CN114814208A - Molecular marker combination for identifying human blood mast cell precursor cells, detection reagent and application - Google Patents
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Abstract
The invention relates to a molecular marker combination for identifying human blood mast cell precursor cells, a detection reagent and application, and belongs to the technical field of cell detection. The molecular marker combination can realize the high-efficiency identification of human blood mast cell precursor cells, provides a new means for the diagnosis of allergic diseases, and provides a new target for the intervention of the allergic diseases. The invention provides a combination of molecular markers for identifying human mast cell precursor cells, the molecular markers comprising human cell surface molecules including CD34 and fcsria and human intracellular molecules including CPA3, TPSAB1 and GATA 2.
Description
Technical Field
The invention 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
Paul Ehrlich found human mast cells for the first time in 1879, however, it was not recognized until 1994 that mast cells were derived from hematopoietic progenitor cells of bone marrow, and then entered peripheral tissues after briefly entering the blood circulation to differentiate into mast cell precursor cells, and further differentiated to develop into mature mast cells. Mature mast cells are the core effector cells of allergic reactions. When the organism is allergic, the immunoglobulin and the non-immunoglobulin are used for controlling and inducing the activation, synthesis and release of a plurality of inflammatory mediators and cytokines by mast cells, and finally the clinical manifestation of the allergy is caused.
Kirshenbaum et al found that the molecular marker for identifying the precursor cells of the peripheral blood mast cells was CD34 + c-kit(CD117) + CD13 + (Blood, 1999, Vol 94, No 7), Dahlin et al (BLOOD, 2016, VOL127, No 4) reported in 2016 the identification of molecules of peripheral Blood mast cell precursors in healthy and allergic asthmatic patients: linkage (CD4, CD8, CD19, CD14) - CD34 + CD117 + FcεRIα + . It is noteworthy that the proportion of the above-mentioned cells in the peripheral blood cells of healthy humans is only 0.0053%, wherein about 75% of the above-mentioned cells finally differentiate into CD117 + FcεRIα + Tryptase + The mast cell of (a). The existing marker molecules have the problem of poor cell specificity, and the existing technology is still lack of the marker molecules for efficiently defining cell populations.
Disclosure of Invention
The invention aims to provide a molecular marker combination for identifying human blood mast cell precursor cells, a detection reagent and application. The molecular marker combination can realize the high-efficiency identification of human blood mast cell precursor cells, provides a new means for the diagnosis of allergic diseases, and provides a new target for the intervention of the allergic diseases.
The present invention provides a combination of molecular markers for identifying human mast cell precursor cells, the molecular markers including human cell surface molecules including CD34 and fcsria and human intracellular molecules including CPA3, TPSAB1 and GATA2 (figure 1 is a flow chart of an assay using the above combination of molecular markers).
The invention also provides application of a reagent for detecting the molecular marker combination in the technical scheme in preparing a kit for identifying human mast cell precursor cells, wherein the cells positively expressed by CD34, Fc epsilon RI alpha, CPA3, TPSAB1 and GATA2 are identified as the human mast cell precursor cells.
The invention also provides an antibody for detecting the molecular marker combination in the technical scheme, wherein the antibody comprises an antibody for resisting human cell surface molecules and an antibody for resisting human cell internal molecules; the antibody against the human cell surface molecule comprises: anti-human CD34 antibodies and anti-human fcsria antibodies; the antibody against the human intracellular molecule comprises: anti-human CPA3 antibody, anti-human TPSAB1 antibody and anti-human GATA2 antibody.
The invention also provides a flow cytometry-based fluorescence labeled antibody for detecting the molecular marker combination in the technical scheme, wherein the fluorescence labeled antibody comprises a fluorescence labeled antibody for resisting human cell surface molecules and a fluorescence labeled antibody for resisting human cell internal molecules; the fluorescence labeled antibody for resisting the human cell surface molecule comprises: a fluorescently labeled anti-human CD34 antibody and a fluorescently labeled anti-human fcepsilonra antibody; the fluorescence labeled antibody for resisting the human intracellular molecules comprises: fluorescently labeled anti-human CPA3 antibody, fluorescently labeled anti-human TPSAB1 antibody, and fluorescently labeled anti-human GATA2 antibody.
Preferably, the different fluorescently labeled antibodies use different colors of the labeling fluorescent molecules.
The invention also provides a kit for detecting the molecular marker combination based on the technical scheme by using a flow cytometry method, wherein the kit comprises the fluorescence-labeled antibody, blood diluent, mononuclear cell separation liquid, cell culture liquid, dead cell removal dye, FcR blocker, washing buffer, cell staining buffer, cell fixing liquid and transmembrane washing liquid.
The invention also provides the use of the antibody of the above technical scheme or the fluorescently-labeled antibody of the above technical scheme or the kit of the above technical scheme in the preparation of a kit for identifying human mast cell precursor cells, wherein the cells positively expressed by CD34, Fc epsilon RI alpha, CPA3, TPSAB1 and GATA2 are identified as human mast cell precursor cells.
The invention also provides application of the antibody in the technical scheme or the fluorescence labeled antibody in the technical scheme or the kit in the technical scheme in preparation of a kit for diagnosing allergic diseases.
The invention also provides application of the antibody in the technical scheme or the fluorescence labeling antibody in the technical scheme or the kit in the technical scheme in preparing a kit for diagnosing diseases related to human blood mast cell precursor cells, wherein the diseases comprise allergic diseases.
The invention also provides application of the antibody in the technical scheme, the fluorescence labeled antibody in the technical scheme or the kit in the technical scheme in preparation of a kit for diagnosing allergic rhinitis.
The invention provides a molecular marker combination for identifying human blood mast cell precursor cells. The molecular marker combination can realize the high-efficiency identification of human blood mast cell precursor cells, provides a new means for the diagnosis of allergic diseases, and provides a new target for the intervention of the allergic diseases. The test result shows that compared with a healthy control group, the number of blood mast cell precursor cells of the allergic rhinitis patient is increased, and the method is beneficial to realizing the diagnosis of the allergic rhinitis.
Drawings
FIG. 1 is a flow chart of the test provided by the present invention;
FIG. 2 is a representative diagram of a flow cytometry analysis provided herein; wherein A is a result representative diagram of a human peripheral blood mononuclear cell population after debris and swollen cells are removed; b is a result graph of human peripheral blood mononuclear cells after adherent cells are removed; c is a representative diagram of the result of human peripheral blood mononuclear cells after dead cells are removed; d is CD34 in peripheral blood mononuclear cell population of allergic rhinitis patient + Represents a graph of expression of (a); e is CD34 of allergic rhinitis patient + FceRI in peripheral blood mononuclear cell populations + Represents a graph of expression of (a); f is CD34 of allergic rhinitis patient + FcεRI + TPSAB1 in peripheral blood mononuclear cell populations + Represents a graph of expression of (a); g is CD34 for allergic rhinitis patient + FcεRI + TPSAB1 + CPA3 in peripheral blood mononuclear cell populations + Expression ofA situation representative diagram; h is CD34 of allergic rhinitis patient + FcεRI + TPSAB1 + CPA3 + GATA2 in peripheral blood mononuclear cell populations + Represents a graph of expression of (a); i is CD34 in healthy human peripheral blood mononuclear cell population + Represents a graph of expression of (a); j is healthy human CD34 + FceRI in peripheral blood mononuclear cell populations + Represents a graph of expression of (a); k is healthy human CD34 + FcεRI + TPSAB1 in peripheral blood mononuclear cell population + Represents a graph of expression of (a); l is healthy human CD34 + FcεRI + TPSAB1 + CPA3 in peripheral blood mononuclear cell populations + Represents a graph of expression of (a); m is healthy human CD34 + FcεRI + TPSAB1 + CPA3 + GATA2 in peripheral blood mononuclear cell populations + Represents a graph of expression of (a);
FIG. 3 is a graph of flow cytometry analysis results provided herein; note: healthy Human (HC), n ═ 11; allergic rhinitis patients (AR), n ═ 16; p < 0.05 is significant difference; wherein A is the cell number of human peripheral blood mononuclear cell population from healthy Human (HC) and patients with Allergic Rhinitis (AR) after removal of debris and swollen cells; b is CD34 in peripheral blood mononuclear cell population of healthy Human (HC) and allergic rhinitis patient (AR) + (ii) percent (d); c is CD34 of healthy people (HC) and patients with Allergic Rhinitis (AR) + FceRI in peripheral blood mononuclear cell populations + (ii) percent (d); d is CD34 of healthy people (HC) and patients with Allergic Rhinitis (AR) + FcεRI + TPSAB1 in peripheral blood mononuclear cell population + (ii) percent (d); e is CD34 of healthy people (HC) and patients with Allergic Rhinitis (AR) + FcεRI + TPSAB1 + CPA3 in peripheral blood mononuclear cell populations + (ii) percent (d); f is CD34 of healthy people (HC) and patients with Allergic Rhinitis (AR) + FcεRI + TPSAB1 + CPA3 + GATA2 in peripheral blood mononuclear cell populations + (ii) percent (d); g is mast cell precursor (CD 34) in peripheral blood mononuclear cell population of healthy Human (HC) and allergic rhinitis patients (AR) + FcεRI + TPSAB1 + CPA3 + GATA2 + ) Expression profile of the cell.
Detailed Description
The invention provides a combination of molecular markers for identifying human mast cell precursor cells, the molecular markers including human cell surface molecules including CD34 and FceRI α and human intracellular molecules including CPA3, TPSAB1 and GATA 2.
The invention discovers that 1 cell subgroup which differentially expresses CD34, Fc epsilon RI alpha, CPA3, TPSAB1 and GATA2 genes exists in the human blood mononuclear cell population at the transcriptome level. In this subset, CD117 is not the top 100 differential gene and, without cell specificity, CD117 cannot serve as a marker molecule defining the cell population. The invention identifies the new cell population by using flow cytometry and immunofluorescence technology, and finds that the new cell population which simultaneously expresses mast cell specific molecules CD34, Fc epsilon RI alpha, CPA3, TPSAB1 and GATA2 is a mast cell precursor cell population. The results of the examples show that mononuclear cells (PBMCs) from 0.039% (n ═ 11) in healthy humans, and from 0.71% (n ═ 16) in patients with allergic rhinitis, express molecules CD34, fcsria, CPA3, TPSAB1, and GATA2, as mast cell precursors. Since there are no mature mast cells in blood and mast cell precursors in the blood circulation can differentiate into mature mast cells after entering peripheral tissues, mast cells have been considered as cells in peripheral tissues. The invention realizes the detection of the mast cell precursor cells in the blood circulation, and the molecular marker combination of the human blood mast cell precursor cells of the invention is to accurately recognize the group of cells for the first time. The mature mast cells of 0.039% in PBMCs of healthy people and 0.71% in patients with allergic rhinitis indicate that the number of mast cell precursor cells in human peripheral blood is quite large, and the number of the mast cell precursor cells in the peripheral blood of the patients with allergic rhinitis is increased by 18.2 times compared with that of the healthy people, so that the diagnosis value of the allergic rhinitis is realized. Since mast cells are the core effector cells (primary effector cells) of allergic reactions, accurate detection of mast cell precursor cells in peripheral blood is of great importance in clinical diagnosis of patients with allergic rhinitis.
The invention also provides application of a reagent for detecting the molecular marker combination in the technical scheme in preparing a kit for identifying human mast cell precursor cells, wherein the cells positively expressed by CD34, Fc epsilon RI alpha, CPA3, TPSAB1 and GATA2 are identified as the human mast cell precursor cells.
In the invention, the kit preferably applies an immunofluorescence detection method and a flow cytometry detection method to identify mast cell precursor cells in human blood. When the kit identifies human mast cell precursor cells using immunofluorescence and flow cytometry assays, the method of identification preferably comprises the steps of: monocyte isolation, dead cell removal, FcR blocking, addition of a fluorescently-labeled antibody against a human cell surface molecule, addition of a fluorescently-labeled antibody against a human intracellular molecule, the human mast cell precursor cell being CD34 + FcεRIα + CPA3 + TPSAB1 + GATA2 + Cells (+ indicating positive expression).
The invention also provides an antibody for detecting the molecular marker combination in the technical scheme, wherein the antibody comprises an antibody for resisting human cell surface molecules and an antibody for resisting human cell internal molecules; the antibody against the human cell surface molecule comprises: anti-human CD34 antibodies and anti-human fcsria antibodies; the antibody against the human intracellular molecule comprises: anti-human CPA3 antibody, anti-human TPSAB1 antibody and anti-human GATA2 antibody. The source of the antibody is not particularly limited in the present invention, and a conventional commercially available product known to those skilled in the art may be used.
The invention also provides a flow cytometry-based fluorescence labeled antibody for detecting the molecular marker combination in the technical scheme, wherein the fluorescence labeled antibody comprises a fluorescence labeled antibody for resisting human cell surface molecules and a fluorescence labeled antibody for resisting human cell internal molecules; the fluorescence labeled antibody for resisting the human cell surface molecule comprises: a fluorescently labeled anti-human CD34 antibody and a fluorescently labeled anti-human fcepsilonra antibody; the fluorescence labeled antibody for resisting the human intracellular molecules comprises: fluorescently labeled anti-human CPA3 antibody, fluorescently labeled anti-human TPSAB1 antibody, and fluorescently labeled anti-human GATA2 antibody. In the present invention, the colors of the labeling fluorescent molecules used for different fluorescently labeled antibodies are preferably different.
The invention also provides a kit for detecting the molecular marker combination based on the technical scheme by using a flow cytometry method, wherein the kit comprises the fluorescence-labeled antibody, blood diluent, mononuclear cell separation liquid, cell culture liquid, dead cell removal dye, FcR blocker, washing buffer, cell staining buffer, cell fixing liquid and transmembrane washing liquid.
The source of the blood diluent is not particularly limited in the present invention. In the present invention, the blood diluent is preferably a solution having an osmotic pressure equal to that of human plasma, and more preferably an aqueous NaCl solution or a PBS buffer solution. In the present invention, the mass percentage of NaCl in the NaCl aqueous solution is preferably 0.9%. In the present invention, the PBS buffer preferably includes the following components in parts by weight: 8.0 parts of NaCl, 0.20 part of KCl and 1.16 parts of Na 2 HPO 4 And 0.20 part of KH 2 PO 4 (ii) a The pH value of the PBS buffer solution is preferably 7.0-7.6, and more preferably 7.2-7.5.
In the present invention, the effective components of the mononuclear cell separation solution preferably include: polysucrose and diatrizoate, or diatrizoate and polysaccharide, or iodixanol, and the solvent of the mononuclear cell fraction is preferably distilled water. The source and variety of the mononuclear cell separation solution are not particularly limited in the present invention. In the invention, the density of the mononuclear cell separation liquid is preferably 1.076-1.078 g/mL, and the osmotic pressure is preferably 275-305 mOsm.
In the present invention, the type and source of the cell culture medium are not particularly limited, and a culture medium for culturing human peripheral blood mononuclear cells is preferred. In the invention, the cell culture solution preferably takes RPMI1640 as a basic culture medium and comprises fetal bovine serum with the volume concentration of 0.1-20% and penicillin-streptomycin solution with the volume concentration of 0.1-3%.
In the present invention, the dead cell removal dye preferably includes an amine-reactive fluorescent dye, and more preferably a solution obtained by dissolving an amine-reactive fluorescent dye in DMSO.
In the present invention, the fluorescent species of the dead cell removal dye, the fluorescent-labeled antibody preferably include: alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 700, 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/Cy5, PE/Cy7, PE/Dazle, PerCP/Cy5.5. With regard to the selection of the kind of fluorescence, the present invention is preferably freely combined depending on the arrangement of the laser and the filter of the flow cytometer.
In the present invention, the effective component of the FcR blocker preferably includes human immunoglobulin or an unrelated immunoglobulin of the same species and subtype as the flow antibody (the above-mentioned fluorescent-labeled antibody) used. In the invention, the mass concentration of the FcR blocker working solution is preferably 0.1-100 mug/mL, and more preferably 1-10 mug/mL.
In the present invention, the washing buffer preferably includes a PBS buffer.
In the present invention, the cell staining buffer is preferably a calcium-magnesium-free Dulbecco's PBS (DPBS) solution, and the effective component of the DPBS solution is fetal bovine serum. In the present invention, the working concentration of the fetal calf serum is preferably 0.1 to 20%, more preferably 0.5 to 15%. In the invention, the pH value of the cell staining buffer solution is preferably 7.0-7.6.
In the present invention, the effective components of the cell fixing solution preferably include formaldehyde and methanol, and the working concentration of the formaldehyde is preferably 0.5 to 10% (mass/mass), more preferably 0.8 to 2% (mass/mass); the working concentration of methanol is preferably 0.1 to 2% (mass/mass), more preferably 0.2 to 1% (mass/mass).
In the present invention, the effective components of the membrane-penetrating lotion preferably include saponin and fetal bovine serum, or saponin and bovine serum albumin. In the invention, 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%.
The invention also provides application of the antibody in the technical scheme or the fluorescently-labeled antibody in the technical scheme or the kit in the technical scheme in preparing a kit for identifying human mast cell precursor cells, wherein the cells positively expressed by CD34, Fc epsilon RI alpha, CPA3, TPSAB1 and GATA2 are identified as the human mast cell precursor cells.
In the present invention, in the application, the method for identification preferably comprises the steps of: monocyte isolation, dead cell removal, FcR blocking, addition of a fluorescently-labeled antibody against a human cell surface molecule, addition of a fluorescently-labeled antibody against a human intracellular molecule, the human mast cell precursor cell being CD34 + FcεRIα + CPA3 + TPSAB1 + GATA2 + Cells (+ indicating positive expression).
Specifically, the method more preferably comprises the steps of:
mixing venous blood and blood diluent, placing the mixture on the upper layer of the mononuclear cell separating medium, centrifuging, and sucking mononuclear cells. In the invention, the volume ratio of the venous blood, the blood diluent and the mononuclear cell separation solution is preferably 1 (0.5-2) to 0.5-2. In the invention, the time for centrifugation is preferably 15-35 min.
After the mononuclear cells are aspirated, the present invention preferably dilutes the mononuclear cells with a blood diluent, centrifuges, and collects the cell pellet. In the invention, the time for centrifugation is preferably 4-30 min.
After obtaining the cell sediment, the invention preferably re-suspends the cell sediment by using a washing buffer solution to obtain a first cell suspension; in the invention, the concentration of the cells is adjusted to be (1-10) multiplied by 10 by the heavy suspension 6 One per mL.
After the first cell suspension is obtained, the first cell suspension, the dead cell removal dye and the FcR blocker are mixed and incubated to obtain a second cell suspension. In the present invention, it is preferable that the first cell suspension is prepared in the range of (0.1 to 1). times.10 6 One cell per 100. mu.L system and 0.1-10. mu.L dead tissueThe cell-removing dye is mixed with 0.1-10 mu LFcR blocker. In the invention, the incubation temperature is preferably 4-40 ℃, and the incubation time is preferably 5-30 min.
After the second cell suspension is obtained, the invention mixes the second cell suspension with cell staining buffer solution, centrifugalizes, abandons the supernatant, mixes the cell sediment with the antibody of the fluorescent-labeled anti-human cell surface molecule, and incubates to obtain a third cell suspension. In the present invention, it is preferable that the second cell suspension is prepared in the range of (0.1 to 1). times.10 6 Each cell/100. mu.L system was mixed with 0.5-5 mL of cell staining buffer. In the invention, the time for centrifugation is preferably 4-30 min. In the present invention, after discarding the supernatant, it is preferable to precipitate the cells in the range of (0.1 to 1). times.10 6 Mixing the system of each cell per 100 mu L and 0.5-20 mu L of fluorescence labeled antibody resisting the surface molecules of the human cells, wherein the mixing is preferably uniform. In the invention, the incubation temperature is preferably 4-40 ℃, and the incubation time is preferably 5-30 min.
After the third cell suspension is obtained, the third cell suspension and the cell staining buffer solution are mixed and centrifuged, and the cell fixing solution is added into the cell sediment for heavy suspension to obtain a fourth cell suspension. In the present invention, the mixing is preferably performed by mixing the third cell suspension with 0.5-5 mL of cell staining buffer. In the present invention, the resuspension is preferably performed in the order of (0.1 to 1). times.10 to the cell pellet 6 0.1-5 mL of cell fixing solution is added into each cell/100 mu L system for resuspension.
After the fourth cell suspension is obtained, the fourth cell suspension and the permeable membrane washing liquid are mixed, centrifuged, the supernatant is discarded, the permeable membrane washing liquid is added again, and the fifth cell suspension is obtained by resuspension. In the invention, the time for centrifugation is preferably 4-30 min.
After the fifth cell suspension is obtained, the fifth cell suspension is preferably centrifuged, the supernatant is discarded, and then the fluorescently-labeled antibody resisting the human intracellular molecules is added, and after mixing, the sixth cell suspension is obtained by incubation in a dark place. In the invention, the time for centrifugation is preferably 4-30 min. In the invention, the addition amount of the antibody is preferably 0.5-20 mu L. In the invention, the incubation time is preferably 10-120 min.
After the sixth cell suspension is obtained, the sixth cell suspension is mixed by a permeable membrane washing solution, centrifuged, the supernatant is discarded, the cell concentration is adjusted by a washing buffer solution, and then the expression condition of each antibody in the cell is detected by a flow cytometer. In the invention, the time for centrifugation is preferably 4-30 min. In the present invention, the cell concentration is preferably adjusted to (0.1 to 10). times.10 6 one/mL.
The present invention identifies CD34 + FcεRIα + CPA3 + TPSAB1 + GATA2 + Cells (+ indicating positive expression) are mast cell precursors.
The invention also provides application of the antibody in the technical scheme or the fluorescence labeled antibody in the technical scheme or the kit in the technical scheme in preparation of a kit for diagnosing allergic diseases.
The invention also provides application of the antibody in the technical scheme or the fluorescence labeling antibody in the technical scheme or the kit in the technical scheme in preparing a kit for diagnosing diseases related to human blood mast cell precursor cells, wherein the diseases comprise allergic diseases.
The invention also provides application of the antibody in the technical scheme, the fluorescence labeled antibody in the technical scheme or the kit in the technical scheme in preparation of a kit for diagnosing allergic rhinitis. The detection of human mast cell precursor cell population enables the diagnosis of allergic diseases typified by allergic rhinitis. The method has important scientific significance for understanding the occurrence and development of allergic rhinitis by carrying out identification research on the peripheral blood mast cell precursor cells based on the allergic rhinitis, more importantly, provides a new method for diagnosing allergic diseases and provides a new target for intervention of the allergic diseases. The results of the embodiment of the invention show that compared with a healthy control group, the number of blood mast cell precursor cells of allergic rhinitis patients is increased by 18.2 times, and statistically significant differences exist.
The molecular marker combination, the detection reagent and the application for identifying human blood mast cell precursor cells according to the present invention are further described in detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.
Example 1
A method for detecting human mast cell precursor cells comprising the steps of:
1. mixing venous blood and blood diluent, placing the mixture on the upper layer of a mononuclear cell separation solution, centrifuging for 15-35 min, and then sucking mononuclear cells, wherein the volume ratio of the venous blood to the blood diluent to the mononuclear cell separation solution is 1: (0.5-2): (0.5-2);
2. diluting the mononuclear cells obtained in the step 1 with a blood diluent, centrifuging for 4-30 min, and collecting cell precipitates;
3. resuspending the cell precipitate obtained in the step 2 with a washing buffer solution, and adjusting the concentration of the cells to be (1-10) x 10 6 Obtaining a first cell suspension liquid after cell/mL;
4. the first cell suspension obtained in the step 3 is subjected to (0.1-1) x 10 6 Mixing the system with each cell per 100 mu L, 0.1-10 mu L of dead cell removal dye and 0.1-10 mu L of FcR blocker, and incubating at 4-40 ℃ for 5-30 min to obtain a second cell suspension;
5. the second cell suspension obtained in the step 4 is subjected to (0.1-1) x 10 6 Mixing the system of each cell per 100 mu L with 0.5-5 mL of cell staining buffer solution, centrifuging for 4-30 min, discarding the supernatant, and precipitating the cells according to the formula of (0.1-1) x 10 6 Adding 0.5-20 mu L of fluorescence labeled anti-human cell surface molecule antibody into each cell/100 mu L system, uniformly mixing, and incubating at 4-40 ℃ for 5-30 min to obtain a third cell suspension;
6. mixing and centrifuging the third cell suspension obtained in the step 5 by using 0.5-5 mL of cell staining buffer solution, and then adding the mixture into cell sediment according to the ratio of (0.1-1) x 10 6 Adding 0.1-5 mL of cell fixing solution into the system with each cell per 100 mu L for resuspension to obtain a fourth cell suspension;
7. and (4) mixing the fourth cell suspension obtained in the step (6) with a proper amount of transmembrane lotion, centrifuging for 4-30 min, discarding the supernatant, and adding a proper amount of transmembrane lotion again to resuspend the mixture to obtain a fifth cell suspension.
8. And (3) centrifuging the fifth cell suspension obtained in the step (7) for 4-30 min, removing the supernatant, adding 0.5-20 mu L of a fluorescence-labeled antibody resisting the human intracellular molecules, mixing, and incubating for 10-120 min in a dark place to obtain a sixth cell suspension.
9. Mixing the sixth cell suspension obtained in the step 8 with a proper amount of transmembrane washing liquid, centrifuging for 4-30 min, discarding the supernatant, and adjusting the cell concentration to (0.1-10) x 10 by using a washing buffer solution 6 And (4) detecting the expression of each antibody in the cells by using a flow cytometer.
10、CD34 + FcεRIα + CPA3 + TPSAB1 + GATA2 + Cells (+ indicating positive expression) are mast cell precursors.
Example 2
1) Collecting peripheral venous blood of healthy people (HC, n-11) and patients with allergic rhinitis (AR, n-16);
2) 1mL of fresh peripheral venous blood (blood in vitro is less than or equal to 2h) is taken and added with 1mL of blood diluent to dilute the blood;
3) 1mL of the mononuclear cell separation solution is taken at the bottom of a centrifuge tube, and diluted blood is carefully superposed on the mononuclear cell separation solution to avoid mixing of the blood and the mononuclear cell separation solution;
4) placing in a centrifuge, centrifuging at 800g for 20min, setting the temperature of the centrifuge at 18 deg.C, setting the acceleration at 9, and setting the deceleration at 0;
5) sucking out the tunica albuginea (mononuclear cells) on the interface by using a dropper, and trying not to suck out the liquid on the tunica albuginea (mononuclear cells) on the interface;
6) diluting the mononuclear cells by 2mL of blood diluent, and centrifuging for 10min at room temperature of 250 g; collecting cell precipitate, resuspending mononuclear cells in 100. mu.L of cell culture medium, counting cells, and adjusting cell density to 4X 10 with cell culture medium 6 counts/mL, obtained mononuclear cells are shown as a in fig. 2;
7) adding 100 mu L of cell suspension into each tube, then adding 1 mu L of dead cell removal dye Zombie NIR and 5 mu L of FcR blocker dissolved in DMSO, and incubating for 15min at room temperature in a dark place;
8) adding 1mL of cell staining buffer solution into each tube, centrifuging for 6min at 200g, and removing supernatant; the obtained desmeared cells (B in fig. 2, approximately 99.05% of the cells in the human peripheral blood mononuclear cell population are mononuclear cells after the removal of the desmeared cells) and the human peripheral blood mononuclear cell population after the removal of the dead cells (C in fig. 2, approximately 99.90% of the human peripheral blood mononuclear cells after the removal of the dead cells are viable cells);
9) adding 100 μ L of cell staining buffer into each tube to resuspend the cells, then adding 5 μ L each of anti-human CD34 labeled by Brilliant Violet 421 and anti-human Fc epsilon RI α labeled by PE/Cy7, and incubating for 15min at room temperature in the dark;
10) adding 1mL of cell staining buffer solution into each tube, centrifuging for 6min at 200g, and removing supernatant;
11) adding 1mL of fixed permeable membrane washing solution into each tube, and keeping the tube away from light for 30min at room temperature;
12) adding 2mL of transmembrane lotion into each tube, centrifuging for 6min at 200g, and removing supernatant;
13) adding 100 mu L of transmembrane lotion into each tube to resuspend cells, then adding 5 mu L of each of the APC-labeled anti-human TPSAB1, FITC-labeled anti-human CPA3 and PE-labeled anti-human GATA2 antibodies, and incubating for 1h at room temperature in a dark place;
14) adding 2mL of transmembrane lotion into each tube, washing once, resuspending cells by 200 mu L of washing buffer solution, and detecting the expression condition of each molecule by a flow cytometer; note: arrows represent the order of analysis of results (FIG. 2).
15) The expression of the obtained antibodies of allergic rhinitis patients is shown as D in FIG. 2 to H in FIG. 2, and the expression of the antibodies of healthy people is shown as I in FIG. 2 to M in FIG. 2; d in 2 is CD34 in peripheral blood mononuclear cell population of allergic rhinitis patients + Expression of (2): about 15.85% of mononuclear cells express CD 34; in FIG. 2, E is CD34 of allergic rhinitis patient + Expression of fceri in peripheral blood mononuclear cell populations: CD34 + About 42.2% of the mononuclear cells express fceri; f in figure 2 is CD34 of allergic rhinitis patient + FcεRI + TPSAB1 in peripheral blood mononuclear cell population + Expression of (2): CD34 + FcεRI + TPSAB1 was expressed in about 83.4% of mononuclear cells; g in figure 2 is CD34 of allergic rhinitis patient + FcεRI + TPSAB1 + CPA3 in peripheral blood mononuclear cell populations + Expression of (2): CD34 + FcεRI + TPSAB1 + About 12.5% of mononuclear cells express CPA 3; h in figure 2 is CD34 of allergic rhinitis patient + FcεRI + TPSAB1 + CPA3 + GATA2 in peripheral blood mononuclear cell populations + Expression of (2): CD34 + FcεRI + TPSAB1 + CPA3 + About 68.5% of the mononuclear cell populations express GATA 2.
FIG. 2 shows the expression I of CD34 in healthy human peripheral blood mononuclear cell population + Expression of (2): about 9.09% of mononuclear cells express CD 34; j in FIG. 2 is a healthy human CD34 + FceRI in peripheral blood mononuclear cell populations + Expression of (2): CD34 + About 44.9% of mononuclear cells express fceri; k in figure 2 is healthy human CD34 + FcεRI + TPSAB1 in peripheral blood mononuclear cell population + Expression of (2): CD34 + FcεRI + About 85.9% of mononuclear cells express TPSAB 1; l in FIG. 2 is a healthy human CD34 + FcεRI + TPSAB1 + CPA3 in peripheral blood mononuclear cell populations + Expression of (2): CD34 + FcεRI + TPSAB1 + About 6.92% of mononuclear cells express CPA 3; m in FIG. 2 is CD34 of healthy human + FcεRI + TPSAB1 + CPA3 + GATA2 in peripheral blood mononuclear cell populations + Expression of (2): CD34 + FcεRI + TPSAB1 + CPA3 + About 19.1% of the mononuclear cell populations express GATA 2.
16) The mast cell precursor cells in the peripheral venous blood mononuclear cells of the obtained healthy human and allergic rhinitis patients are shown in FIG. 3. Note: HC; n is 11; AR; n is 16.
A in fig. 3: the number of human peripheral blood mononuclear cells after removal of debris and swollen cells in healthy Humans (HC) and patients with Allergic Rhinitis (AR);
b in fig. 3: CD34 + Percentage of cells to PBMC;
c in fig. 3: fc epsilon RI + Cell occupancy of CD34 in PBMCs + Percentage of cells;
d in fig. 3: TPSAB1 + Cell occupancy of CD34 in PBMCs + FcεRI + Percentage of cells;
e in fig. 3: CPA3 + Cell occupancy of CD34 in PBMCs + FcεRI + TPSAB1 + Percentage of cells;
f in fig. 3: GATA2 + Cell occupancy of CD34 in PBMCs + FcεRI + TPSAB1 + CPA3 + Percentage of cells;
g in fig. 3: CD34 + FcεRI + TPSAB1 + CPA3 + GATA2 + Mast cell precursors account for PBMC percentage.
And (4) conclusion: percentage of CD34 positive cells (B in FIG. 3), mast cell precursor CD34 in mononuclear cell population of allergic rhinitis patients + FcεRI + TPSAB1 + CPA3 + Percentage of cells (F in fig. 3), mast cell precursor CD34 + FcεRI + TPSAB1 + CPA3 + GATA2 + The proportion of cells (G in fig. 3) is significantly higher than in healthy people. The ratio of the mast cell precursor cells to the peripheral blood mononuclear cells is proved to have significance for diagnosing the allergic rhinitis.
From the above examples, it can be seen that the kit and the detection method of the present invention can rapidly detect mast cell precursor cells in human peripheral blood and can be used for the auxiliary diagnosis of allergic rhinitis.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A combination of molecular markers for the identification of human mast cell precursor cells, wherein said molecular markers comprise human cell surface molecules including CD34 and fcsria and human intracellular molecules including CPA3, TPSAB1 and GATA 2.
2. Use of reagents for detecting the combination of molecular markers of claim 1 in the preparation of a kit for identifying human mast cell precursor cells, said identification identifying cells positively expressing CD34, fcsria, CPA3, TPSAB1 and GATA2 as human mast cell precursor cells.
3. Detecting the antibody of the molecular marker combination of claim 1, wherein the antibody comprises an antibody against a human cell surface molecule and an antibody against a human intracellular molecule; the antibody against the human cell surface molecule comprises: anti-human CD34 antibodies and anti-human fcsria antibodies; the antibody against the human intracellular molecule includes: anti-human CPA3 antibody, anti-human TPSAB1 antibody, and anti-human GATA2 antibody.
4. Detecting a fluorescently-labeled antibody of the molecular marker combination of claim 1 based on flow cytometry, wherein said fluorescently-labeled antibody comprises a fluorescently-labeled antibody against a human cell surface molecule and a fluorescently-labeled antibody against a human intracellular molecule; the fluorescence labeled antibody for resisting the human cell surface molecule comprises: a fluorescently labeled anti-human CD34 antibody and a fluorescently labeled anti-human fcepsilonra antibody; the fluorescence labeled antibody for resisting the human intracellular molecules comprises: 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, wherein the color of the labeling fluorescent molecule used for different fluorescently labeled antibodies is different.
6. A kit for detecting the combination of molecular markers of claim 1 by flow cytometry, wherein the kit comprises the fluorescently labeled antibody of claim 4 or 5, a blood diluent, a mononuclear cell separation solution, a cell culture solution, a dead cell removal dye, an FcR blocker, a washing buffer, a cell staining buffer, a cell fixative solution, and a membrane permeation washing solution.
7. 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 mast cell precursor cells as identifying cells positively expressing CD34, fcsria, CPA3, TPSAB1, and GATA2 as human mast cell precursor cells.
8. Use of the antibody of claim 3 or the fluorescently labeled antibody of claim 4 or 5 or the kit of claim 6 for the preparation of a kit for diagnosing allergic diseases.
9. Use of the antibody of claim 3 or the fluorescently labeled antibody of claim 4 or 5 or the kit of claim 6 for the preparation of a kit for diagnosing a disease associated with human mast cell precursor cells, including allergic diseases.
10. Use of the antibody of claim 3 or the fluorescently labeled antibody of claim 4 or 5 or the kit of claim 6 for preparing a kit for diagnosing allergic rhinitis.
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