Molecular marker combination and detection reagent for identifying human blood mast cell precursor cells and application

Abstract

The present application relates to a molecular marker combination and detection reagent for identifying human blood mast cell precursor cells and application, and belongs to the technical field of cell detection. The molecular marker combination can realize efficient identification of human blood mast cell precursor cells, provide a new means for diagnosis of allergic diseases, and provide a new target for intervention of allergic diseases. The present application provides a molecular marker combination for identifying human blood mast cell precursor cells, wherein the molecular marker comprises a human cell surface molecule and a human intracellular molecule, the human cell surface molecule comprises CD34 and Fc epsilon R1 alpha, and the human intracellular molecule comprises CPA3, TPSAB1 and GATA2.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell detection, in particular to a molecular marker combination for identifying human blood mast cell precursor cells, a detection reagent and application. BACKGROUND

[0002] Paul Ehrlich first discovered human mast cells in 1879. However, it was not until 1994 that people realized that mast cells were derived from hematopoietic progenitor cells in bone marrow, and then entered the peripheral tissue after a short period of differentiation into mast cell precursor cells and further differentiation into mature mast cells. Mature mast cells are the core effector cells of allergic reactions. When the body is allergic, mast cells can be activated, synthesize and release various inflammatory mediators and cytokines through immunoglobulin and non-immunoglobulin related mechanisms, ultimately causing the clinical manifestations of allergy.

[0003] Kirshenbaum et al. found that the identification molecular marker of peripheral blood mast cell precursor cells was CD34 + c-kit (CD117) + CD13 + (Blood, 1999, Vol 94, No 7), Dahlin et al. (BLOOD, 2016, VOL 127, No 4) reported in 2016 that the identification molecules of peripheral blood mast cell precursor cells in healthy people and allergic asthma patients were lineage (CD4, CD8, CD19, CD14) - CD34 + CD117 + FcεRIα + . It is worth noting that the proportion of the above-mentioned cells in the peripheral blood cells of healthy people is only 0.0053%, and about 75% of the above-mentioned cells are eventually differentiated into CD117 + FcεRIα + Tryptase + Mast cells. The existing marker molecules have the problem of poor cell specificity, and the existing technology still lacks marker molecules that can efficiently define cell populations. SUMMARY

[0004] The purpose of the present application is to provide a molecular marker combination for identifying human blood mast cell precursor cells, a detection reagent and application. The molecular marker combination described in the present application can efficiently identify human blood mast cell precursor cells, providing a new means for the diagnosis of allergic diseases and a new target for the intervention of allergic diseases.

[0005] The present application provides a molecular marker combination for identifying human blood mast cell precursor cells, the molecular marker combination comprising human cell surface molecules and human intracellular molecules, the human cell surface molecules comprising CD34 and FcεRIα, and the human intracellular molecules comprising CPA3, TPSAB1 and GATA2. Figure 1 A test flow chart for detecting the molecular marker combination described above.

[0006] The present application also provides a use of a reagent for detecting the molecular marker combination described in the above technical solution in the preparation of a kit for identifying human blood mast cell precursor cells, wherein the identification is to identify cells positively expressing CD34, FcεRIα, CPA3, TPSAB1 and GATA2 as human blood mast cell precursor cells.

[0007] The present application also provides antibodies for detecting the molecular marker combination described in the above technical solution, the antibodies comprising antibodies against human cell surface molecules and antibodies against human intracellular molecules; the antibodies against human cell surface molecules comprising: anti-human CD34 antibodies and anti-human FcεRIα antibodies; and the antibodies against human intracellular molecules comprising: anti-human CPA3 antibodies, anti-human TPSAB1 antibodies and anti-human GATA2 antibodies.

[0008] The present application also provides fluorescently labeled antibodies for detecting the molecular marker combination described in the above technical solution based on flow cytometry, the fluorescently labeled antibodies comprising fluorescently labeled antibodies against human cell surface molecules and fluorescently labeled antibodies against human intracellular molecules; the fluorescently labeled antibodies against human cell surface molecules comprising: fluorescently labeled anti-human CD34 antibodies and fluorescently labeled anti-human FcεRIα antibodies; and the fluorescently labeled antibodies against human intracellular molecules comprising: fluorescently labeled anti-human CPA3 antibodies, fluorescently labeled anti-human TPSAB1 antibodies and fluorescently labeled anti-human GATA2 antibodies.

[0009] Preferably, the markers used by different fluorescently labeled antibodies are different in color.

[0010] The present application also provides a kit for detecting the molecular marker combination described in the above technical solution based on flow cytometry, the kit comprising the fluorescently labeled antibodies described in the above technical solution, blood diluent, mononuclear cell separation solution, cell culture solution, dead cell removal dye, FcR blocker, washing buffer, cell staining buffer, cell fixing solution and membrane permeable washing solution.

[0011] This invention also provides the application of the antibody described in the above-mentioned technical solution, or the fluorescently labeled antibody described in the above-mentioned technical solution, or the kit described in the above-mentioned technical solution, in the preparation of a kit for identifying human blood mast cell precursor cells, wherein the identification is to identify cells that are positively expressed by CD34, FcεRIα, CPA3, TPSAB1, and GATA2 as human blood mast cell precursor cells.

[0012] The present invention also provides the application of the antibody described in the above technical solution, or the fluorescently labeled antibody described in the above technical solution, or the kit described in the above technical solution in the preparation of a kit for diagnosing allergic diseases.

[0013] The present invention also provides the application of the antibody described in the above technical solution, or the fluorescently labeled antibody described in the above technical solution, or the kit described in the above technical solution in the preparation of a kit for diagnosing diseases related to human blood mast cell precursor cells, including allergic diseases.

[0014] The present invention also provides the application of the antibody described in the above technical solution, or the fluorescently labeled antibody described in the above technical solution, or the kit described in the above technical solution in the preparation of a kit for diagnosing allergic rhinitis.

[0015] This invention provides a combination of molecular markers for identifying human blood mast cell precursor cells. This combination of molecular markers enables highly efficient identification of human blood mast cell precursor cells, providing a new method for the diagnosis of allergic diseases and offering new targets for intervention. Experimental results show that, compared with healthy controls, patients with allergic rhinitis have an increased number of blood mast cell precursor cells, indicating that this invention is beneficial for the diagnosis of allergic rhinitis. Attached Figure Description

[0016] Figure 1 The experimental flowchart provided for this invention;

[0017] Figure 2 The following are representative flow cytometry analysis results provided by this invention: A represents the results of human peripheral blood mononuclear cell populations after removing debris and swollen cells; B represents the results of human peripheral blood mononuclear cell populations after removing adhesion cells; C represents the results of human peripheral blood mononuclear cell populations after removing dead cells; D represents the CD34 content in the peripheral blood mononuclear cell populations of patients with allergic rhinitis. + The expression status is represented in the diagram; E represents CD34 in patients with allergic rhinitis. + FcεRI in peripheral blood mononuclear cell population + The expression status is represented by the graph; F represents the CD34 expression of patients with allergic rhinitis. + FcεRI + TPSAB1 in peripheral blood mononuclear cell population +representative diagram of expression of CD34; G is CD34 + FcεRI + TPSAB1 + CPA3 + representative diagram of expression of CD34; H is CD34 + FcεRI + TPSAB1 + CPA3 + GATA2 + representative diagram of expression of CD34; I is CD34 + representative diagram of expression of CD34; J is CD34 + FcεRI + representative diagram of expression of CD34; K is CD34 + FcεRI + TPSAB1 + representative diagram of expression of CD34; L is CD34 + FcεRI + TPSAB1 + CPA3 + representative diagram of expression of CD34; M is CD34 + FcεRI + TPSAB1 + CPA3 + GATA2 + representative diagram of expression of CD34;

[0018] Figure 3 representative diagram of flow cytometry analysis result provided by the present application; note: healthy person (HC), n = 11; allergic rhinitis patient (AR), n = 16; P < 0.05 is significant difference; wherein, A is the number of cells of human peripheral blood mononuclear cell group after removing debris and swollen cells of healthy person (HC) and allergic rhinitis patient (AR); B is the percentage of CD34 + in peripheral blood mononuclear cell group of healthy person (HC) and allergic rhinitis patient (AR); C is the percentage of CD34 + FcεRI + in peripheral blood mononuclear cell group of healthy person (HC) and allergic rhinitis patient (AR); D is the percentage of CD34 + FcεRI + TPSAB1 + in peripheral blood mononuclear cell group of healthy person (HC) and allergic rhinitis patient (AR); E is the percentage of CD34+ FcεRI + TPSAB1 + CPA3 in peripheral blood mononuclear cell population + Percentage; F represents healthy individuals (HC) and patients with allergic rhinitis (AR) CD34. + FcεRI + TPSAB1 + CPA3 + GATA2 in peripheral blood mononuclear cell population + The percentage; G represents the percentage of mast cell precursors (CD34) in the peripheral blood mononuclear cell population of healthy individuals (HC) and patients with allergic rhinitis (AR). + FcεRI + TPSAB1 + CPA3 + GATA2 + ) Expression status of cells. Detailed Implementation

[0019] This invention provides a combination of molecular markers for identifying human blood mast cell precursor cells, the molecular markers including human cell surface molecules and human cell intracellular molecules, the human cell surface molecules including CD34 and FcεRIα, and the human cell intracellular molecules including CPA3, TPSAB1 and GATA2.

[0020] The present application discovers that there is a subpopulation of cells differentially expressing CD34, FcεRIα, CPA3, TPSAB1 and GATA2 at the level of transcriptome in human blood mononuclear cell population. In this subpopulation, CD117 is not among the top 100 differentially expressed genes, and CD117 is not cell specific, and CD117 cannot be used as a marker molecule to define the cell population. The present application uses flow cytometry and immunofluorescence techniques to identify this new cell population, and discovers that the new cell population simultaneously expressing mast cell specific molecules CD34, FcεRIα, CPA3, TPSAB1 and GATA2 is a mast cell precursor cell population. The results of the examples show that 0.039% (n=11) of mononuclear cells (PBMC) of healthy people and 0.71% (n=16) of mononuclear cells (PBMC) of patients with allergic rhinitis express CD34, FcεRIα, CPA3, TPSAB1 and GATA2 molecules, and are mast cell precursor cells. Since there are no mature mast cells in blood, mast cell precursor cells in blood circulation can differentiate into mature mast cells after entering peripheral tissues, and people have always believed that mast cells are cells in peripheral tissues. The present application realizes the detection of mast cell precursor cells in blood circulation, and the molecular marker combination of human blood mast cell precursor cells of the present application should be the first accurate understanding of this cell population. The present application discovers that 0.039% of mature mast cells in PBMC of healthy people and 0.71% of mature mast cells in PBMC of patients with allergic rhinitis not only show that there is a considerable number of mast cell precursor cells in human peripheral blood, but also suggest that the number of mast cell precursor cells in peripheral blood of patients with allergic rhinitis is increased by 18.2 times compared with healthy people, which has diagnostic value for allergic rhinitis. Since mast cells are the core effector cells (primary effector cells) of allergic reactions, accurate detection of mast cell precursor cells in peripheral blood has very important significance for the clinical diagnosis of patients with allergic rhinitis.

[0021] The present application also provides the use of a reagent for detecting the molecular marker combination in the technical solution in the preparation of a kit for identifying human blood mast cell precursor cells, and the identification is that the cells positively expressing CD34, FcεRIα, CPA3, TPSAB1 and GATA2 are identified as human blood mast cell precursor cells.

[0022] In the present application, the kit is preferably used for identifying human blood mast cell precursor cells by immunofluorescence detection method and flow cytometry detection method. When the kit is used for identifying human blood mast cell precursor cells by immunofluorescence detection method and flow cytometry detection method, the identification method preferably comprises the following steps: mononuclear cell separation, dead cell removal, FcR blocking, adding a fluorescently labeled anti-human cell surface molecule antibody, and adding a fluorescently labeled anti-human intracellular molecule antibody, and the human blood mast cell precursor cells are CD34 + FcεRIα + CPA3+ TPSAB1 + GATA2 + Cells (+ means positive expression).

[0023] The present application also provides antibodies for detecting the molecular marker combination of the above technical solution, which include antibodies against human cell surface molecules and antibodies against human intracellular molecules; the antibodies against human cell surface molecules include antibodies against human CD34 and antibodies against human FcεRIα; the antibodies against human intracellular molecules include antibodies against human CPA3, antibodies against human TPSAB1 and antibodies against human GATA2. The present application does not have special limitations on the source of the above antibodies, and conventional commercially available products known to those skilled in the art can be used.

[0024] The present application also provides fluorescently labeled antibodies for detecting the molecular marker combination of the above technical solution based on flow cytometry, which include fluorescently labeled antibodies against human cell surface molecules and fluorescently labeled antibodies against human intracellular molecules; the fluorescently labeled antibodies against human cell surface molecules include fluorescently labeled antibodies against human CD34 and fluorescently labeled antibodies against human FcεRIα; the fluorescently labeled antibodies against human intracellular molecules include fluorescently labeled antibodies against human CPA3, fluorescently labeled antibodies against human TPSAB1 and fluorescently labeled antibodies against human GATA2. In the present application, the labels used by different fluorescently labeled antibodies preferably have different colors.

[0025] The present application also provides a kit for detecting the molecular marker combination of the above technical solution based on flow cytometry, which includes the fluorescently labeled antibodies of the above technical solution, a blood diluent, a mononuclear cell separation solution, a cell culture solution, a dead cell removal dye, an FcR blocking agent, a washing buffer, a cell staining buffer, a cell fixing solution and a membrane permeable washing solution.

[0026] The present application does not have special limitations on the source of the blood diluent. In the present application, the blood diluent is preferably a solution with an osmotic pressure equal to that of human plasma, and more preferably a NaCl aqueous solution or a PBS buffer. In the present application, the mass percentage content of NaCl in the NaCl aqueous solution is preferably 0.9%. In the present application, the PBS buffer preferably includes the following components by weight: 8.0 parts of NaCl, 0.20 parts of KCl, 1.16 parts of Na2HPO4 and 0.20 parts of KH2PO4; the pH value of the PBS buffer is preferably 7.0-7.6, and more preferably 7.2-7.5.

[0027] In the present application, the effective component of the single-nucleus cell separation solution preferably comprises: polyfructose and diatrizoate, or sodium amidotrizoate and polysaccharide, or iodixanol, and the solvent of the single-nucleus cell separation solution is preferably distilled water. The present application does not have special limitations on the source and type of the single-nucleus cell separation solution. In the present application, the density of the single-nucleus cell separation solution is preferably 1.076-1.078 g / mL, and the osmotic pressure is preferably 275-305 mOsm.

[0028] The present application does not have special limitations on the type and source of the cell culture solution, and preferably is a culture solution for human peripheral blood mononuclear cells. In the present application, the cell culture solution preferably uses RPMI1640 as the basic medium, and includes fetal bovine serum with a volume concentration of 0.1-20% and penicillin-streptomycin solution with a volume concentration of 0.1-3%.

[0029] In the present application, the dead cell removal dye preferably comprises an amine-active fluorescent dye, and more preferably is a solution obtained by dissolving an amine-active fluorescent dye with DMSO.

[0030] In the present application, the fluorescence species of the dead cell removal dye and the fluorescently labeled antibody preferably include: Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 700, APC, APC / Cy7, APC / Cy7, APC / H7, Brilliant Violet 421, Brilliant Violet 510, Brilliant Blue 515, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 785, FITC, LEAF, Pacific Blue, PE, PE / Cy5, PE / Cy7, PE / Dazzle, PerCP, PerCP / Cy5.5. As for the selection of fluorescence species, the present application preferably freely combines according to the configuration of the laser and filter of the flow cytometer.

[0031] In the present application, the effective component of the FcR blocker preferably comprises human immunoglobulin, or an irrelevant immunoglobulin of the same genus and subtype as the flow cytometry antibody (the above-mentioned fluorescently labeled antibody) used. In the present application, the mass concentration of the FcR blocker working solution is preferably 0.1-100 μg / mL, and more preferably 1-10 μg / mL.

[0032] In the present application, the washing buffer preferably comprises a PBS buffer.

[0033] In the present application, the cell staining buffer is preferably a calcium and magnesium free Dulbecco's PBS (DPBS) solution, and the effective component of the DPBS solution is fetal bovine serum. In the present application, the working concentration of the fetal bovine serum is preferably 0.1-20%, more preferably 0.5-15%. In the present application, the pH value of the cell staining buffer is preferably 7.0-7.6.

[0034] In the present application, the effective components of the cell fixing solution preferably include formaldehyde and methanol, the working concentration of the formaldehyde is preferably 0.5-10% (mass / mass), more preferably 0.8-2% (mass / mass), and the working concentration of the methanol is preferably 0.1-2% (mass / mass), more preferably 0.2-1% (mass / mass).

[0035] In the present application, the effective components of the membrane permeation washing solution preferably include saponin and fetal bovine serum, or saponin and bovine serum albumin. In the present application, the concentration of the saponin is preferably 0.1-5% (w / v), the volume percentage content of the fetal bovine serum is preferably 0.1-20%, and the mass volume concentration of the bovine serum albumin is preferably 0.1-20%.

[0036] The present application also provides the use of the antibody of the above technical solution, the fluorescently labeled antibody of the above technical solution, or the kit of the above technical solution in the preparation of a kit for identifying human blood mast cell precursor cells, wherein the cells that positively express CD34, FcεRIα, CPA3, TPSAB1 and GATA2 are identified as human blood mast cell precursor cells.

[0037] In the present application, in the use, the identification method preferably includes the following steps: single nuclear cell separation, dead cell removal, FcR blocking, adding a fluorescently labeled antibody against a human cell surface molecule, and adding a fluorescently labeled antibody against a human intracellular molecule, wherein the human blood mast cell precursor cells are CD34 + FcεRIα + CPA3 + TPSAB1 + GATA2 + Cells (+ indicates positive expression).

[0038] Specifically, the method more preferably includes the following steps:

[0039] The intravenous blood mixed with the blood diluent is placed on the upper layer of the mononuclear cell separation solution, centrifuged, and the mononuclear cells are aspirated. In the present application, the volume ratio of the intravenous blood, the blood diluent, and the mononuclear cell separation solution is preferably 1 : (0.5-2) : (0.5-2). In the present application, the time of centrifugation is preferably 15-35 min.

[0040] After the mononuclear cells are aspirated, the present application preferably dilutes the mononuclear cells with the blood diluent, centrifuges, and collects the cell pellet. In the present application, the time of centrifugation is preferably 4-30 min.

[0041] After the cell pellet is obtained, the present application preferably resuspends the cell pellet with the washing buffer to obtain a first cell suspension; in the present application, the resuspension adjusts the concentration of the cells to be (1-10) x 10 6 cells / mL.

[0042] After the first cell suspension is obtained, the present application mixes the first cell suspension, the dead cell removal dye, and the FcR blocking agent, incubates, to obtain a second cell suspension. In the present application, the first cell suspension is preferably mixed at (0.1-1) x 10 6 cells / 100 μL of the system with 0.1-10 μL of the dead cell removal dye and 0.1-10 μL of the FcR blocking agent. In the present application, the temperature of incubation is preferably 4-40°C, and the time of incubation is preferably 5-30 min.

[0043] After the second cell suspension is obtained, the present application mixes the second cell suspension and the cell staining buffer, centrifuges, mixes the cell pellet with the fluorescently labeled antibody against the human cell surface molecule after the supernatant is discarded, incubates, to obtain a third cell suspension. In the present application, the second cell suspension is preferably mixed at (0.1-1) x 10 6 cells / 100 μL of the system with 0.5-5 mL of the cell staining buffer. In the present application, the time of centrifugation is preferably 4-30 min. In the present application, after the supernatant is discarded, the cell pellet is preferably mixed at (0.1-1) x 10 6 cells / 100 μL of the system with 0.5-20 μL of the fluorescently labeled antibody against the human cell surface molecule, and the mixing is preferably uniform mixing. In the present application, the temperature of incubation is preferably 4-40°C, and the time of incubation is preferably 5-30 min.

[0044] After the third cell suspension is obtained, the present application mixes the third cell suspension with the cell staining buffer, centrifuges, resuspends the cell pellet by adding the cell fixing solution, to obtain a fourth cell suspension. In the present application, the mixing is preferably mixing the third cell suspension with 0.5-5 mL of the cell staining buffer. In the present application, the resuspension is preferably resuspending the cell pellet at (0.1-1) x 106 0.1-5 mL cell fixation solution is added to resuspend the cells.

[0045] After obtaining the fourth cell suspension, the fourth cell suspension is mixed with a membrane permeation washing solution, centrifuged, and the supernatant is discarded, and then the membrane permeation washing solution is added again to resuspend the fifth cell suspension. In the present application, the centrifugation time is preferably 4-30 min.

[0046] After obtaining the fifth cell suspension, the fifth cell suspension is preferably centrifuged, the supernatant is discarded, and then a fluorescently labeled anti-human intracellular molecule antibody is added, mixed, and incubated in the dark to obtain the sixth cell suspension. In the present application, the centrifugation time is preferably 4-30 min. In the present application, the amount of the antibody added is preferably 0.5-20 μL. In the present application, the incubation time is preferably 10-120 min.

[0047] After obtaining the sixth cell suspension, the sixth cell suspension is mixed with a membrane permeation washing solution, centrifuged, and the supernatant is discarded, and then the cell concentration is adjusted with a washing buffer, and then a flow cytometer is used to detect the expression of each antibody on the cells. In the present application, the centrifugation time is preferably 4-30 min. In the present application, the cell concentration is preferably adjusted to (0.1-10) × 10 6 cells / mL.

[0048] The present application identifies CD34 + FcεRIα + CPA3 + TPSAB1 + GATA2 + The cells (+ indicates positive expression) are mast cell precursor cells.

[0049] The present application also provides the use of the antibody described in the above technical solution, or the fluorescently labeled antibody described in the above technical solution, or the kit described in the above technical solution in the preparation of a kit for diagnosing an allergic disease.

[0050] The present application also provides the use of the antibody described in the above technical solution, or the fluorescently labeled antibody described in the above technical solution, or the kit described in the above technical solution in the preparation of a kit for diagnosing a disease related to human blood mast cell precursor cells, and the disease includes an allergic disease.

[0051] The application further provides application of the antibody, the fluorescently labeled antibody or the kit in preparation of a kit for diagnosing allergic rhinitis. Detection of the human blood mast cell precursor cell group can realize diagnosis of allergic diseases represented by allergic rhinitis. The application has important scientific significance for understanding the occurrence and development of allergic rhinitis, and more importantly, provides a new method for diagnosis of allergic diseases and a new target for intervention of allergic diseases. The results of the embodiments of the application show that the number of blood mast cell precursor cells of the allergic rhinitis patients is increased by 18.2 times compared with the healthy control group, and the difference is statistically significant.

[0052] The molecular marker combination for identifying human blood mast cell precursor cells, the detection reagent and the application will be further described in detail below in combination with specific embodiments. The technical scheme of the application includes but is not limited to the following embodiments.

[0053] Embodiment 1

[0054] The method for detecting human mast cell precursor cells comprises the following steps:

[0055] 1. After mixing the venous blood with a blood diluent, the mixture is placed on the upper layer of a mononuclear cell separation medium, and after centrifugation for 15-35 min, the mononuclear cells are absorbed. The volume ratio of the venous blood, the blood diluent and the mononuclear cell separation medium is 1:(0.5-2):(0.5-2).

[0056] 2. After diluting the mononuclear cells obtained in step 1 with the blood diluent, centrifugation is performed for 4-30 min, and the cell precipitate is collected.

[0057] 3. The cell precipitate obtained in step 2 is resuspended with a washing buffer, and the concentration of the cells is adjusted to (1-10)×10 6 cells / mL to obtain a first cell suspension.

[0058] 4. The first cell suspension obtained in step 3 is mixed with 0.1-10 μL of dead cell removal dye and 0.1-10 μL of FcR blocking agent at a system of (0.1-1)×10 6 cells / 100 μL, and then incubated at 4-40 °C for 5-30 min to obtain a second cell suspension.

[0059] 5. The second cell suspension obtained in step 4 is mixed with 0.5-5 mL of cell staining buffer at a system of (0.1-1)×10 6 cells / 100 μL, centrifuged for 4-30 min, and then the supernatant is discarded, and the cell precipitate is mixed with 0.5-5 mL of cell staining buffer at a system of (0.1-1)×10 60.5-20 μL of fluorescently labeled antibodies against human cell surface molecules were added to the 100 μL of the cell suspension, and the mixture was incubated at 4-40 °C for 5-30 min to obtain a third cell suspension;

[0060] 6. The third cell suspension obtained in step 5 was mixed with 0.5-5 mL of cell staining buffer, and then centrifuged to obtain a cell pellet. The cell pellet was resuspended in 0.1-1 mL of cell permeabilization buffer, and then incubated at 4-40 °C for 5-30 min to obtain a fourth cell suspension. 6 0.1-5 mL of cell fixation solution was added to the 100 μL of the cell suspension to obtain a fourth cell suspension;

[0061] 7. The fourth cell suspension obtained in step 6 was mixed with an appropriate amount of cell permeabilization buffer, and then centrifuged for 4-30 min. The supernatant was discarded, and the cell pellet was resuspended in an appropriate amount of cell permeabilization buffer to obtain a fifth cell suspension.

[0062] 8. The fifth cell suspension obtained in step 7 was centrifuged for 4-30 min, and the supernatant was discarded. Then, 0.5-20 μL of fluorescently labeled antibodies against human intracellular molecules were added to the cell pellet, and the mixture was incubated in the dark for 10-120 min to obtain a sixth cell suspension.

[0063] 9. The sixth cell suspension obtained in step 8 was mixed with an appropriate amount of cell permeabilization buffer, and then centrifuged for 4-30 min. The supernatant was discarded, and the cell concentration was adjusted to (0.1-10) x 10 6 The expression of each antibody on the cells was detected by flow cytometry.

[0064] 10. CD34 + FcεRIα + CPA3 + TPSAB1 + GATA2 + The cells (+) are mast cell precursor cells.

[0065] Example 2

[0066] 1) Peripheral venous blood was collected from healthy people (HC, n = 11) and allergic rhinitis patients (AR, n = 16);

[0067] 2) 1 mL of fresh peripheral venous blood (blood was separated from the body for ≤ 2 h) was diluted with 1 mL of blood diluent;

[0068] 3) 1 mL of mononuclear cell separation solution was placed at the bottom of a centrifuge tube, and the diluted blood was carefully overlaid on the mononuclear cell separation solution to avoid mixing of the blood and the mononuclear cell separation solution;

[0069] 4) The centrifuge was set to 800 g for 20 min, and the temperature was set to 18 °C, the speed was set to 9, and the deceleration was set to 0.

[0070] 5) Use a dropper to suck out the white membrane layer (mononuclear cells) on the interface, trying not to suck out the liquid above it;

[0071] 6) Dilute the mononuclear cells with 2 mL of blood diluent, centrifuge at 250g for 10 min at room temperature; collect the cell precipitate, resuspend the mononuclear cells with 100 μL of cell culture medium, and then perform cell counting, adjust the cell density to 4 x 10 6 Figure 2

[0072] 7) Add 100 μL of cell suspension to each tube, then add 1 μL of DMSO-dissolved dead cell removal dye ZombieNIR and 5 μL of FcR blocker, incubate at room temperature for 15 min in the dark;

[0073] 8) Add 1 mL of cell staining buffer to each tube, centrifuge at 200g for 6 min, discard the supernatant; the obtained human peripheral blood mononuclear cell population after removing adherent cells Figure 2 (B, human peripheral blood mononuclear cells after removing adherent cells, about 99.05% of the cells in the human peripheral blood mononuclear cell population are mononuclear cells) and dead cells Figure 2 (C, human peripheral blood mononuclear cells after removing dead cells, about 99.90% of the cells in the human peripheral blood mononuclear cell population after removing adherent cells are live cells);

[0074] 9) Resuspend the cells by adding 100 μL of cell staining buffer to each tube, then add 5 μL of Brilliant Violet 421-labeled anti-human CD34 and PE / Cy7-labeled anti-human FcεRIα antibodies, incubate at room temperature for 15 min in the dark;

[0075] 10) Add 1 mL of cell staining buffer to each tube, centrifuge at 200g for 6 min, discard the supernatant;

[0076] 11) Resuspend the cells by adding 1 mL of fixation and membrane permeabilization wash solution to each tube, incubate at room temperature for 30 min in the dark;

[0077] 12) Add 2 mL of membrane permeabilization wash solution to each tube, centrifuge at 200g for 6 min, discard the supernatant;

[0078] 13) Resuspend the cells by adding 100 μL of membrane permeabilization wash solution to each tube, then add 5 μL of APC-labeled anti-human TPSAB1, FITC-labeled anti-human CPA3, and PE-labeled anti-human GATA2 antibodies, incubate at room temperature for 1 h in the dark;

[0079] ​​14) Add 2 mL of permeabilization washing buffer to each tube, wash once, resuspend cells in 200 μL of washing buffer, and analyze the expression of each molecule by flow cytometry; Note: Arrows indicate the order of result analysis ( Figure 2 ).

[0080] 15) The expression of various antibodies in patients with allergic rhinitis was obtained as follows: Figure 2 D in Figure 2 As shown by H in the figure, the expression of various antibodies in healthy individuals is as follows: Figure 2 I in Figure 2 M in Figure 2 represents CD34 in the peripheral blood mononuclear cell population of patients with allergic rhinitis; D in Figure 2 represents CD34 in the peripheral blood mononuclear cell population of patients with allergic rhinitis. + CD34 expression status: Approximately 15.85% of mononuclear cells express CD34; Figure 2 E in the text represents CD34 in patients with allergic rhinitis. + Expression of FcεRI in peripheral blood mononuclear cell populations: CD34 + Approximately 42.2% of mononuclear cells express FcεRI; Figure 2 F in the text represents CD34 in patients with allergic rhinitis. + FcεRI + TPSAB1 in peripheral blood mononuclear cell population + Expression status: CD34 + FcεRI + Approximately 83.4% of mononuclear cells express TPSAB1; Figure 2 G in the text represents CD34 in patients with allergic rhinitis. + FcεRI + TPSAB1 + CPA3 in peripheral blood mononuclear cell population + Expression status: CD34 + FcεRI + TPSAB1 + Approximately 12.5% ​​of mononuclear cells express CPA3; Figure 2 The H in the text represents CD34 in patients with allergic rhinitis. + FcεRI + TPSAB1 + CPA3 + GATA2 in peripheral blood mononuclear cell population + Expression status: CD34 + FcεRI + TPSAB1 + CPA3 + Approximately 68.5% of the mononuclear cell population expresses GATA2.

[0081] Figure 2 In this context, I represents CD34 in the peripheral blood mononuclear cell population of healthy individuals. +CD34 expression in mononuclear cells: about 9.09% expressed CD34; Figure 2 J in Table 1 is CD34 expression in healthy people + FcεRI expression in peripheral blood mononuclear cell population + CD34 expression in mononuclear cells: about 9.09% expressed CD34; + FcεRI expression in mononuclear cells: about 44.9% expressed FcεRI; Figure 2 K in Table 1 is CD34 expression in healthy people + FcεRI expression in mononuclear cells: about 44.9% expressed FcεRI; + TPSAB1 expression in peripheral blood mononuclear cell population + CD34 expression in mononuclear cells: about 9.09% expressed CD34; + FcεRI expression in mononuclear cells: about 44.9% expressed FcεRI; + TPSAB1 expression in mononuclear cells: about 85.9% expressed TPSAB1; Figure 2 L in Table 1 is CD34 expression in healthy people + FcεRI expression in mononuclear cells: about 44.9% expressed FcεRI; + TPSAB1 expression in mononuclear cells: about 85.9% expressed TPSAB1; + CPA3 expression in peripheral blood mononuclear cell population + CD34 expression in mononuclear cells: about 9.09% expressed CD34; + FcεRI expression in mononuclear cells: about 44.9% expressed FcεRI; + TPSAB1 expression in mononuclear cells: about 85.9% expressed TPSAB1; + CPA3 expression in mononuclear cells: about 6.92% expressed CPA3; Figure 3 M in Table 1 is CD34 expression in healthy people + FcεRI expression in mononuclear cells: about 44.9% expressed FcεRI; + TPSAB1 expression in mononuclear cells: about 85.9% expressed TPSAB1; + CPA3 expression in mononuclear cells: about 6.92% expressed CPA3; + GATA2 expression in peripheral blood mononuclear cell population + CD34 expression in mononuclear cells: about 9.09% expressed CD34; + FcεRI expression in mononuclear cells: about 44.9% expressed FcεRI; + TPSAB1 expression in mononuclear cells: about 85.9% expressed TPSAB1; + CPA3 expression in mononuclear cells: about 6.92% expressed CPA3; + GATA2 expression in mononuclear cells: about 19.1% expressed GATA2.

[0082] 16) The obtained mast cell precursor cells in peripheral venous blood mononuclear cells of healthy people and allergic rhinitis patients are shown in Table 1. Figure 3 Note: HC; n = 11; AR; n = 16.

[0083] Figure 3 A in Table 1: the number of human peripheral blood mononuclear cells after removing debris and swollen cells of healthy people (HC) and allergic rhinitis patients (AR);

[0084] Figure 3 B in Table 1: the percentage of CD34 + cells in PBMC;

[0085] Figure 3 C in Table 1: the percentage of FcεRI + cells in CD34+ Cell percentage;

[0086] Figure 3 D in: TPSAB1 + CD34 cells account for a significant portion of PBMCs + FcεRI + Cell percentage;

[0087] Figure 3 E in CPA3 + CD34 cells account for a significant portion of PBMCs + FcεRI + TPSAB1 + Cell percentage;

[0088] Figure 3 F in: GATA2 + CD34 cells account for a significant portion of PBMCs + FcεRI + TPSAB1 + CPA3 + Cell percentage;

[0089] Figure 3 G in CD34 + FcεRI + TPSAB1 + CPA3 + GATA2 + Percentage of mast cell precursor cells in PBMCs.

[0090] Conclusion: The percentage of CD34-positive cells in the mononuclear cell population of patients with allergic rhinitis ( Figure 3 B) CD34 of mast cell precursor cells + FcεRI + TPSAB1 + CPA3 + Percentage of cells ( Figure 3 F in F), mast cell precursor cells CD34 + FcεRI + TPSAB1 + CPA3 + GATA2 + Cell ratio ( ​ The proportion of mast cell precursor cells in peripheral blood mononuclear cells was significantly higher than in healthy individuals. This indicates that the proportion of mast cell precursor cells in peripheral blood mononuclear cells is significant for diagnosing allergic rhinitis.

[0091] As can be seen from the above embodiments, the kit and detection method of the present invention can be used to quickly detect mast cell precursor cells in human peripheral blood and can be used as an auxiliary diagnostic tool for allergic rhinitis.

[0092] The above merely preferred embodiments of the present application, it should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the scope of protection of the present application.

Claims

1. A molecular marker combination for identifying human blood mast cell precursor cells, characterized in that, The molecular markers are human cell surface molecules and human cell intracellular molecules. The human cell surface molecules are CD34 and FcεRIα, and the human cell intracellular molecules are CPA3, TPSAB1, and GATA2. Cells that are positive for CD34, FcεRIα, CPA3, TPSAB1, and GATA2 are identified as human blood mast cell precursor cells.

2. The use of the reagent for detecting the molecular marker combination described in claim 1 in the preparation of a kit for identifying human blood mast cell precursor cells.

3. An antibody for detecting the molecular marker combination of claim 1, characterized in that, The antibodies are antibodies against human cell surface molecules and antibodies against human intracellular molecules; the antibodies against human cell surface molecules are: anti-human CD34 antibody and anti-human FcεRIα antibody; the antibodies against human intracellular molecules are: anti-human CPA3 antibody, anti-human TPSAB1 antibody and anti-human GATA2 antibody.

4. Detection of the fluorescently labeled antibody of the molecular marker combination of claim 1 by flow cytometry, characterized in that, The fluorescently labeled antibodies are fluorescently labeled antibodies against human cell surface molecules and fluorescently labeled antibodies against human intracellular molecules; the fluorescently labeled antibodies against human cell surface molecules are: fluorescently labeled anti-human CD34 antibody and fluorescently labeled anti-human FcεRIα antibody; the fluorescently labeled antibodies against human intracellular molecules are: fluorescently labeled anti-human CPA3 antibody, fluorescently labeled anti-human TPSAB1 antibody and fluorescently labeled anti-human GATA2 antibody.

5. The fluorescently labeled antibody according to claim 4, characterized in that, Different fluorescently labeled antibodies use different colors of fluorescent molecules for labeling.

6. A kit for detecting the molecular marker combination of claim 1 using flow cytometry, characterized in that, The kit includes the fluorescently labeled antibody as described in claim 4 or 5, blood diluent, mononuclear cell separation medium, cell culture medium, dead cell dye removal medium, FCR blocking agent, washing buffer, cell staining buffer, cell fixation medium, and permeabilization wash solution.

7. The use of the antibody of claim 3, or the fluorescently labeled antibody of claim 4 or 5, or the kit of claim 6 in the preparation of a kit for identifying human blood mast cell precursor cells.

8. The use of the antibody of claim 3, the fluorescently labeled antibody of claim 4 or 5, or the kit of claim 6 in the preparation of a kit for diagnosing allergic diseases.

9. The use of the antibody of claim 3, or the fluorescently labeled antibody of claim 4 or 5, or the kit of claim 6, in the preparation of a kit for diagnosing diseases associated with human blood mast cell precursor cells, said diseases including allergic diseases.

10. The use of the antibody of claim 3, the fluorescently labeled antibody of claim 4 or 5, or the kit of claim 6 in the preparation of a kit for diagnosing allergic rhinitis.

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