WO2019196194A1 - 一种检测人调节性t细胞亚型的试剂盒及检测方法 - Google Patents

一种检测人调节性t细胞亚型的试剂盒及检测方法 Download PDF

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WO2019196194A1
WO2019196194A1 PCT/CN2018/091106 CN2018091106W WO2019196194A1 WO 2019196194 A1 WO2019196194 A1 WO 2019196194A1 CN 2018091106 W CN2018091106 W CN 2018091106W WO 2019196194 A1 WO2019196194 A1 WO 2019196194A1
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cell
human
antibody
cells
solution
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何韶衡
何萍
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何韶衡
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4704—Inhibitors; Supressors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5428—IL-10
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70517—CD8
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

  • the invention belongs to the technical field of cell subtype detection, and particularly relates to a kit for detecting human regulatory T cell subtypes and a detection method thereof.
  • Naive T cells can differentiate into a variety of regulatory T (Treg) cells under different conditions, and at least 7 Treg cell subtypes have been identified: (1) nTregs: CD4 + CD25 + Foxp3 secreting IL-10 and TGF- ⁇ + T cells are the most abundant Treg cell subtypes; (2) iTregs: foreign antigens can stimulate peripheral blood naive CD4 + T cells to induce expression of Foxp3, and Th3 cells secreting TGF- ⁇ and IL-10 also belong to this Subtype; (3) CD127 low/- Treg: CD4 + CD25 + CD127 low/- Foxp3 + Treg cells were found in neonatal thymus; (4) ICOS + Treg: ICOS + Treg cells expressed IL-10, IL-17 And interferon (IFN)- ⁇ ; (5) Tr1 cells: Tr1 cells do not express Foxp3 but secrete IL-10 and inhibit effector Th cell function CD4 + T cells; (6) CD8 + Tregs: OT-1CD8 cells Under
  • Treg cells have multiple subtypes and their roles in the body are different, it is particularly important to distinguish them separately for individual studies.
  • the currently marketed Treg kit only defines CD4 + CD25 + Foxp3 + T cells as Treg cells, does not include CD8 + Treg cells and CD127 low / - Treg cells, nor can it further identify other Tregs in CD4 + Treg cells. Cell subtype.
  • the present invention provides a kit for detecting a human regulatory T cell subtype, comprising the following components: a blood dilution solution, a mononuclear cell separation solution, a cell culture solution, Lymphocyte activating solution, dead cell removing dye, FcR blocking agent, fluorescently labeled antibody against human cell surface marker molecule, fluorescently labeled antibody against human intracellular molecule, lipopolysaccharide, washing buffer, cell staining buffer, a cell fixative and a permeabilized lotion;
  • the fluorescently labeled anti-human cell surface marker molecule antibody comprises: anti-human CD3 or CD4 antibody, anti-human CD8 antibody, anti-human CD25 antibody, anti-human CD127 antibody, anti-human ICOS antibody
  • the fluorescently labeled antibody against a human intracellular molecule comprises: an anti-human IL-10 antibody, an anti-human IL-17 antibody, and
  • the fluorescent cell type of the dead cell removal dye, the fluorescently labeled anti-human cell surface marker molecule, and the fluorescent species of the fluorescently labeled anti-human intracellular molecule are independently: 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 or PerCP/Cy 5.5.
  • the type of fluorescence is freely combined according to the configuration of the laser and the filter of the flow cytometer.
  • the active ingredient of the FcR blocker comprises a human immunoglobulin or an unrelated immunoglobulin of the same genus and subtype as the flow antibody used.
  • the present invention provides a method for detecting a human regulatory T cell subtype using the kit described above, comprising the steps of:
  • the mononuclear cells obtained in the step 1) are diluted with a blood dilution solution, and centrifuged at 90 to 600 g for 4 to 30 minutes, and then the cell pellet is collected;
  • the cell pellet obtained in the step 2) is resuspended in the cell culture medium, and the density of the cells is adjusted to be 0.5 ⁇ 10 6 to 4 ⁇ 10 6 cells/mL to obtain a first cell suspension;
  • the first cell suspension obtained in the step 3) is uniformly mixed with the lymphocyte activation solution and the lipopolysaccharide, and cultured in an incubator containing CO 2 for 4 to 6 hours to obtain a first incubation cell;
  • step 6) taking the second cell suspension obtained in the step 5) according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L system and 0.1-10 ⁇ L dead dye-removing DMSO solution and 0.1-10 ⁇ L Lc blocker. After mixing, incubating at 4 to 40 ° C for 5 to 30 minutes to obtain a second incubation cell;
  • the second incubation cells obtained in the step 6) are mixed with 0.5 to 5 mL of cell staining buffer according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L of the system, centrifuged at 90 to 600 g for 4 to 30 minutes, and discarded. After clearing, the cell pellet was added with 0.5 to 20 ⁇ L of each fluorescently labeled antibody against the human cell surface marker molecule in a system of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L, and uniformly mixed, and then incubated at 4 to 40° C. in the dark at 5 to 40° C. 30min;
  • the third incubation cell obtained in the step 7) is mixed with 0.5 to 5 mL of cell staining buffer according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L of the system, and then centrifuged at 90 to 600 g for 4 to 30 minutes, and discarded. After clearing, add 0.1 ⁇ 5mL cell fixative to the sediment according to 1 ⁇ 10 5 ⁇ 10 ⁇ 10 6 cells/100 ⁇ L system, incubate at 4 ⁇ 40°C for 10 ⁇ 120min in the dark, and obtain the third cultured cells resuspended. ;
  • the resuspended third incubation cells obtained in the step 8) are mixed with 0.5 to 5 mL of the permeated membrane washing solution according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L, and centrifuged at 90 to 600 g for 4 to 30 minutes. After discarding the supernatant, the third incubation cell pellet was obtained, and the third incubation cell pellet was resuspended by adding 0.5 to 5 mL permeabilized washing solution according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L system, and centrifuged at 90-600 g. ⁇ 30min, discard the supernatant, and obtain the third incubation cell pellet;
  • the third incubation cell pellet obtained in the step 9) is added with 0.5 to 20 ⁇ L of each fluorescently labeled anti-intracellular molecule antibody in a system of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L, and uniformly mixed. Incubate at ⁇ 40 ° C for 10 to 120 min in the dark to obtain a fourth incubation cell;
  • the fourth incubation cell pellet obtained in the step 10) is added to a 0.5 to 5 mL membrane permeate according to a system of 10 5 to 10 6 cells/100 ⁇ L, uniformly mixed, and centrifuged at 90 to 600 g for 4 to 30 minutes, and discarded. Clear, the fourth incubation cell pellet was obtained, and the cell concentration was adjusted to (0.1-10) ⁇ 10 6 /mL with PBS, and the expression of the antibody was detected by flow cytometry; the detection result of the Treg cell subtype was obtained according to the expression of the antibody, The relationship between the Treg cell subset and antibody expression is as follows:
  • the conditions of the centrifugation in the step 1) include: a centrifugal force of 200 to 1000 g, and a centrifugation temperature of 18 to 22 °C.
  • the cell culture solution according to the step 3) is based on RPMI 1640 medium, and comprises fetal bovine serum in a volume concentration of 0.1% to 20% and a penicillin-streptomycin double-antibody solution in a volume concentration of 0-3%. .
  • the conditions of the incubation culture in the step 4) include: culturing in an incubator containing 5% CO 2 at a culture temperature of 35 to 40 °C.
  • the amount of each antibody in the antibodies of the fluorescently labeled anti-human cell surface marker molecule and the antibody against the human intracellular molecule of step 7) and step 9) is determined according to the number of cells to be incubated and the working concentration of the antibody.
  • the invention provides a kit for detecting human regulatory T cell subtypes and a detection method thereof.
  • the kit component is simple and easy to obtain, and can simultaneously identify 2-6 kinds of Treg cell subtypes in human blood.
  • Figure 1 is a population of human peripheral blood mononuclear cells
  • Figure 2 is a view of a human peripheral blood mononuclear cell population after removal of dead cells
  • Figure 3 shows the expression of CD4 + (CD3 + CD8 - ) and CD8 + (CD3 + CD8 - ) in human peripheral blood mononuclear cell population
  • Figure 4 shows the expression of CD8 + Treg:CD8 + CD25 + Foxp3 + in human peripheral blood CD3 + CD8 + cell population
  • Figure 5 shows the expression of CD25 and Foxp3 in human peripheral blood CD3 + CD8 - cell population
  • Figure 6 shows the expression of inducible (or adaptive) Tregs (iTreg): CD4 + Foxp3 + IL - 10 + TGF- ⁇ + in human peripheral blood CD3 + CD8 - Foxp3 + cell population;
  • Figure 7 shows the expression of IL-10 - derived type 1 Treg(Tr1):CD4 + CD25 + Foxp3 - IL-10 + in human peripheral blood CD3 + CD8 - CD25 + Foxp3 - cell population;
  • Figure 8 shows the expression of CD127 low expression and negative Treg (CD127 (low&-) Treg): CD4 + CD25 + Foxp3 + CD127 (low&-) in human peripheral blood CD3 + CD8 - CD25 + Foxp3 + cell population;
  • Figure 9 shows the expression of ICOS-positive Treg, (ICOS+Treg): CD4 + CD25 + Foxp3 + ICOS + in human peripheral blood CD3 + CD8 - CD25 + Foxp3 + cell population;
  • Figure 10 shows the expression of IL-17-producing Treg (IL-17 + Treg): CD4 + Foxp3 + CCR6 + IL-17 + in human peripheral blood CD3 + CD8 - Foxp3 + cell population.
  • the invention provides a kit for detecting human regulatory T cell subtypes, comprising the following components: blood dilution liquid, mononuclear cell separation liquid, cell culture liquid, lymphocyte activation liquid, dead cell removal dye, FcR blocking Agents, fluorescently labeled antibodies against human cell surface marker molecules, antibodies to fluorescently labeled anti-human intracellular molecules, PBS buffer, lipopolysaccharide, wash buffer, cell staining buffer, cell fixative, and permeabilized lotion.
  • the fluorescently labeled antibody against human cell surface marker molecule comprises: anti-human CD3 or CD4 antibody, anti-human CD8 antibody, anti-human CD25 antibody, anti-human CD127 antibody, anti-human TGF- ⁇ antibody, anti-antibody Human ICOS antibody and anti-human CCR6 antibody;
  • the fluorescently labeled anti-human intracellular molecular antibody comprises: an anti-human IL-10 antibody, an anti-human IL-17 antibody, and an anti-human Foxp3 antibody.
  • the fluorescent cell-removing dye, the fluorescent-labeled anti-human cell extracellular domain molecule fluorescent species, and the fluorescently labeled anti-human intracellular molecule are preferably independent of the antibody fluorescent species: 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 or PerCP/Cy 5.5.
  • Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 700 APC, APC/Cy7, APC/Cy7, APC/H7
  • the kind of the fluorescence can be freely combined according to the configuration of the laser and the filter of the flow cytometer.
  • the blood diluent preferably has a solution having an osmotic pressure equal to that of the human body, and the blood diluent preferably includes a NaCl solution or a PBS buffer, and the mass concentration of the NaCl solution is preferably 0.9%;
  • the PBS buffer preferably comprises the following components by weight: 8.0 parts NaCl, 0.20 parts KCl, 1.16 parts Na 2 HPO4 and 0.20 parts KH 2 PO4; pH of the PBS buffer The value is preferably 7.0 to 7.6, more preferably 7.2 to 7.5.
  • the source of the blood diluent of the present invention is not particularly limited.
  • the active ingredient of the mononuclear cell separation solution preferably comprises: polysucrose and diatrizoate, or diatom sodium and polysaccharide, or iodixanol, the mononuclear cell separation solution
  • the solvent is preferably distilled water.
  • the source and variety of the single nuclear cell separation solution are not particularly limited in the present invention.
  • the density of the single nuclear cell separation solution is 1.076 to 1.078 g/mL, and the osmotic pressure is 275 to 305 mOsm.
  • the cultivar and the source of the cell culture fluid are not particularly limited, and a culture solution for culturing human peripheral blood mononuclear cells, which is more preferably based on RPMI 1640 medium, contains fetal bovine serum and Penicillin-streptomycin double antibody solution.
  • the final concentration of the fetal calf serum is preferably from 0.1 to 20%, more preferably from 0.5 to 15%, most preferably from 1 to 10%.
  • the final concentration of the penicillin-streptomycin double-antibody solution in the present invention is preferably from 0 to 3%, more preferably from 0.5 to 2%.
  • the active ingredient of the lymphocyte activating solution preferably comprises: stearic acid phorbol ethyl ester, ionomycin and monensin, or myristoyl phorbol ethyl ester, ionomycin and Brefe Decoction A, or stearyl phorbol ethyl ester, calcium ionophore and monensin, or myristoyl phorbol ethyl ester, calcium ionophore and Brefeldin A; in the present invention, the lymph
  • the concentration of the cell activating solution is 500 to 5000 times the concentration of the working solution at the time of use;
  • the working concentration of the myristoyl phorbol ethyl ester is preferably 0.01 to 0.50 ⁇ M, more preferably 0.050 to 0.2 ⁇ M;
  • the working concentration of the prime is preferably from 0.5 to 5.0 ⁇ M, more preferably from 1.0 to 4.0 ⁇ M; the working concentration of the monensin is
  • the dead cell removing dye preferably includes an amine-active fluorescent dye.
  • the amine-active fluorescent dye can penetrate the membrane of damaged cells and cannot penetrate the membrane of intact cells.
  • the dead cell removing dye is preferably a solution obtained by dissolving an amine-active fluorescent dye in DMSO.
  • the fluorescently labeled antibody against human cell surface marker molecule comprises: anti-human CD3 or CD4 antibody, anti-human CD8 antibody, anti-human CD25 antibody, anti-human CD127 antibody, anti-human ICOS antibody, anti-human TGF - ⁇ and an anti-human CCR6 antibody;
  • the fluorescently labeled anti-human intracellular molecular antibody comprises: an anti-human IL-10 antibody, an anti-human IL-17 antibody, and an anti-human Foxp3 antibody.
  • the fluorescent species of the dead cell removal dye, the fluorescently labeled antibody against the human extracellular domain molecule, and the fluorescently labeled antibody against the human intracellular molecule 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 or PerCP/Cy 5.5.
  • the kind of the fluorescence can be freely combined according to the configuration of the laser and the filter of the flow cytometer.
  • the washing buffer preferably includes a PBS buffer.
  • the cell staining buffer is preferably a solution of calcium-free magnesium in Dulbecco's PBS (DPBS), the active ingredient of the DPBS solution is fetal bovine serum, and the working concentration of the fetal bovine serum of the present invention is preferably 0.1 to 20%, more preferably 0.5 to 15%, most preferably 1 to 10%.
  • the pH of the cell staining buffer in the present invention is preferably 7.0 to 7.6.
  • the active ingredient of the cell fixing solution preferably includes 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 the methanol is preferably from 0.1% to 2% (mass/mass), more preferably from 0.2% to 1% (mass/mass).
  • the active ingredient of the permeate lotion preferably includes saponin and fetal bovine serum, or saponin and bovine serum albumin, the concentration of the saponin is preferably 0.1 to 5% (w / v);
  • the volume concentration of the fetal bovine serum is preferably 0.1 to 20%; and the mass concentration of the bovine serum albumin is preferably 0.1 to 20%.
  • the active ingredient of the FcR blocker preferably includes a human immunoglobulin, or an unrelated immunoglobulin of the same genus and subtype as the flow antibody used.
  • the mass concentration of the working solution of the FcR blocking agent is preferably 0.1 to 100 ⁇ g/mL, and more preferably 1 to 10 ⁇ g/mL.
  • the present invention also provides a method for detecting a human regulatory T cell subtype using the above kit, comprising the steps of:
  • the mononuclear cells obtained in the step 1) are diluted with a blood dilution solution, and centrifuged at 90 to 600 g for 4 to 30 minutes, and then the cell pellet is collected;
  • the cell pellet obtained in the step 2) is resuspended in the cell culture medium, and the density of the cells is adjusted to be 0.5 ⁇ 10 6 to 4 ⁇ 10 6 cells/mL to obtain a first cell suspension;
  • the first cell suspension obtained in the step 3) is uniformly mixed with the lymphocyte activation solution and the lipopolysaccharide, and cultured in an incubator containing CO 2 for 4 to 6 hours to obtain a first incubation cell;
  • step 6) taking the second cell suspension obtained in the step 5) according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L system and 0.1-10 ⁇ L dead dye-removing DMSO solution and 0.1-10 ⁇ L Lc blocker. After mixing, incubating at 4 to 40 ° C for 5 to 30 minutes to obtain a second incubation cell;
  • the second incubation cells obtained in the step 6) are mixed with 0.5 to 5 mL of cell staining buffer according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L of the system, centrifuged at 90 to 600 g for 4 to 30 minutes, and discarded. After clearing, the cell pellet was added to the antibody of fluorescently labeled anti-human cell surface marker molecule in an amount of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L, 0.5 to 20 ⁇ L of each antibody, and uniformly mixed at 4 to 40° C. ⁇ 30min;
  • the third incubation cell obtained in the step 7) is mixed with 0.5 to 5 mL of cell staining buffer according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L of the system, and then centrifuged at 90 to 600 g for 4 to 30 minutes, and discarded. After clearing, add 0.1 ⁇ 5mL cell fixative to the sediment according to 1 ⁇ 10 5 ⁇ 10 ⁇ 10 6 cells/100 ⁇ L system, incubate at 4 ⁇ 40°C for 10 ⁇ 120min in the dark, and obtain the third cultured cells resuspended. ;
  • the resuspended third incubation cells obtained in the step 8) are mixed with 0.5 to 5 mL of the permeated membrane washing solution according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L, and centrifuged at 90 to 600 g for 4 to 30 minutes. After discarding the supernatant, the third incubation cell pellet was obtained, and the third incubation cell pellet was resuspended by adding 0.5 to 5 mL permeabilized washing solution according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L system, and centrifuged at 90-600 g. ⁇ 30min, discard the supernatant, and obtain the third incubation cell pellet;
  • the third incubation cell pellet obtained in the step 9) is added with 0.5 to 20 ⁇ L of each fluorescently labeled anti-intracellular molecule antibody in a system of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L, and uniformly mixed. Incubate at ⁇ 40 ° C for 10 to 120 min in the dark to obtain a fourth incubation cell;
  • the fourth incubation cell pellet obtained in the step 10) is added to a 0.5 to 5 mL membrane permeate according to a system of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L, uniformly mixed, and centrifuged at 90 to 600 g. After 30 min, the supernatant was discarded, and the fourth incubation cell pellet was obtained.
  • the cell concentration was adjusted to (0.1-10) ⁇ 10 6 /mL with PBS, and the expression of the antibody was detected by flow cytometry; the Treg cell subtype was obtained according to the expression of the antibody.
  • the relationship between the Treg cell subset and antibody expression is as follows:
  • the venous blood is mixed with the blood diluent and placed in the upper layer of the single nuclear cell separation solution, and after centrifugation for 15 to 35 minutes, the single nuclear cells are aspirated.
  • the volume ratio of the venous blood to the blood diluent and the mononuclear cell separation liquid in the present invention is preferably 1: (0.5 to 2.0): (0.5 to 2.0), more preferably 1: (0.9 to 1.1): 0.9 to 1.1), most preferably 1:1:1.
  • the mixing according to the present invention mixes the venous blood and the blood diluent in a volume ratio of 1:1, and then is placed on the upper layer of the mononuclear cell separation solution.
  • the centrifugal force of the centrifugation is preferably 200 to 1000 g, more preferably 250 to 950 g, and most preferably 300 to 900 g.
  • the time of centrifugation is preferably 15 to 35 min, and when the venous blood is separated by > 2 h, the preferred selective centrifugation is 20 to 40 min; and the temperature of the centrifugation is preferably 18 to 22 °C. After centrifugation, the liquid is stratified. Carefully use a dropper to aspirate the white layer on the interface, and try not to aspirate the upper layer of liquid to obtain mononuclear cells.
  • the mononuclear cells are diluted with a blood dilution solution and centrifuged for 4 to 30 minutes, and then the cell pellet is collected.
  • the volume ratio of the mononuclear cells to the blood diluent in the present invention is preferably 1: (0.5 to 50).
  • the centrifugation time in the present invention is preferably 4 to 30 minutes, and the centrifugal centrifugal force is preferably 90 to 600 g, more preferably 100 to 550 g, and most preferably 150 to 500 g.
  • the cell pellet obtained in the present invention is resuspended in the cell culture medium, and the density of the cells is adjusted to be 0.5 ⁇ 10 6 to 4 ⁇ 10 6 /mL to obtain a first cell suspension.
  • the cell culture solution is preferably added in two portions, and the first time for resuspending the cell pellet, the volume of the first medium added is 0.1 to 1.0 mL / (1 mL of peripheral blood single blood prepared by whole blood)
  • the nucleated cells are then counted, and then the medium is added a second time, and finally the density of the cells is 0.5 ⁇ 10 6 to 4 ⁇ 10 6 /mL.
  • the first cell suspension is mixed with 0.1-10 ⁇ L lymphocyte activation solution and 0.1-10 ⁇ L lipopolysaccharide according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L system, and then incubated. After culturing for 4-6 hours, the first incubation cells were obtained.
  • the mixing is preferably blown with a pipette, and the number of times the pipette is blown is preferably 5 to 20 times, more preferably 5 to 15 times, and most preferably 8 to 12 times. .
  • a CO 2 incubator volume of CO 2 concentration is preferably 3% to 8%, more preferably 4.5% - 5.5% .
  • the temperature of the CO 2 incubator of the present invention is preferably 35 to 40 ° C, more preferably 36 to 38 ° C, and most preferably 36.5 to 37.5 ° C.
  • the cultured cells are mixed with the PBS buffer in a volume ratio of 1: (0.5 to 50), centrifuged for 4 to 30 minutes, and the supernatant is discarded to obtain a second cell suspension.
  • the rotational speed of the centrifugation is preferably from 90 to 600 g, more preferably from 100 to 550 g, most preferably from 150 to 500 g; and the centrifugation time is preferably from 4 to 30 min, more preferably 5 ⁇ 20min.
  • the density of the first incubation cells is adjusted after the centrifugation, and the adjustment is preferably adjusted using a PBS buffer solution.
  • the second cell suspension according to the present invention is in a range of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L system and 0.1-10 ⁇ L of a dead cell-removing dye-containing DMSO solution and 0.1-10 ⁇ L.
  • the FcR blocker is mixed, it is incubated for 5 to 30 minutes to obtain a second incubation cell.
  • the dead cell-removing dye after dissolution is preferably dissolved in DMSO.
  • the incubation temperature of the present invention is preferably 4 to 40 ° C, more preferably 15 to 35 ° C, and the incubation is preferably incubated in a dark environment, preferably 5 to 30 minutes, more preferably It is 10 to 20 minutes.
  • the second incubation cell is mixed with 0.5 to 5 mL of cell staining buffer according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L system, centrifuged for 4 to 30 minutes, and discarded.
  • the cell pellet was added with 0.5 to 20 ⁇ L of each fluorescently labeled antibody against the human cell surface marker molecule in a ratio of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells / 100 ⁇ L, uniformly mixed, and then incubated to obtain a third incubation cell.
  • the rotation speed at the time of centrifugation according to the present invention is preferably from 90 to 600 g, more preferably from 100 to 550 g, most preferably from 150 to 500 g, and the centrifugation time is preferably from 4 to 30 min, more preferably from 5 to 20 min.
  • the incubation temperature of the present invention is preferably 4 to 40 ° C, more preferably 15 to 35 ° C, most preferably 18 to 28 ° C; the incubation is preferably carried out in a dark environment; the incubation The time is preferably from 5 to 30 min, more preferably from 10 to 20 min.
  • the third incubation cell is mixed with 0.5 to 5 mL of cell staining buffer according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L, and centrifuged at 90 to 600 g for 4 to 30 minutes. After discarding the supernatant, the cell suspension was added to the cell pellet and resuspended, and incubated for 10 to 120 minutes to obtain a resuspended third incubation cell.
  • the volume ratio of the second incubation cell to the cell fixation solution is preferably from 1 ⁇ 10 5 to 10 ⁇ 10 6 cells / 100 ⁇ L of the system: (0.1 to 5.0) mL.
  • the rotation speed of the centrifugation of the present invention is preferably from 90 to 600 g, more preferably from 100 to 550 g, most preferably from 150 to 500 g, and the centrifugation time is preferably from 4 to 30 min, more preferably from 10 to 20 min.
  • the incubation temperature of the present invention is preferably 4 to 40 ° C, more preferably 15 to 35 ° C, most preferably 18 to 28 ° C; the incubation is preferably carried out in a dark environment; the incubation time It is preferably from 10 to 150 min, more preferably from 20 to 120 min.
  • the present invention resuspends the third incubation cell and the permeation lotion according to a system of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L: (0.1-5 mL). The volume ratio was mixed, centrifuged for 4 to 30 minutes, and after the supernatant was discarded, the third incubation cell pellet was obtained.
  • the volume ratio of the resuspended third incubation cell to the permeation lotion is preferably 1 ⁇ 10 5 to 10 ⁇ 10 6 cells / 100 ⁇ L of the system: (0.1 to 5 mL), more preferably It is a system of 1 ⁇ 10 5 to 1 ⁇ 10 6 cells / 100 ⁇ L: (0.2 to 4 mL).
  • the rotation speed of the centrifugation according to the present invention is preferably from 90 to 600 g, more preferably from 100 to 550 g, most preferably from 150 to 500 g, and the centrifugation time is preferably from 4 to 30 min, more preferably from 5 to 20 min.
  • the third incubation cell is precipitated according to the invention according to 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L system and (0.1-20 ⁇ L) each fluorescently labeled anti-human intracellular molecule.
  • the antibody was mixed and incubated for 10 to 120 min to obtain a fourth incubation cell.
  • the third incubation cell pellet is preferably washed before the third incubation cell pellet is mixed with the permeabilization lotion and the fluorescently labeled anti-human intracellular molecular antibody.
  • the number of times of washing in the present invention is preferably 1 to 3 times, more preferably 2 times, and each time washing, it is preferably washed with a permeated membrane washing liquid, and the amount of the permeating membrane washing liquid is 1 ⁇ 10 5 to 10 ⁇ 10 6 cells / 100 ⁇ L of the cell system is preferably 0.5 to 5 mL, more preferably 0.5 to 4 mL, and most preferably 1 to 3 mL.
  • the supernatant is centrifuged to obtain a third incubation cell after washing. precipitation.
  • the rotation speed of the centrifugation according to the present invention is preferably from 90 to 600 g, more preferably from 100 to 550 g, most preferably from 150 to 500 g, and the centrifugation time is preferably from 4 to 30 min, more preferably from 5 to 20 min.
  • the third incubation cell after washing and the fluorescently labeled anti-human intracellular molecule antibody are preferably 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L of the antibody.
  • System Mix in a ratio of (0.1 to 20) ⁇ L and incubate for 10 to 120 minutes to obtain a fourth incubation cell.
  • the volume ratio of the third incubation cell pellet to the fluorescently labeled intrabody after washing is preferably 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L of the cell system: (0.1-20) ⁇ L, More preferably, it is a cell system of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells / 100 ⁇ L: (0.5 to 15) ⁇ L.
  • the incubation is preferably carried out in the dark, preferably at a temperature of 4 to 40 ° C, more preferably 15 to 35 ° C, most preferably 18 to 28 ° C;
  • the incubation time is preferably from 10 to 120 min, more preferably from 20 to 100 min.
  • the fourth incubation cell and the permeate wash solution are mixed according to a ratio of 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L of the cell system: (0.5-5) mL, and then centrifuged.
  • the expression of the antibody was detected by flow cytometry.
  • the volume ratio of the fourth incubation cell to the permeate washing solution and the cell staining buffer is preferably 1 ⁇ 10 5 to 10 ⁇ 10 6 cells/100 ⁇ L of the cell system: (0.5-5) mL. More preferably, it is 1 ⁇ 10 5 - 10 ⁇ 10 6 cells / 100 ⁇ L of the cell system: (0.5 - 4) mL.
  • the expression of the antibody is detected by flow cytometry, and the cell subtype of Treg is distinguished according to the expression of the antibody, as shown in Table 1:
  • kits for detecting human regulatory T cell subsets provided by the present invention are described in detail below with reference to the examples, but they are not to be construed as limiting the scope of the invention.
  • Fig. 1 Diluting the mononuclear cell solution with 2 mL of blood dilution solution, and centrifuging at room temperature for 250 min at room temperature for 10 min; collecting the cell pellet, and resuspending the mononuclear cells in 100 ⁇ L of the cell culture medium, and the obtained mononuclear cells are shown in Fig. 1 .
  • the kit of the present invention and the detection method thereof can rapidly detect 2-6 kinds of Treg cell subtypes in human blood, and the expression of inducible (or adaptive) Tregs is shown in FIG. 6;
  • the expression of IL-10-producing Treg is shown in Figure 7;
  • the expression of CD127 low expression and negative Treg is shown in Figure 8;
  • the expression of ICOS-positive Treg is shown in Figure 9;
  • the expression of IL-17-producing Treg is shown in Figure 10. Shown.

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Abstract

一种检测人调节性T细胞亚型的试剂盒及其检测方法，属于细胞亚型检测技术领域。试剂盒，包括以下组分：血液稀释液，单个核细胞分离液，细胞培养液，淋巴细胞活化液，死细胞去除染料，FcR阻断剂、荧光标记的抗人细胞表面标记分子的抗体、荧光标记的抗人细胞内分子的抗体、PBS缓冲液、脂多糖、洗涤缓冲液、细胞染色缓冲液、细胞固定液和透膜洗液。检测时，首先分离全血中的单个核细胞，其次刺激人外周血单个核细胞，最后荧光抗体染色即可检测调节性T细胞亚型。利用所述试剂盒及检测方法可简单快捷的分辨2-6种调节性T细胞亚型。

Description

一种检测人调节性T细胞亚型的试剂盒及检测方法
本申请要求于2018年04月13日提交中国专利局、申请号为201810331146.2、发明名称为“一种检测人调节性T细胞亚型的试剂盒及检测方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于细胞亚型检测技术领域，具体涉及一种检测人调节性T细胞亚型的试剂盒及检测方法。
背景技术
19世纪90年代中期，人们提出CD4 +抑制性T细胞，即调节性T(Treg)细胞，可以抑制效应性T细胞如Th细胞对机体的免疫损伤效应。目前，越来越多的资料显示人体内有几种不同亚型的Treg，且其在免疫系统中的作用不同，使得Treg的作用更加复杂。
幼稚T细胞在不同条件可分化为多种调节性T(Treg)细胞，目前已鉴定出至少7种Treg细胞亚型：(1)nTregs：分泌IL-10和TGF-β的CD4 +CD25 +Foxp3 +的T细胞，是数量最多的Treg细胞亚型；(2)iTregs：外来抗原可刺激外周血幼稚CD4 +T细胞诱导其表达Foxp3，此外分泌TGF-β和IL-10的Th3细胞也属于该亚型；(3)CD127 low/-Treg：在新生儿胸腺发现了CD4 +CD25 +CD127 low/-Foxp3 +Treg细胞；(4)ICOS +Treg：ICOS +Treg细胞表达IL-10、IL-17和干扰素(IFN)-γ；(5)Tr1细胞：Tr1细胞是不表达Foxp3但分泌IL-10并可抑制效应性Th细胞功能CD4 +T细胞；(6)CD8 +Tregs：OT-1CD8细胞在IL-4和IL-12作用下可迅速分化为表达CD8、分泌IL-10的Treg细胞(CD8 +Treg细胞)，且共表达活化和幼稚细胞相关的标记物；(7)产IL-17的Foxp3 +Tregs。
鉴于Treg细胞有多种亚型，且其在机体中的作用不同，将其分辨开进行单独研究显得尤为重要。但是目前市售的Treg试剂盒仅将CD4 +CD25 +Foxp3 +T细胞定义为Treg细胞，并不包括CD8 +Treg细胞和CD127 low/-Treg细胞，也不能进一步鉴定CD4 +Treg细胞中的其它Treg细 胞亚型。
发明内容
有鉴于此，本发明的目的在于提供一种可同时分辨2-6种人调节性T细胞亚型的试剂盒。
为了实现上述发明目的，本发明提供以下技术方案：本发明提供了一种检测人调节性T细胞亚型的试剂盒，包括以下组分：血液稀释液、单个核细胞分离液、细胞培养液、淋巴细胞活化液、死细胞去除染料、FcR阻断剂、荧光标记的抗人细胞表面标记分子的抗体、荧光标记的抗人细胞内分子的抗体、脂多糖、洗涤缓冲液、细胞染色缓冲液、细胞固定液和透膜洗液；所述荧光标记的抗人细胞表面标记分子的抗体包括：抗人CD3或CD4抗体，抗人CD8抗体，抗人CD25抗体，抗人CD127抗体，抗人ICOS抗体，抗人TGF-β抗体和抗人CCR6抗体；所述荧光标记的抗人细胞内分子的抗体包括：抗人IL-10抗体，抗人IL-17抗体和抗人Foxp3抗体。
优选的，所述死细胞去除染料、荧光标记的抗人细胞表面标记分子的抗体荧光种类和荧光标记的抗人细胞内分子的抗体荧光种类独立的包括：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 Violet650、Brilliant Violet711、Brilliant Violet785、FITC、LEAF、Pacific Blue、PE、PE/Cy5、PE/Cy7、PE/Dazzle、PerCP或PerCP/Cy 5.5。
优选的，所述荧光的种类根据流式细胞仪的激光和滤光片的配置进行自由组合。
优选的，所述FcR阻断剂的有效成分包括人免疫球蛋白或与使用的流式抗体同一种属且亚型相同的不相关的免疫球蛋白。
本发明提供了一种利用上述试剂盒检测人调节性T细胞亚型的方法，包括以下步骤：
1)将静脉血与血液稀释液混合后置于单个核细胞分离液上层，离心15～35min后，吸取单个核细胞，所述静脉血、血液稀释液及单个核细胞分离液的体积比为1：(0.5～2)：(0.5～2)；
2)将步骤1)得到的所述单个核细胞用血液稀释液稀释后，90～600g离心4～30min后，收集细胞沉淀；
3)将步骤2)得到的所述细胞沉淀用细胞培养液重悬，调整所述细胞的密度为0.5×10 6～4×10 6个/mL，得第一细胞悬液；
4)将步骤3)得到的所述第一细胞悬液与淋巴细胞活化液和脂多糖均匀混合，置于含CO 2的培养箱中培养4～6h，得第一孵育细胞；
5)将步骤4)得到的所述第一孵育细胞与PBS缓冲液按照1：(0.5～10)的体积比混合后，90～600g离心4～30min，弃去上清后得第二细胞悬液；
6)取步骤5)得到的所述第二细胞悬液按照1×10 5～10×10 6个细胞/100μL的体系与0.1～10μL含死细胞去除染料的DMSO溶液和0.1～10μLFcR阻断剂混合后，4～40℃孵育5～30min，得第二孵育细胞；
7)将步骤6)得到的所述第二孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL细胞染色缓冲液混合，90～600g离心4～30min，弃上清后将细胞沉淀按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～20μL各荧光标记的抗人细胞表面标记分子的抗体，均匀混合后4～40℃避光孵育5～30min；
8)将步骤7)得到的所述第三孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL细胞染色缓冲液混合后90～600g离心4～30min，弃上清后向沉淀中按照1×10 5～10×10 6个细胞/100μL的体系加入0.1～5mL细胞固定液重悬，4～40℃避光孵育10～120min，得重悬的第三孵育细胞；
9)将步骤8)得到的所述重悬的第三孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL透膜洗液混合，90～600g离心4～30min，弃上清后得第三孵育细胞沉淀，再次按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～5mL透膜洗液重悬第三孵育细胞沉淀，90～600g离心4～30min，弃上清，得第三孵育细胞沉淀；
10)将步骤9)得到的所述第三孵育细胞沉淀按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～20μL各荧光标记的抗细胞内分子的抗体，均匀混合后4～40℃避光孵育10～120min，得第四孵育细胞；
11)将步骤10)得到的所述第四孵育细胞沉淀按照10 5～10 6个细胞/100μL的体系加入0.5～5mL透膜洗液，均匀混合后，90～600g离心4～30min， 弃上清，得第四孵育细胞沉淀，用PBS调整细胞浓度为(0.1-10)×10 6个/mL后用流式细胞仪检测抗体的表达；根据抗体的表达得到Treg细胞亚型的检测结果，所述Treg细胞亚型与抗体表达的关系对应如下：
Figure PCTCN2018091106-appb-000001
注：+为阳性；low为低表达；-为阴性。
优选的，步骤1)所述离心的条件包括：离心力为200～1000g，离心温度为18～22℃。
优选的，步骤3)所述的细胞培养液以RPMI 1640培养基为基础，包括体积浓度为0.1％～20％的胎牛血清和体积浓度为0-3％的青霉素-链霉素双抗溶液。
优选的，步骤4)所述孵育培养的条件包括：在含5％CO 2的培养箱中进行培养，培养温度为35～40℃。
优选的，步骤7)和步骤9)所述荧光标记的抗人细胞表面标记分子的抗体和抗人细胞内分子的抗体中各抗体的添加量根据孵育细胞的数量和抗体的工作浓度而定。
本发明提供了一种检测人调节性T细胞亚型的试剂盒及其检测方法，试剂盒成分简单易得，可同时鉴别人体血液中的2～6种Treg细胞亚型。
说明书附图
图1为人外周血单个核细胞群；
图2为去除死细胞后的人外周血单个核细胞群；
图3为人外周血单个核细胞群中CD4 +(CD3 +CD8 -)和CD8 +(CD3 +CD8 -)的表达情况；
图4为人外周血CD3 +CD8 +细胞群中CD8 +Treg：CD8 +CD25 +Foxp3 +的表达情况；
图5为人外周血CD3 +CD8 -细胞群中CD25和Foxp3表达情况；
图6为人外周血CD3 +CD8 -Foxp3 +细胞群中诱导型(或适应型)Treg (iTreg)：CD4 +Foxp3 +IL -10 +TGF-β +的表达情况；
图7为人外周血CD3 +CD8 -CD25 +Foxp3 -细胞群中产IL-10的1型Treg(Tr1)：CD4 +CD25 +Foxp3 -IL-10 +的表达情况；
图8为人外周血CD3 +CD8 -CD25 +Foxp3 +细胞群中CD127低表达和阴性Treg(CD127 (low&-)Treg)：CD4 +CD25 +Foxp3 +CD127 (low&-)的表达情况；
图9为人外周血CD3 +CD8 -CD25 +Foxp3 +细胞群中ICOS阳性Treg，(ICOS+Treg)：CD4 +CD25 +Foxp3 +ICOS +的表达情况；
图10为人外周血CD3 +CD8 -Foxp3 +细胞群中产IL-17的Treg(IL-17 +Treg)：CD4 +Foxp3 +CCR6 +IL-17 +的表达情况。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
本发明提供了一种检测人调节性T细胞亚型的试剂盒，包括以下组分：血液稀释液、单个核细胞分离液、细胞培养液、淋巴细胞活化液、死细胞去除染料、FcR阻断剂、荧光标记的抗人细胞表面标记分子的抗体、荧光标记的抗人细胞内分子的抗体、PBS缓冲液、脂多糖、洗涤缓冲液、细胞染色缓冲液、细胞固定液和透膜洗液。
在本发明中，所述荧光标记的抗人细胞表面标记分子的抗体包括：抗人CD3或CD4抗体、抗人CD8抗体、抗人CD25抗体、抗人CD127抗体、抗人TGF-β抗体、抗人ICOS抗体和抗人CCR6抗体；所述荧光标记的抗人细胞内分子抗体包括：抗人IL-10抗体、抗人IL-17抗体和抗人Foxp3抗体。
在本发明中，所述死细胞去除染料、荧光标记的抗人细胞胞膜外段分子的抗体荧光种类和荧光标记的抗人细胞内分子的抗体荧光种类独立的优选包括：Alexa Fluor 488、Alexa Fluor 594、Alexa Fluor 647、Alexa Fluor700、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 Violet711、Brilliant Violet785、FITC、LEAF、Pacific Blue、PE、PE/Cy5、PE/Cy7、PE/Dazzle、PerCP或PerCP/Cy 5.5。
在本发明中，所述荧光的种类可以根据流式细胞仪的激光和滤光片的配置进行自由组合。
在本发明中，所述血液稀释液优选的渗透压与人体血浆渗透压相等的溶液，具体的所述血液稀释液优选的包括NaCl溶液或PBS缓冲液，所述NaCl溶液的质量浓度优选的为0.9％；在本发明中，所述PBS缓冲液优选的包括以下重量份的组分：8.0份NaCl，0.20份KCl，1.16份Na 2HPO4和0.20份KH 2PO4；所述PBS缓冲液的pH值优选的为7.0～7.6，更优选的为7.2～7.5。本发明对血液稀释液的来源没有特殊限定。
在本发明中，所述单个核细胞分离液的有效成分优选的包括：聚蔗糖和泛影葡胺，或者泛影钠和多聚糖，或者碘克沙醇，所述单个核细胞分离液的溶剂优选的为蒸馏水。本发明对所述单个核细胞分离液的来源及品种没有特殊限定，优选的所述单个核细胞分离液的密度是1.076～1.078g/mL，渗透压是275～305mOsm。
本发明对所述细胞培养液的品种及来源没有特殊限定，优选的为培养人外周血单个核细胞的培养液，所述培养液更优选的以RPMI 1640培养基为基础，包含胎牛血清和青霉素-链霉素双抗溶液。本发明中，所述胎牛血清的体积终浓度优选的为0.1～20％，更优选的为0.5～15％，最优选的为1～10％。在本发明中所述青霉素-链霉素双抗溶液的体积终浓度优选的为0～3％，更优选的为0.5～2％。
在本发明中，所述淋巴细胞活化液的有效成分优选的包括：豆蔻酰佛波醇乙酯、离子霉素和莫能菌素，或者豆蔻酰佛波醇乙酯、离子霉素和布雷非德菌素A，或者豆蔻酰佛波醇乙酯、钙离子载体和莫能菌素，或者豆蔻酰佛波醇乙酯、钙离子载体和布雷非德菌素A；本发明中，所述淋巴细胞活化液的浓度是使用时工作液浓度的500～5000倍；所述豆蔻酰佛波醇乙酯的工作浓度优选的为0.01～0.50μM，更优选的为0.050～0.2μM；所述离子霉素的工作浓度优选的为0.5～5.0μM，更优选的为1.0～4.0μM；所述莫能菌素的的工作浓度优选的为0.5～6.0μM，更优选的为1.0～3.0μM；所述布雷非德菌素A的工作浓度优选的为0.5～30μg/mL，更优选的为1～20μg/mL；所述钙离子载体的工作浓度优选的为25～1000ng/mL，更优选的为100～500ng/mL。本发明所述淋巴细胞活化液的溶剂 优选的为DMSO。
在本发明中，所述死细胞去除染料优选的包括胺活性荧光染料。所述胺活性荧光染料可以透过受损细胞的胞膜而不能透过完整细胞的胞膜。本发明中，所述死细胞去除染料优选的用DMSO溶解胺活性荧光染料得到的溶液。
在本发明中，所述荧光标记的抗人细胞表面标记分子的抗体包括：抗人CD3或CD4抗体、抗人CD8抗体、抗人CD25抗体、抗人CD127抗体、抗人ICOS抗体、抗人TGF-β和抗人CCR6抗体；所述荧光标记的抗人细胞内分子抗体包括：抗人IL-10抗体、抗人IL-17抗体和抗人Foxp3抗体。
在本发明中，所述死细胞去除染料、荧光标记的抗人细胞胞膜外段分子的抗体和荧光标记的抗人细胞内分子的抗体的荧光种类分别包括：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 Violet650、Brilliant Violet711、Brilliant Violet785、FITC、LEAF、Pacific Blue、PE、PE/Cy5、PE/Cy7、PE/Dazzle、PerCP或PerCP/Cy 5.5。
在本发明中，所述荧光的种类可以根据流式细胞仪的激光和滤光片的配置进行自由组合。
在本发明中，所述洗涤缓冲液优选的包括PBS缓冲液。
在本发明中，所述细胞染色缓冲液优选的为无钙镁的Dulbecco’s PBS(DPBS)溶液，所述DPBS溶液的有效成分是胎牛血清，本发明所述胎牛血清的工作浓度优选的为0.1～20％，更优选的为0.5～15％，最优选的为1～10％。本发明中所述细胞染色缓冲液的pH值优选的为7.0～7.6。
在本发明中，所述细胞固定液的有效成分优选的包括甲醛和甲醇，所述甲醛的工作浓度优选的为0.5％～10％(质量/质量)，更优选的为0.8％～2％(质量/质量)；所述甲醇的工作浓度优选的为0.1％～2％(质量/质量)，更优选的为0.2％～1％(质量/质量)。
在本发明中，所述透膜洗液的有效成分优选的包括皂素和胎牛血清，或者皂素和牛血清白蛋白，所述皂素的浓度优选为0.1～5％(w/v)；所述 胎牛血清的体积浓度优选为0.1～20％；所述牛血清白蛋白的质量体积浓度优选为0.1～20％。
在本发明中，所述FcR阻断剂的有效成分优选的包括人免疫球蛋白，或与使用的流式抗体同一种属且亚型相同的不相关的免疫球蛋白。所述FcR阻断剂的工作液的质量浓度优选为0.1～100μg/mL，更优选为1～10μg/mL。
本发明还提供了一种利用上述试剂盒检测人调节性T细胞亚型的方法，包括以下步骤：
1)将静脉血与血液稀释液混合后置于单个核细胞分离液上层，离心15～35min后，吸取单个核细胞，所述静脉血、血液稀释液及单个核细胞分离液的体积比为1：(0.5～2)：(0.5～2)；
2)将步骤1)得到的所述单个核细胞用血液稀释液稀释后，90～600g离心4～30min后，收集细胞沉淀；
3)将步骤2)得到的所述细胞沉淀用细胞培养液重悬，调整所述细胞的密度为0.5×10 6～4×10 6个/mL，得第一细胞悬液；
4)将步骤3)得到的所述第一细胞悬液与淋巴细胞活化液和脂多糖均匀混合，置于含CO 2的培养箱中培养4～6h，得第一孵育细胞；
5)将步骤4)得到的所述第一孵育细胞与PBS缓冲液按照1：(0.5～10)的体积比混合后，90～600g离心4～30min，弃去上清后得第二细胞悬液；
6)取步骤5)得到的所述第二细胞悬液按照1×10 5～10×10 6个细胞/100μL的体系与0.1～10μL含死细胞去除染料的DMSO溶液和0.1～10μLFcR阻断剂混合后，4～40℃孵育5～30min，得第二孵育细胞；
7)将步骤6)得到的所述第二孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL细胞染色缓冲液混合，90～600g离心4～30min，弃上清后将细胞沉淀按照1×10 5～10×10 6个细胞/100μL的体系加入荧光标记的抗人细胞表面标记分子的抗体每种0.5～20μL，均匀混合后4～40℃避光孵育5～30min；
8)将步骤7)得到的所述第三孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL细胞染色缓冲液混合后90～600g离心4～30min，弃上清后向沉淀中按照1×10 5～10×10 6个细胞/100μL的体系加入 0.1～5mL细胞固定液重悬，4～40℃避光孵育10～120min，得重悬的第三孵育细胞；
9)将步骤8)得到的所述重悬的第三孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL透膜洗液混合，90～600g离心4～30min，弃上清后得第三孵育细胞沉淀，再次按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～5mL透膜洗液重悬第三孵育细胞沉淀，90～600g离心4～30min，弃上清，得第三孵育细胞沉淀；
10)将步骤9)得到的所述第三孵育细胞沉淀按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～20μL各荧光标记的抗细胞内分子的抗体，均匀混合后4～40℃避光孵育10～120min，得第四孵育细胞；
11)将步骤10)得到的所述第四孵育细胞沉淀按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～5mL透膜洗液，均匀混合后，90～600g离心4～30min，弃上清，得第四孵育细胞沉淀，用PBS调整细胞浓度为(0.1-10)×10 6个/mL后用流式细胞仪检测抗体的表达；根据抗体的表达得到Treg细胞亚型的检测结果，所述Treg细胞亚型与抗体表达的关系对应如下：
Figure PCTCN2018091106-appb-000002
注：+为阳性；low为低表达；-为阴性。
本发明将静脉血与血液稀释液混合后置于单个核细胞分离液上层，离心15～35min后，吸取单个核细胞。本发明中所述静脉血与血液稀释液及单个核细胞分离液的体积比优选的为1：(0.5～2.0)：(0.5～2.0)，更优选的为1：(0.9～1.1)：(0.9～1.1)，最优选的为1:1:1。本发明所述混合优选的为将静脉血和血液稀释液按照1:1的体积比混合，而后置于单个 核细胞分离液上层。在本发明中，所述离心时优选的设置升速为9，降速为0，所述离心的离心力优选的为200～1000g，更优选的为250～950g，最优选的为300～900g，所述离心的时间优选的为15～35min，当所述静脉血离体时间＞2h时，优选的选择离心为20～40min；所述离心的温度优选的为18～22℃。离心后，液体分层，小心用滴管吸出界面上的白膜层，尽量不要吸出其上层液体，得单个核细胞。
得单个核细胞后，本发明将所述单个核细胞用血液稀释液稀释后离心4～30min后，收集细胞沉淀。本发明中所述单个核细胞与血液稀释液的体积比优选的为1：(0.5～50)。本发明中所述离心的时间优选的为4～30min，所述离心的离心力优选的为90～600g，更优选的为100～550g，最优选的为150～500g。
收集细胞沉淀后，本发明将得到的所述细胞沉淀用细胞培养液重悬，调整所述细胞的密度为0.5×10 6～4×10 6个/mL，得第一细胞悬液。在本发明中，所述细胞培养液优选的分两次添加，第一次用于重悬细胞沉淀，第一次加入培养基的体积为0.1～1.0mL/(1mL全血制备的外周血单个核细胞)，然后进行细胞计数，然后第二次添加培养基，最终使细胞的密度为0.5×10 6～4×10 6个/mL。
得第一细胞悬液后，本发明将所述第一细胞悬液按照1×10 5～10×10 6个细胞/100μL体系与0.1～10μL淋巴细胞活化液和0.1～10μL脂多糖混合，孵育培养4～6h，得第一孵育细胞。在本发明中，所述混合优选的为用移液器吹打，所述移液器吹打的次数优选的为5～20次，更优选的为5～15次，最优选的为8～12次。在本发明中，所述孵育培养优选的为在CO 2培养箱中进行，所述CO 2培养箱中CO 2的体积浓度优选的为3％～8％，更优选的为4.5％～5.5％。本发明所述CO 2培养箱的温度优选的为35～40℃，更优选的为36～38℃，最优选的为36.5～37.5℃。
得第一孵育细胞后，本发明将所述培养细胞与PBS缓冲液按照1：(0.5～50)的体积比混合后离心4～30min，弃上清后得第二细胞悬液。在本发明中，所述离心的转速优选的为90～600g，更优选的为100～550g，最优选的为150～500g；所述离心的时间优选的为4～30min，更优选的为5～20min。在本发明中，所述离心后调整第一孵育细胞的密度，所述调整 优选的利用PBS缓冲溶液调整。
得第二细胞悬液后，本发明将所述第二细胞悬液按照1×10 5～10×10 6个细胞/100μL体系与0.1～10μL含死细胞去除染料的DMSO溶液和0.1～10μL的FcR阻断剂混合后，孵育5～30min，得第二孵育细胞。在本发明中，溶解后的死细胞去除染料优选的为DMSO溶解。本发明所述孵育的温度优选的为4～40℃，更优选的为15～35℃，所述孵育优选的在避光环境中孵育，所述孵育的时间优选的为5～30min，更优选的为10～20min。
得第二孵育细胞后，本发明将所述第二孵育细胞按照1×10 5～10×10 6个细胞/100μL体系与0.5～5mL细胞染色缓冲液混合，离心4～30min，弃上清后细胞沉淀按照1×10 5～10×10 6个细胞/100μL体系加入0.5～20μL各荧光标记的抗人细胞表面标记分子的抗体，均匀混合后孵育，得第三孵育细胞。本发明所述离心时的转速优选的为90～600g，更优选的为100～550g，最优选的为150～500g，所述离心的时间优选的为4～30min，更优选的为5～20min。本发明所述孵育的温度优选的为4～40℃，更优选的为15～35℃，最优选的为18～28℃；所述孵育优选的为在避光的环境中孵育；所述孵育的时间优选的为5～30min，更优选的为10～20min。
得第三孵育细胞后，本发明将所述第三孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL细胞染色缓冲液混合，90～600g离心4～30min，弃上清后向细胞沉淀中加入细胞固定液重悬，孵育10～120min，得重悬的第三孵育细胞。在本发明中，所述第二孵育细胞与细胞固定液的体积比优选的为按照1×10 5～10×10 6个细胞/100μL的体系：(0.1～5.0)mL。本发明所述离心的转速优选的为90～600g，更优选的为100～550g，最优选的为150～500g，所述离心的时间优选的为4～30min，更优选的为10～20min。本发明所述孵育的温度优选的为4～40℃，更优选的为15～35℃，最优选的为18～28℃；所述孵育优选的为避光环境中孵育；所述孵育的时间优选的为10～150min，更优选的为20～120min。
得重悬的第三孵育细胞后，本发明将所述重悬的第三孵育细胞与透膜洗液按照1×10 5～10×10 6个细胞/100μL的体系：(0.1～5mL)的体积比混合，离心4～30min，弃上清后，得第三孵育细胞沉淀。在本发明中， 所述重悬的第三孵育细胞与透膜洗液的体积比优选的为1×10 5～10×10 6个细胞/100μL的体系：(0.1～5mL)，更优选的为1×10 5～1×10 6个细胞/100μL的体系：(0.2～4mL)。本发明所述离心的转速优选的为90～600g，更优选的为100～550g，最优选的为150～500g，所述离心的时间优选的为4～30min，更优选的为5～20min。
得第三孵育细胞沉淀后，本发明将所述第三孵育细胞沉淀按照1×10 5～10×10 6个细胞/100μL的体系与(0.1～20)μL各荧光标记的抗人细胞内分子抗体混合，孵育10～120min，得第四孵育细胞。
在本发明中，将所述第三孵育细胞沉淀与透膜洗液及荧光标记抗人细胞内分子抗体进行混合前，优选的清洗所述第三孵育细胞沉淀。在本发明所述清洗的次数优选的为1～3次，更优选的为2次，每次清洗时，优选的利用透膜洗液进行清洗，所述透膜洗液的用量按照1×10 5～10×10 6个细胞/100μL的细胞体系优选的为0.5～5mL，更优选的为0.5～4mL，最优选的为1～3mL，清洗后离心弃上清，得清洗后第三孵育细胞沉淀。本发明所述离心的转速优选的为90～600g，更优选的为100～550g，最优选的为150～500g，所述离心的时间优选的为4～30min，更优选的为5～20min。
得清洗后第三孵育细胞沉淀后，本发明优选将所述清洗后第三孵育细胞沉淀与荧光标记的抗人细胞内分子的抗体按照1×10 5～10×10 6个细胞/100μL的细胞体系：(0.1～20)μL的比例混合，孵育10～120min，得第四孵育细胞。在本发明中，所述清洗后第三孵育细胞沉淀与荧光标记胞内抗体的体积比优选的为1×10 5～10×10 6个细胞/100μL的细胞体系：(0.1～20)μL，更优选的为1×10 5～10×10 6个细胞/100μL的细胞体系：(0.5～15)μL。在本发明中，所述孵育优选的在避光条件下进行，所述孵育的温度优选的为4～40℃，更优选的为15～35℃，最优选的为18～28℃；所述孵育的时间优选的为10～120min，更优选的为20～100min。
得第四孵育细胞后，本发明将所述第四孵育细胞与透膜洗液按照1×10 5～10×10 6个细胞/100μL的细胞体系：(0.5～5)mL的比例混合后离心，采用流式细胞仪检测抗体的表达。在本发明中，所述第四孵育细胞与透膜洗液及细胞染色缓冲液的体积比优选的为1×10 5～10×10 6个细胞 /100μL的细胞体系：(0.5～5)mL，更优选的为1×10 5～10×10 6个细胞/100μL的细胞体系：(0.5～4)mL。在本发明中，采用流式细胞仪检测抗体的表达，根据抗体的表达分辨Treg的细胞亚型，具体如表1所示：
表1.Treg细胞亚型与抗体表达对应表
Figure PCTCN2018091106-appb-000003
注：+为阳性；low为低表达；-为阴性。
下面结合实施例对本发明提供的检测人调节性T细胞亚型的试剂盒进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
分离全血中的单个核细胞：
1)取1mL新鲜静脉血(血液离体≤2h)加入1mL血液稀释液稀释血液；
2)取1mL单个核细胞分离液于离心管底部，将稀释后的血液小心地叠加于单个核细胞分离液的上面，避免血液和单个核细胞分离液混合；
3)离心机中，800g离心20min，离心机的温度设为18℃，升速设为9，降速设为0；
4)用滴管吸出界面上的白膜层(单个核细胞)，尽量不要吸出其上层液体；
5)2mL血液稀释液稀释单个核细胞溶液，室温250g离心10min；收集细胞沉淀，100μL细胞培养液重悬单个核细胞，获得的单个核细胞如图1所示。
实施例2
刺激人外周血单个核细胞：
1)细胞计数，用细胞培养液调整细胞密度为1×10 6/mL；
2)每1mL细胞悬液加入2μL淋巴细胞活化液和1μL LPS，均匀混合后接种于细胞培养板上，置于CO 2培养箱中37℃孵育4h；
3)每管收集细胞1mL，加入1mL磷酸盐缓冲液，200g离心6min；
4)细胞计数，磷酸盐缓冲液调整细胞密度为1×10 7/mL细胞悬液。
实施例3
荧光抗体染色：
1)每管加入100μL细胞悬液，然后加入1uL溶解后的死细胞去除染料和5uL FcR阻断剂，室温避光孵育15min；
2)每管加入1mL细胞染色缓冲液，200g离心6min，弃去上清液；得到的去除死细胞后的人外周血单个核细胞群如图2所示；
3)每管加入100μL细胞染色缓冲液重悬细胞，然后加入PerCP/Cy 5.5标记的抗人CD3、FITC标记的抗人CD8、PE标记的抗人CD25、Brilliant Violet 421标记的抗人TGF-β和Brilliant Violet 510标记的抗人CD127抗体各5uL，或者PerCP/Cy 5.5标记的抗人CD3、FITC标记的抗人CD8、BrilliantViolet 510标记的抗人CD25、PE/Cy7标记的抗人CD278(ICOS)和PE标记的抗人CCR6抗体各5uL室温避光孵育15min；
4)每管加入1mL细胞染色缓冲液，200g离心6min，弃去上清液；
5)每管加入1mL固定透膜洗液重悬细胞，室温避光30min；
6)每管加入2mL透膜洗液，200g离心6min，弃去上清液；
7)每管加入100uL透膜洗液重悬细胞，然后Alexa Fluor 647标记的抗人Foxp3、PE/Cy 7标记的抗人IL-10各5μL，或者Brilliant Violet 421标记的抗人IL-17A和Alexa Fluor 647标记的抗人Foxp3抗体各5μL，室温避光孵育1h；
8)每管加入2mL透膜洗液，清洗一次，200uL细胞染色缓冲液重悬细胞，上流式细胞仪检测各分子的表达情况，得到的各抗体的表达情况如图3～10所示。
由上述实施例可知，利用本发明的试剂盒及其检测方法，可以快速的同时检测人体血液中2-6种Treg细胞亚型，诱导型(或适应型)Treg的 表达如图6所示；产IL-10的Treg的表达如图7所示；CD127低表达和阴性Treg的表达如图8所示；ICOS阳性Treg的表达如图9所示；产IL-17的Treg的表达如图10所示。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (15)

  1. 一种检测人调节性T细胞亚型的试剂盒，包括以下组分：血液稀释液、单个核细胞分离液、细胞培养液、淋巴细胞活化液、死细胞去除染料、FcR阻断剂、荧光标记的抗人细胞表面标记分子的抗体、荧光标记的抗人细胞内分子的抗体、脂多糖、洗涤缓冲液、细胞染色缓冲液、细胞固定液和透膜洗液；所述荧光标记的抗人细胞表面标记分子的抗体包括：抗人CD3或CD4抗体，抗人CD8抗体，抗人CD25抗体，抗人CD127抗体，抗人ICOS抗体，抗人TGF-β抗体和抗人CCR6抗体；所述荧光标记的抗人细胞内分子的抗体包括：抗人IL-10抗体，抗人IL-17抗体和抗人Foxp3抗体。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述死细胞去除染料、荧光标记的抗人细胞表面标记分子的抗体荧光种类和荧光标记的抗人细胞内分子的抗体荧光种类包括：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 Violet711、Brilliant Violet785、FITC、LEAF、Pacific Blue、PE、PE/Cy5、PE/Cy7、PE/Dazzle、PerCP或PerCP/Cy 5.5。
  3. 根据权利要求2所述的试剂盒，其特征在于，所述荧光的种类根据流式细胞仪的激光和滤光片的配置进行自由组合。
  4. 根据权利要求1所述的试剂盒，其特征在于，所述血液稀释液包括与人体血浆渗透压相等的溶液。
  5. 根据权利要求4所述的试剂盒，其特征在于，所述血液稀释液包括NaCl溶液或PBS缓冲液。
  6. 根据权利要求1所述的试剂盒，去其特征在于，所述单个核细胞分离液的有效成分包括：聚蔗糖和泛影葡胺，或者泛影钠和多聚糖，或者碘克沙醇。
  7. 根据权利要求1所述的试剂盒，去其特征在于，所述细胞固定液的有效成分包括甲醛和甲醇。
  8. 根据权利要求1所述的试剂盒，去其特征在于，所述透膜洗液的有效成分优选的包括皂素和胎牛血清，或者皂素和牛血清白蛋白。
  9. 根据权利要求1所述的试剂盒，其特征在于，所述FcR阻断剂的有效成分包括人免疫球蛋白或与使用的流式抗体同一种属且亚型相同的不相关的免疫球蛋白。
  10. 根据权利要求1～9任一项所述的试剂盒检测人调节性T细胞亚型的方法，包括以下步骤：
    1)将静脉血与血液稀释液混合后置于单个核细胞分离液上层，离心15～35min后，吸取单个核细胞，所述静脉血、血液稀释液及单个核细胞分离液的体积比为1：(0.5～2)：(0.5～2)；
    2)将步骤1)得到的所述单个核细胞用血液稀释液稀释后，90～600g离心4～30min后，收集细胞沉淀；
    3)将步骤2)得到的所述细胞沉淀用细胞培养液重悬，调整所述细胞的密度为0.5×10 6～4×10 6个/mL，得第一细胞悬液；
    4)将步骤3)得到的所述第一细胞悬液与淋巴细胞活化液和脂多糖均匀混合，置于含CO 2培养箱中培养4～6h，得第一孵育细胞；
    5)将步骤4)得到的所述第一孵育细胞与PBS缓冲液按照1：(0.5～10)的体积比混合后，90～600g离心4～30min，弃去上清后得第二细胞悬液；
    6)取步骤5)得到的所述第二细胞悬液按照1×10 5～10×10 6个细胞/100μL的体系与0.1～10μL含死细胞去除染料的DMSO溶液和0.1～10μLFcR阻断剂混合后，4～40℃孵育5～30min，得第二孵育细胞；
    7)将步骤6)得到的所述第二孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL细胞染色缓冲液混合，90～600g离心4～30min，弃上清后将细胞沉淀按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～20μL每种荧光标记的抗人细胞表面标记分子的抗体，均匀混合后4～40℃避光孵育5～30min；
    8)将步骤7)得到的所述第三孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL细胞染色缓冲液混合后90～600g离心4～30min，弃上清后向沉淀中按照1×10 5～10×10 6个细胞/100μL的体系加入0.1～5mL细胞固定液重悬，4～40℃避光孵育10～120min，得重悬的第三孵育细胞；
    9)将步骤8)得到的所述重悬的第三孵育细胞按照1×10 5～10×10 6个细胞/100μL的体系与0.5～5mL透膜洗液混合，90～600g离心4～30min，弃上清后得第三孵育细胞沉淀，再次按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～5mL透膜洗液重悬第三孵育细胞沉淀，90～600g离心4～30min，弃上清，得第三孵育细胞沉淀；
    10)将步骤9)得到的所述第三孵育细胞沉淀按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～20μL每种荧光标记的抗细胞内分子的抗体，混合后4～40℃避光孵育10～120min，得第四孵育细胞；
    11)将步骤10)得到的所述第四孵育细胞沉淀按照1×10 5～10×10 6个细胞/100μL的体系加入0.5～5mL透膜洗液，均匀混合后，90～600g离心4～30min，弃上清，得第四孵育细胞沉淀，用PBS调整细胞浓度为(0.1-10)×10 6个/mL后用流式细胞仪检测抗体的表达；根据抗体的表达得到Treg细胞亚型的检测结果，所述Treg细胞亚型与抗体表达的关系对应如下：
    Figure PCTCN2018091106-appb-100001
    注：+为阳性；low为低表达；-为阴性。
  11. 根据权利要求10所述的检测人调节性T细胞亚型的方法，其特征在于，步骤1)所述离心的条件包括：离心力为200～1000g，离心温度为18～22℃。
  12. 根据权利要求10所述的检测人调节性T细胞亚型的方法，其特征在于，步骤2)所述单个核细胞与血液稀释液的体积比优选的为1：(0.5～50)。
  13. 根据权利要求10所述的检测人调节性T细胞亚型的方法，其特征在于，步骤3)所述的细胞培养液以RPMI 1640培养基为基础，包括体积浓度为0.1％～20％的胎牛血清和体积浓度为0～3％的青霉素-链霉素双抗溶液。
  14. 根据权利要求10所述的检测人调节性T细胞亚型的方法，其特征在于，步骤4)所述孵育培养的条件包括：在含5％CO 2的培养箱中进行培养，培养温度为35～40℃。
  15. 根据权利要求10所述的检测人调节性T细胞亚型的方法，其特征在于，步骤7)和步骤9)所述荧光标记的抗人细胞表面标记分子的抗体和抗人细胞内分子的抗体中各抗体的添加量根据孵育细胞的数量和抗体的工作浓度而定。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120088678A1 (en) * 2010-09-08 2012-04-12 Sanford-Burnham Medical Research Institute Method for prediction of response to rheumatoid arthritis therapeutics
US20130225427A1 (en) * 2010-09-08 2013-08-29 Sanford-Burnham Medical Research Institute Method for prediction of response to immune mediated disease therapeutics
WO2017072251A1 (en) * 2015-10-28 2017-05-04 Life Technologies As Selective expansion of different subpopulations of t cells by the alteration of cell surfacing signals and signal ratio

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7351546B2 (en) * 1998-09-22 2008-04-01 Becton, Dickinson And Company Flow cytometric, whole blood dendritic cell immune function assay
CA2702230A1 (en) * 2007-10-12 2009-04-16 Max-Delbrueck-Centrum Fuer Molekulare Medizin (Mdc) Method and kit for rapid isolation of human foxp3+ treg cells
US9910039B2 (en) * 2011-07-01 2018-03-06 Beckman Coulter, Inc. Regulatory T cells and methods of identifying, obtaining and using to treat immuno-based disorders
EP2682750A1 (en) * 2012-07-02 2014-01-08 Sotio a.s. In vitro method for the diagnosis and surveillance of cancer
CN102766597A (zh) * 2012-07-31 2012-11-07 中国人民解放军第三军医大学 利用FOXP3为标记物分选Treg细胞的方法
CN103235135B (zh) * 2013-04-26 2015-02-04 史其新 一种用于表征外周调节性t细胞抑制活性的特征细胞群的检测方法及其应用
US10578619B2 (en) * 2013-07-31 2020-03-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and kits for identifying effector Treg cells
CN104651309A (zh) * 2015-02-04 2015-05-27 中山大学孙逸仙纪念医院 人外周血CD4+CD25+Foxp3+调节性T细胞的分选扩增方法
CN106932576A (zh) * 2017-03-22 2017-07-07 山东大学深圳研究院 一种人调节性t细胞的免疫抑制功能的检测方法
CN107314965B (zh) * 2017-04-26 2020-07-31 马鞍山普梅森医学检验实验室有限公司 基于流式结合icp-ms单细胞蛋白检测的样本前处理方法
CN107478563A (zh) * 2017-07-19 2017-12-15 浙江省人民医院 一种基于流式细胞术检测结核特异性双细胞因子的方法
CN107603948B (zh) * 2017-08-14 2019-07-05 武汉圣济科技有限公司 一种直接体外诱导人外周血单个核细胞成为复合型抗原非特异性调节性t细胞的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120088678A1 (en) * 2010-09-08 2012-04-12 Sanford-Burnham Medical Research Institute Method for prediction of response to rheumatoid arthritis therapeutics
US20130225427A1 (en) * 2010-09-08 2013-08-29 Sanford-Burnham Medical Research Institute Method for prediction of response to immune mediated disease therapeutics
WO2017072251A1 (en) * 2015-10-28 2017-05-04 Life Technologies As Selective expansion of different subpopulations of t cells by the alteration of cell surfacing signals and signal ratio

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SERVE, R.: "Comparative Analysis of the Regulatory T cells Dynamics in Pe- ripheral Blood in Human and Porcine Polytrauma", FRONTIERS IN IMMUNOLOGY, vol. 9, 13 March 2018 (2018-03-13), pages 435, XP055645683, DOI: 10.3389/fimmu.2018.00435 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113899893A (zh) * 2021-09-30 2022-01-07 杭州联科生物技术股份有限公司 一种ivd流式产品的开发方法
CN113899893B (zh) * 2021-09-30 2024-01-30 杭州联科生物技术股份有限公司 一种ivd流式产品的开发方法
CN114112567A (zh) * 2021-10-29 2022-03-01 郑州金域临床检验中心有限公司 一种结核感染t细胞斑点试验样本的制备方法

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