。 样本中的 TRPC5越多， 相对检测值 就越高， TRPC5就表达为阳性， 那么提供血液样本者发生耐药的可能性也就越 高。 [0082] The content of TRPC5 in blood sample EVs was quantitatively determined by the double antibody sandwich method (Elisa method). The commercially available or manually coated Elisa assay kit for the detection of human TRPC5 protein is used to detect samples of blood EVs lysed with protein lysate, and the content of TRP C5 in the sample is determined according to the conventional Elisa assay. , use the corresponding detection instrument to detect the signal strength. The more TRPC5 in the sample, the higher the relative detection value, and the higher the TRPC5 expression, the more likely the person who provides the blood sample will become resistant. High.
[0083]  [0083]
[0084] 二、 MVs-BCRP， MVs-MRPl在检测乳腺癌耐药程度中的应用  [0084] Second, MVs-BCRP, MVs-MRPl in the detection of breast cancer resistance
[0085] 实施例中所使用的材料来源或制备方法如下： [0085] The source or preparation method of the materials used in the examples are as follows:
[0086] 1、 抗体及 siRNA [0086] 1. Antibodies and siRNA
[0087] MRP1 antibody (ab24102)和 BCRP antibody (ab3380)购自美国 Abeam公司； goat anti-MUCl monoclonal antibody (sc-6825)购自美国 Santa Cruz公司； Alexa Fluor 488 labeled Donkey Anti-Mouse IgG (H+L) Antibody和 Alexa Fluor 546-labeled donkey anti-goat IgG (H+L) Antibody购自美国 Invitrogen公司。  [0087] MRP1 antibody (ab24102) and BCRP antibody (ab3380) were purchased from Abeam, USA; goat anti-MUCl monoclonal antibody (sc-6825) was purchased from Santa Cruz, USA; Alexa Fluor 488 labeled Donkey Anti-Mouse IgG (H+ L) Antibody and Alexa Fluor 546-labeled donkey anti-goat IgG (H+L) Antibody was purchased from Invitrogen, USA.
[0088] 2、 病人  [0088] 2, the patient
[0089] 肿瘤样本选取的是术前接受 2-4个化疗疗程或术前未经治疗的肿瘤， 血液样本 采自 55位乳腺癌患者， 包括 34个在手术后接受 3-6个周期蒽环类紫杉类辅助化疗 (下一个周期之前提取） 的患者， 以及 21个在手术前未接受化疗的患者。  [0089] The tumor samples were selected before surgery for 2-4 chemotherapy courses or preoperative untreated tumors. Blood samples were taken from 55 breast cancer patients, including 34 patients receiving 3-6 cycles after surgery. Patients with taxoid-like adjuvant chemotherapy (extracted before the next cycle) and 21 patients who did not receive chemotherapy before surgery.
[0090] 3、 实验仪器 [0090] 3. Experimental instrument
[0091] 激光共聚焦显微镜购自德国 Zeiss公司； PCR仪购自美国 Bio-Rad公司； CO 2恒 温细胞培养箱购自美国 Thermo公司； 核酸电泳仪系统购自美国 Bio-Rad公司； 流 式细胞仪购自美国 BD公司。 [0091] Laser confocal microscope was purchased from Zeiss, Germany; PCR instrument was purchased from Bio-Rad, USA; CO 2 thermostatic cell culture incubator was purchased from Thermo Corporation, USA; nucleic acid electrophoresis system was purchased from Bio-Rad, USA; flow cytometry The instrument was purchased from BD Corporation of the United States.
[0092]  [0092]
[0093] 实施例 4: FISH法检测血液样本 MVs中 MRP1及 BCRP的表达情况。  Example 4: FISH method was used to detect the expression of MRP1 and BCRP in MVs of blood samples.
[0094] 方法： 将患者肿瘤细胞以 3000个 /孔接种于共聚焦小皿中， 待细胞贴壁后进行 免疫染色， 样本经 BSA封闭、 MRP1/BCRP—抗、 红色荧光二抗孵育， 并对细 胞核用 PADI复染后， 在激光共聚焦显微镜下观察 MRP1， BCRP的表达及分布情 况， 并拍照记录。 结果如图 3、 图 4所示。  [0094] Method: The patient tumor cells were inoculated into a confocal dish at 3000 cells/well, and immunostained after the cells were attached, and the samples were incubated with BSA blocking, MRP1/BCRP-anti, red fluorescent secondary antibody, and the nuclei were incubated. After counterstaining with PADI, the expression and distribution of MRP1 and BCRP were observed under laser confocal microscopy and photographed. The results are shown in Figure 3 and Figure 4.
[0095] 将患者肿瘤细胞以 3000个 /孔接种于共聚焦小皿中， 待细胞贴壁后进行免疫染 色， 样本经 BSA封闭、 MRP1/BCRP和 Flotillin-2—抗、 绿色荧光二抗孵育， 并 对细胞核用 PADI复染后， 在激光共聚焦显微镜下观察 MRP1， BCRP, Flotillin-2 的表达及分布情况， 并拍照记录。 结果如图 5、 图 6所示。  [0095] The patient tumor cells were seeded in a confocal dish at 3000 cells/well, and immunostained after the cells were attached, and the samples were incubated with BSA blocking, MRP1/BCRP and Flotillin-2 anti-green fluorescent secondary antibody, and After counterstaining the nucleus with PADI, the expression and distribution of MRP1, BCRP and Flotillin-2 were observed under laser confocal microscopy and photographed. The results are shown in Figure 5 and Figure 6.
[0096] 结果： [0097] 免疫荧光染色结果显示， MRP1和 BCRP均在化疗患者肿瘤样本的 MVs上高量表 达 （图 3、 图 4) ， MRP1 , BCRP和 Flotillin-2在化疗患者肿瘤样本的 MVs上高量 表达 （图 5、 图 6) 。 这些结果表明， MRP1或者 BCRP的高表达， 可以作为检测 化疗的指标。 [0096] Results: [0097] Immunofluorescence staining showed that both MRP1 and BCRP were highly expressed in MVs of tumor samples from chemotherapy patients (Fig. 3, Fig. 4), and MRP1, BCRP and Flotillin-2 were highly expressed in MVs of tumor samples from chemotherapy patients. (Figure 5, Figure 6). These results indicate that high expression of MRP1 or BCRP can be used as an indicator for detecting chemotherapy.
[0098]  [0098]
[0099] 实施例 5: RT-PCR法， 流式细胞技术检测血液样本 MVs中 MRP1及 BCRP的表达 情况。  Example 5: RT-PCR method, flow cytometry was used to detect the expression of MRP1 and BCRP in MVs of blood samples.
[0100] 方法： 收集血液样本中的 MVs: 血浆样本收集在含 EDTA的聚丙烯离心管中， 首先以 300xg离心 10分钟去除完整细胞， 以 2500xg离心 20分钟去除血小板和细胞 碎片， 重复操作 2次， 然后在 4度条件下以 16000xg离心 1小吋、 lOOOOOxg离心 1小 吋， 沉淀为 MVs， 用 PBS洗涤后， 重悬在 PBS溶液中备用。  Method: Collecting MVs in blood samples: Plasma samples were collected in polypropylene tubes containing EDTA, first centrifuged at 300×g for 10 minutes to remove intact cells, centrifuged at 2500×g for 20 minutes to remove platelets and cell debris, and repeated operations twice. Then, after centrifugation at 16000 xg for 1 hour, centrifuge at 16000 xg for 1 hour, precipitate into MVs, wash with PBS, and resuspend in PBS solution for use.
[0101] RT-PCR: 运用反转录试剂盒 Reverse Transcriptase M-MLV (RNase H-) Kit RT-PCR: Using Reverse Transcription Kit Reverse Transcriptase M-MLV (RNase H-) Kit
(TaKaRa)将 MVs的基因组反转录成 cDNAs, 然后用常规的 RT-PCR方法进行检测 (TaKaRa) Reverse transcribes the genome of MVs into cDNAs, which are then detected by conventional RT-PCR methods.
[0102] 用于检测病人血液样本 MRP1的引物包括： [0102] Primers for detecting a blood sample of a patient MRP1 include:
[0103] GAPDH: forward, 5'-CTCCTGCACCACCAACTGCTTAGC-3'；  [0103] GAPDH: forward, 5'-CTCCTGCACCACCAACTGCTTAGC-3';
[0104] Reverse, 5'-CGCCTGCTTCACCACCTTCTTGAT-3'；  [0104] Reverse, 5'-CGCCTGCTTCACCACCTTCTTGAT-3';
[0105] MRP1 : forward, 5'-CTGCACGACCTCCGCTTCAAGAT-3'；  [0105] MRP1 : forward, 5'-CTGCACGACCTCCGCTTCAAGAT-3';
[0106] Reverse, 5'-GACTGGATGAGGTCGTCCGTTTCC-3'；  [0106] Reverse, 5'-GACTGGATGAGGTCGTCCGTTTCC-3';
[0107] Flotillin-2: forward, 5'-AGATCCGGCAGGAAGAGATT-3'；  [0107] Flotillin-2: forward, 5'-AGATCCGGCAGGAAGAGATT-3';
[0108] Reverse, 5'-GCTTCTGCCTTGAGCTTCAT-3'；  [0108] Reverse, 5'-GCTTCTGCCTTGAGCTTCAT-3';
[0109] MUC1 : forward, 5 ' -CGACTACTACCAAGAGCTGCAGAG AGACAT-3 '； [0109] MUC1 : forward, 5 ' -CGACTACTACCAAGAGCTGCAGAG AGACAT-3 ';
[0110] Reverse, 5 ' -TGTAAGAGAGGCTGCTGCCACC ATT ACCTG-3 ' [0110] Reverse, 5 ' -TGTAAGAGAGGCTGCTGCCACC ATT ACCTG-3 '
[0111]  [0111]
[0112] 用于检测病人血液样本 BCRP的引物包括：  [0112] Primers for detecting a blood sample of a patient BCRP include:
[0113] BCRP: forward, 5'-CAG CCG TGG AAC TCT TTG TGG TAG AGA AG-3'； [0113] BCRP: forward, 5'-CAG CCG TGG AAC TCT TTG TGG TAG AGA AG-3';
[0114] Reverse， 5'-CTG TTG CAT TGA GTC CTG GGC AGA AG-3' Reverse, 5'-CTG TTG CAT TGA GTC CTG GGC AGA AG-3'
[0115] Flotillin-2: forward, 5，-AGATCCGGCAGGAAGAGATT-3，； [0116] Reverse, 5 ' -GCTTCTGCCTTGAGCTTCAT-3 ' [0115] Flotillin-2: forward, 5,-AGATCCGGCAGGAAGAGATT-3,; [0116] Reverse, 5 ' -GCTTCTGCCTTGAGCTTCAT-3 '
[0117] MUCl : forward, 5 ' -CGACTACTACCAAGAGCTGCAGAG AGACAT-3 '； [0118] Reverse, 5 ' -TGTAAGAGAGGCTGCTGCCACCATTACCTG-3 ' .  [0117] MUCl : forward, 5 ' -CGACTACTACCAAGAGCTGCAGAG AGACAT-3 '; [0118] Reverse, 5 ' -TGTAAGAGAGGCTGCTGCCACCATTACCTG-3 ' .
[0119] GAPDH: forward。 5，-CTC CTG CAC CAC CAA CTG CTT AGC-3' ; [0119] GAPDH: forward. 5,-CTC CTG CAC CAC CAA CTG CTT AGC-3';
[0120] Reverse， 5'-CGC CTG CTT CAC CAC CTT CTT GAT-3' Reverse, 5'-CGC CTG CTT CAC CAC CTT CTT GAT-3'
[0121] [0121]
[0122] 流式细胞技术： 将 MVs在 4度条件孵育， 然后经过 BSA封闭、 MRP1/BCRP和 M UC 1一抗、 Alexa Fluor 488和 Alexa Fluor 546标记的二抗孵育后， 用流式细胞仪 检测。  Flow cytometry: MVs were incubated at 4 degrees and then incubated with BSA-blocked, MRP1/BCRP and M UC 1 primary antibodies, Alexa Fluor 488 and Alexa Fluor 546 labeled secondary antibodies, using flow cytometry Detection.
[0123] 结果：  [0123] Results:
[0124] RT-PCR检测病人血液样本 MVs中各蛋白的 mRNA水平， 34位经化疗的病人中 [0124] RT-PCR detection of mRNA levels of various proteins in MVs of blood samples of patients, among 34 patients undergoing chemotherapy
， MRPl , BCRP, Flotillin-2, MUCl和 GAPDH的 mRNA水平均显示高表达 （图 7 和图 8的 A曲线） ， 且 MRPl , BCRP表达为强阳性 （图 7和图 8的 B曲线） ， 而 21 位未经化疗的病人中， 这些蛋白的 mRNA水平均较低。 mRNA levels of MRP1, BCRP, Flotillin-2, MUCl and GAPDH showed high expression (A curve of Fig. 7 and Fig. 8), and MRP1 and BCRP expression were strongly positive (B curves of Fig. 7 and Fig. 8). The mRNA levels of these proteins were lower in 21 patients without chemotherapy.
[0125] 流式细胞术来检测从血液中分离得到的 MVs中 MRPl , BCRP, Flotillin-2, 和 M[0125] Flow cytometry to detect MRP1, BCRP, Flotillin-2, and M in MVs isolated from blood
UCl的表达情况， 化疗病人中 MRPl , BCRP, Flotillin-2, 和 MUCl的表达量高于 未化疗的病人 （图 7和图 8的 C~E曲线） 。 The expression of UCl, the expression of MRP1, BCRP, Flotillin-2, and MUCl in chemotherapy patients was higher than that in patients without chemotherapy (Fig. 7 and Fig. 8 C~E curves).
[0126] 以上结果进一步表明了携带有 MRP1或者 BCRP的 MVs与肿瘤耐药有密切的联系[0126] The above results further indicate that MVs carrying MRP1 or BCRP are closely related to tumor resistance.
， 提示乳腺癌病人血液 MVs中 MRP1或者 BCRP的表达可作为检测指标应用于耐 药乳腺癌的诊断治疗中。 It is suggested that the expression of MRP1 or BCRP in blood MVs of breast cancer patients can be used as a detection index in the diagnosis and treatment of drug-resistant breast cancer.
[0127] 实施例中实验选用乳腺癌肿瘤作为样本， 选取乳腺癌细胞 EVs中特异性表达的 细胞膜蛋白、 RNA及 DNA (TRPC5, BCRP, MRPl, Flotillin-2) ， 用 RT-PCR法， 流式细胞技术， 或者 Elisa法对他们的特异表达情况进行检测， 按照表达量的高 低， 说明乳腺癌肿瘤多药耐药的可能性。 In the examples, breast cancer tumors were selected as samples, and cell membrane proteins, RNA and DNA (TRPC5, BCRP, MRP1, Flotillin-2) specifically expressed in EVs of breast cancer cells were selected, by RT-PCR, flow Cell technology, or Elisa method to detect their specific expression, according to the level of expression, indicating the possibility of multidrug resistance in breast cancer.
[0128] 推广到其他癌症， 只要发现其他癌症肿瘤细胞 EVs中特异性表达的细胞膜蛋白[0128] Generalized to other cancers, as long as other cell membrane proteins specifically expressed in EVs of cancer cells are found
、 RNA及 DNA， 通过上述同样的方法检测这些物质的表达量， 从而说明多药耐 药的可能性。 , RNA and DNA, the amount of expression of these substances was measured by the same method as described above, thereby demonstrating the possibility of multidrug resistance.
[0129] 而实施例中所用到的表达分子都是与所有肿瘤多药耐药相关的分子， 因此， 本 申请所提供的方法具有普适性。 [0129] The expression molecules used in the examples are all molecules related to multidrug resistance of all tumors, therefore, The method provided by the application is universal.

Claims

权利要求书 [权利要求 1] 、 血液 EVs及其关联分子在肿瘤耐药检测中的应用， 其特征在于具 体检测方法为： Claim [Claim 1] Application of blood EVs and their associated molecules in tumor drug resistance detection, characterized in that the specific detection method is:
(1) 收集患有肿瘤的病人或动物的血液样本， 分别通过离心去除 样本中的完整细胞、 血小板及细胞碎片， 最后离心收集样本中的 E Vs; (1) Collect blood samples from patients or animals with tumors, remove intact cells, platelets, and cell fragments from the samples by centrifugation, and finally collect EVs from the samples by centrifugation ;
(2) 采用化学发光法， RT-PCR法、 流式细胞技术、 或者 Elisa法 检测收集到的 EVs中相关细胞膜蛋白、 RNA或 DNA的表达情况， 若表达阳性， 为出现耐药性的可疑病人或动物。 (2) Use chemiluminescence method, RT-PCR method, flow cytometry technology, or Elisa method to detect the expression of relevant cell membrane proteins, RNA or DNA in the collected EVs. If the expression is positive, it is a suspected patient with drug resistance. or animals.
2、 根据权利要求 1所述的血液 EVs及其关联分子在肿瘤耐药检测中 的应用， 其特征在于所述肿瘤为乳腺癌或其他类型肿瘤。 2. Application of blood EVs and their associated molecules in tumor drug resistance detection according to claim 1, characterized in that the tumor is breast cancer or other types of tumors.
3、 根据权利要求 1所述的血液 EVs及其关联分子在肿瘤耐药检测中 的应用， 其特征在于所述相关细胞膜蛋白是指瞬吋受体电位通道 即 TRPC5。 3. The application of blood EVs and their associated molecules in tumor drug resistance detection according to claim 1, characterized in that the related cell membrane protein refers to the transient receptor potential channel, namely TRPC5.
4、 根据权利要求 1所述的血液 EVs及其关联分子在肿瘤耐药检测中 的应用， 其特征在于所述相关细胞膜蛋白是指乳腺癌耐药蛋白即 B CRP。 4. The application of blood EVs and their associated molecules in tumor drug resistance detection according to claim 1, characterized in that the related cell membrane protein refers to the breast cancer drug resistance protein, BCRP.
5、 根据权利要求 1所述的血液 EVs及其关联分子在肿瘤耐药检测中 的应用， 其特征在于所述相关细胞膜蛋白是指多药耐药相关蛋白- 1即 MRP1。 5. Application of blood EVs and their associated molecules in tumor drug resistance detection according to claim 1, characterized in that the related cell membrane protein refers to multidrug resistance-related protein-1, that is, MRP1.
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