Application of CD3+CD4+CCR1 + T cells in diagnosis of allergic respiratory diseases

Abstract

The invention provides application of CD3 + CD4 + CCR1 + T cells in diagnosis of allergic respiratory diseases, and relates to the technical field of biology. Based on the fact that the proportion of CD3 + CD4 + CCR1 + T cells in peripheral blood PBMC (peripheral blood mononuclear cells) of patients with allergic respiratory diseases is higher than that of healthy people, the application comprises the step that substances for detecting the CD3 + CD4 + CCR1 + T cells are applied to preparation of allergic respiratory disease diagnosis products so as to relieve the defects of allergic respiratory disease products in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of CD3+CD4+CCR1+ T cells in the diagnosis of allergic respiratory diseases. Background Art

[0002] The following statements provide only background information related to the present invention and do not necessarily constitute prior art.

[0003] Allergic respiratory diseases are a class of respiratory inflammatory diseases caused by abnormal immune responses, mainly due to the body's over-sensitivity to environmental allergens (such as dust mites, pollen, pet dander, etc.). It is often overlooked, underdiagnosed, misdiagnosed, and not taken seriously, which not only endangers health but also increases social costs. Allergic respiratory diseases have significant clinical significance because they are the basis for many complications, the main risk factor for poor asthma control, and affect quality of life, work, or study efficiency.

[0004] Currently, it is difficult to diagnose allergic respiratory diseases. They are similar in symptoms to colds and COPD (Chronic Obstructive Pulmonary Disease), and it is particularly difficult to distinguish them in elderly patients, which is prone to misdiagnosis or missed diagnosis. For example, vasomotor rhinitis may be misjudged as allergic. Existing diagnostic techniques for allergic respiratory diseases include: allergen skin tests, which are in vivo allergen detections, and the main methods include SPT and intradermal tests. If a patient has a hypersensitivity reaction to a certain allergen, wheals and erythema will appear at the skin prick site within 20 minutes. Compared with the negative control, a wheal with an average diameter > 3 mm is determined as positive for SPT; serum specific IgE detection, which is an in vitro allergen detection. Usually, the critical value of serum specific IgE level is 0.35 kU / L, and a value greater than or equal to this value is positive, indicating that the body is in a sensitized state; nasal provocation test, which directly acts on the nasal mucosa with a certain allergen to simulate the natural disease situation and observes whether relevant symptoms are induced.

[0005] Existing diagnostic techniques for allergic respiratory diseases mainly have the following defects: Skin prick tests are affected by antihistamines and skin conditions (such as dermographism), with a high false positive rate; Serum IgE detection lacks specificity. An increase in total IgE does not necessarily indicate an allergen, and the detection range is limited. Also, due to threshold differences, some patients have positive IgE but no symptoms, making treatment decisions difficult, and non-IgE-mediated allergies cannot be covered, such as some food allergies. And after desensitization treatment, re-evaluation is required; The risk of provocation tests may induce severe reactions, limiting their clinical application; Gene polymorphisms affect the intensity of allergic reactions, and the same test results have different meanings in different individuals. Based on this, there is a need to seek new diagnostic methods and products.

[0006] In view of this, the present invention is hereby provided. Summary of the Invention

[0007] The object of the present invention is to provide the use of a substance for detecting CD3+CD4+CCR1+ T cells in the preparation of a diagnostic product for allergic respiratory diseases, so as to alleviate at least one defect in the existing diagnostic techniques for allergic respiratory diseases described above.

[0008] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0009] In the first aspect, there is provided the use of a substance for detecting CD3+CD4+CCR1+ T cells in the preparation of a diagnostic product for allergic respiratory diseases.

[0010] In the second aspect, there is provided a diagnostic kit for allergic respiratory diseases, wherein the diagnostic kit for allergic respiratory diseases contains the substance for detecting CD3+CD4+CCR1+ T cells described in the first aspect.

[0011] In the third aspect, there is provided a diagnostic device for allergic respiratory diseases, wherein the diagnostic device for allergic respiratory diseases includes a detection module and a prediction and judgment module;

[0012] The detection module obtains the content of CD3+CD4+CCR1+ T cells;

[0013] The prediction and judgment module includes a computer-readable medium on which judgment rules are recorded, and when the computer-readable medium is processed and executed, it realizes comparing the content of CD3+CD4+CCR1+ T cells with a threshold value, and when the content of CD3+CD4+CCR1+ T cells is within the range defined by the threshold value, it is determined that the subject has allergic respiratory diseases.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention discovers that the proportion of CD3+CD4+CCR1+ T cells in peripheral blood PBMC of patients with allergic respiratory diseases is higher than that of healthy people; in a mouse model of allergic respiratory diseases, the proportion of CD3+CD4+CCR1+ T cells in the respiratory mucosa of the model group mice is also higher than that of the control group mice; at the same time, it is found that the expression of Th2 characteristic cytokines of CD3+CD4+CCR1+ T cells increases, and CCR1 can be used as a phenotypic marker of Th2 cells. By constructing an ROC curve, it is verified that CD3+CD4+CCR1+ T cells can be used as a diagnostic marker for allergic respiratory diseases.

[0016] Using CD3+CD4+CCR1+ T cells as a diagnostic marker for allergic airway diseases eliminates the need to challenge subjects with allergens, reducing adverse reactions during diagnosis; moreover, it can improve the diagnostic accuracy of diagnostic products, guide individualized treatment, dynamically monitor disease activity, and reduce the degree of disease risk. Immunocyte detection provides multi-dimensional information from mechanism to clinic for the diagnosis of allergic airway diseases, contributing to the later development of precision medical products and improving disease stratification management and treatment response prediction. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is the proportion of CD3+CD4+CCR1+ T cells in the PBMC of healthy people in Example 1;

[0019] Figure 2 It is the proportion of CD3+CD4+CCR1+ T cells in the PBMC of patients with allergic airway diseases in Example 1;

[0020] Figure 3 It is the proportion of CD3+CD4+CCR1+ T cells in the respiratory mucosa of the mice in the model group in Example 2;

[0021] Figure 4 It is the proportion of CD3+CD4+CCR1+ T cells in the respiratory mucosa of the control group mice in Example 2;

[0022] Figure 5 It is the proportion of CD3+CD4+CCR1+ T cells after stimulating the spleen cells of the mice in the model group with APC cells in Example 2;

[0023] Figure 6 It is the IL-4 expression levels of CD3+CD4+CCR1+ T cells and CD3+CD4+CCR1- T cells after stimulating the spleen cells of the mice in the model group with APC cells presenting antigens in Example 2;

[0024] Figure 7 It is the IL-5 expression levels of CD3+CD4+CCR1+ T cells and CD3+CD4+CCR1- T cells after stimulating the spleen cells of the mice in the model group with APC cells presenting antigens in Example 2;

[0025] Figure 8The IL-13 expression levels of CD3+CD4+CCR1+ T cells and CD3+CD4+CCR1- T cells in the spleen cells of the mouse model group in Example 2 after being stimulated by antigen-presenting APC cells;

[0026] Figure 9 Box plot of the proportion of CD3+CD4+CCR1+ T cells in PBMC of the patient group and the healthy group in Example 3, Tukey method;

[0027] Figure 10 ROC curve constructed with 0.855% of the proportion of CD3+CD4+CCR1+ T cells in PBMC as the Cut-off value in Example 3. Detailed implementation manners

[0028] Next, the technical solutions of the present invention will be described clearly and completely in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0029] Immune cells play a central role in the pathogenesis and diagnosis of allergic airway diseases (such as allergic rhinitis, asthma). They are not only key participants in the occurrence of the diseases, but also can be used as diagnostic markers and therapeutic targets. Th2 cells (T helper 2 cells) secrete IL-4, IL-5, and IL-13, promoting IgE synthesis and eosinophil infiltration. Th2 cell-related cytokines (such as IL-4) or gene expression profiles can be used to evaluate the type and severity of allergic reactions. The present invention finds that the expression of Th2 characteristic cytokines in CD3+CD4+CCR1+ T cells increases, and CCR1 can be used as a phenotypic marker of Th2 cells. At the same time, it is found that the proportion of CD3+CD4+CCR1+ T cells in PBMC of patients with allergic airway diseases is higher than that of healthy people; in a mouse model of allergic airway diseases with OVA as an allergen, the proportion of CD3+CD4+CCR1+ T cells in the respiratory mucosa of the model group mice is also higher than that of the control group mice. Therefore, CD3+CD4+CCR1+ T cells are expected to be used as a diagnostic marker for allergic airway diseases, and finally, it is verified by constructing an ROC curve that CD3+CD4+CCR1+ T cells can be used as a diagnostic marker for allergic airway diseases.

[0030] In the first aspect, there is provided an application of a substance for detecting CD3+CD4+CCR1+ T cells in the preparation of a diagnostic product for allergic airway diseases.

[0031] In an alternative embodiment, the product includes a detection reagent, a kit, an antibody chip, an antibody probe, or a detection device.

[0032] In an alternative embodiment, the CD3+CD4+CCR1+ T cells show increased expression of Th2 signature cytokines.

[0033] In an alternative embodiment, the CD3+CD4+CCR1+ T cells show increased expression of one or more of the Th2 signature cytokines IL-4, IL-5, and IL-13.

[0034] In an alternative embodiment, the substance for detecting CD3+CD4+CCR1+ T cells includes a substance for detecting Th2 signature cytokines of CD3+CD4+CCR1+ T cells; further optionally, the substance for detecting CD3+CD4+CCR1+ T cells includes a substance for detecting one or more of IL-4, IL-5, and IL-13 of CD3+CD4+CCR1+ T cells.

[0035] In an alternative embodiment, the substance for detecting CD3+CD4+CCR1+ T cells includes a substance for detecting one or more of CD3+CD4+CCR1+ T cells in peripheral blood, CD3+CD4+CCR1+ T cells in respiratory mucosa, and CD3+CD4+CCR1+ T cells in splenocytes.

[0036] In an alternative embodiment, the substance for detecting CD3+CD4+CCR1+ T cells includes a substance for detecting CD3+CD4+CCR1+ T cells in PBMC (Peripheral Blood Mononuclear Cells).

[0037] In an alternative embodiment, detecting CD3+CD4+CCR1+ T cells includes detecting the proportion of CD3+CD4+CCR1+ T cells in PBMC.

[0038] In an alternative embodiment, the substance for detecting CD3+CD4+CCR1+ T cells includes a CD3 antibody, a CD4 antibody, and a CCR1 antibody.

[0039] As used herein, the term "antibody" includes any immunoglobulin capable of binding to a specific antigen. The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, full-length antibodies, nanobodies, antigen-binding fragments, provided that they exhibit the desired antigen-binding activity. The term "antigen-binding fragment" refers to a substance that contains all or part of the CDRs of an antibody, lacks at least some of the amino acids present in the full-length chain, but is still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. Examples of antigen-binding fragments include but are not limited to Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), and scFv dimers (bivalent bifunctional antibodies).

[0040] In a second aspect, there is provided a diagnostic kit for allergic respiratory diseases, which kit contains a substance for detecting CD3+CD4+CCR1+ T cells as described in the first aspect.

[0041] In an alternative embodiment, the diagnostic kit for allergic respiratory diseases contains anti-CD3 antibody, anti-CD4 antibody, and anti-CCR1 antibody.

[0042] In an alternative embodiment, the diagnostic kit for allergic respiratory diseases contains reagents for separating blood, reagents for separating PBMCs, reagents for separating respiratory mucosa, and reagents for separating splenocytes.

[0043] In an alternative embodiment, the diagnostic kit for allergic respiratory diseases contains reagents for immunoassay, which immunoassay includes but is not limited to reagents for flow cytometry, chemiluminescence assay, enzyme-linked immunosorbent assay, immunomagnetic particle assay, immunofluorescence assay, or immunoblot assay, preferably containing reagents for flow cytometry.

[0044] In an alternative embodiment, the kit contains reagents and / or consumables for detection, including but not limited to one or more of primers, probes, buffers, dyes, diluents, washing solutions, chromogenic solutions, lysis solutions, secondary antibodies, signal substances, negative controls, positive controls, and blank controls. Those skilled in the art can select the reagent composition of the kit according to the specific detection means, and the present invention does not limit this.

[0045] In a third aspect, there is provided a diagnostic device for allergic respiratory diseases, which diagnostic device contains a detection module and a prediction and judgment module;

[0046] The detection module obtains the content of CD3+CD4+CCR1+ T cells;

[0047] The prediction and judgment module includes a computer-readable medium storing judgment rules. When the computer-readable medium is processed and executed, it realizes comparing the content of CD3+CD4+CCR1+ T cells with a threshold value. When the content of CD3+CD4+CCR1+ T cells is within the range defined by the threshold value, it is determined that the subject has allergic airway disease.

[0048] In an optional embodiment, obtaining the content of CD3+CD4+CCR1+ T cells includes obtaining the proportion of CD3+CD4+CCR1+ T cells in PBMC of the subject; when the value of the proportion of CD3+CD4+CCR1+ T cells in PBMC of the obtained subject ≥ the threshold value, it is determined that the subject has the state of allergic airway disease.

[0049] In an optional embodiment, the threshold value is 0.855%.

[0050] In an optional embodiment, the detection module includes an immunoassay device, such as but not limited to a device for realizing flow cytometry, chemiluminescence detection, enzyme-linked immunosorbent assay, immunomagnetic particle detection, immunofluorescence detection or immunoblot detection, preferably including a device for realizing flow cytometry.

[0051] In an optional embodiment, the device further includes a communication interface. Each module and the communication interface in the device are directly or indirectly electrically connected to each other to realize data transmission or interaction. For example, these components can be electrically connected to each other through one or more buses or signal lines.

[0052] In an optional embodiment, the allergic airway disease diagnosis device further includes the allergic airway disease diagnosis kit described in the second aspect.

[0053] In an optional embodiment, the subjects in the first, second or third aspect above include mammals. Exemplary subjects include but not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, alpacas, poultry, goats and sheep. In some embodiments, the subject is a human. In some embodiments, the subject is an animal model constructed with allergic airway symptoms.

[0054] In an optional embodiment, the subject is an animal model constructed with allergic airway symptoms using OVA (ovalbumin) as an allergen.

[0055] The present invention will be further described below through specific embodiments. However, it should be understood that these embodiments are only for more detailed illustration and should not be construed as limiting the present invention in any way.

[0056] Example 1

[0057] Detect the proportion of CD3+CD4+CCR1+ T cells in the peripheral blood of healthy people and patients with allergic respiratory diseases by flow cytometry.

[0058] Peripheral blood was drawn by experienced nurses and anticoagulant (heparin) was added to prevent coagulation. Detection of lymphocyte subsets: Take 1 mL of whole blood from each sample, dilute the peripheral blood with sterile PBS at a ratio of 1:1, and mix well. Add an appropriate amount of Ficoll-Paque PLUS to the centrifuge tube, and slowly add the diluted blood along the tube wall to the upper layer of Ficoll, avoiding mixing. Centrifuge at 1500 rpm for 20 minutes at room temperature. After centrifugation, the tube is divided into four layers: the upper layer is plasma and PBS, the middle layer is the PBMC (Peripheral Blood Mononuclear Cells) buffy coat, the lower layer is Ficoll, and the bottom layer is red blood cells and granulocytes. Carefully aspirate the PBMC buffy coat with a pipette and transfer it to a new centrifuge tube. Add PBS, centrifuge at 1500 rpm for 10 minutes, and discard the supernatant. Repeat the washing once. The extracted PBMCs are used for flow cytometry detection. Take 1 μL of zombie into 50 μL of PBS to form a 50 μL system, take 50 μL of the mixed system to resuspend the washed cells, and stain at room temperature for 20 minutes. Add 2 mL of PBS to wash the cells twice, centrifuge at 2000 rpm at room temperature for 5 minutes to obtain cell pellets. Add 1 μL of each of the CD3\CD4\CCR1 flow cytometry fluorescent antibodies to 50 μL of PBS to form a 50 μL system. Take 50 μL of the mixed system to resuspend the washed cells, and stain at room temperature for 20 minutes. Add 2 mL of PBS to wash the cells twice, centrifuge at 2000 rpm at room temperature for 5 minutes to obtain cell pellets. Resuspend the cells in 180 μL of PBS and perform on-machine detection. Quantitatively collect 100 μL of resuspended cells and analyze the data. The results are as Figure 1 and Figure 2 shown. The results show that the proportion of CD3+CD4+CCR1+ T cells in the peripheral blood of healthy people is 0.55%, and the proportion of CD3+CD4+CCR1+ T cells in the peripheral blood of patients with allergic respiratory diseases is 1.43%, indicating that the proportion of CD3+CD4+CCR1+ T cells in the peripheral blood of patients with allergic respiratory diseases is higher than that of healthy people.

[0059] Example 2

[0060] Detect the proportion of CD3+CD4+CCR1+ T cells in the respiratory mucosa of mice with allergic airway diseases by establishing a model, and detect the inflammatory factor response of CD3+CD4+CCR1+ T cells by culturing antigen-specific splenocytes.

[0061] (1) Experimental purpose: To establish a mouse model of allergic airway diseases, verify the differences in CD3+CD4+CCR1+ T cells between mice with allergic airway diseases and healthy mice, and study the inflammatory factor response of CD3+CD4+CCR1+ T cells.

[0062] (2) Experimental groups: Group 1 is the control group, and Group 2 is the model establishment group.

[0063] (3) Experimental materials and methods for establishing the model: OVA (ovalbumin) (allergen), Al(OH)3 suspension (adjuvant). On days 0, 7, and 14, Group 1 was intraperitoneally injected with 200 μL of PBS, and Group 2 was intraperitoneally injected with 200 μL (100 μg OVA, 2 mg Al(OH)3) for sensitization. On days 21 - 27, Group 1 was instilled with 20 μL of PBS into the nasal cavity, and Group 2 was instilled with 20 μL of OVA with a concentration of 40 mg / mL for challenge.

[0064] (4) Experimental content: Flow cytometry was used to detect the proportion of CD3+CD4+CCR1+ T cells in the respiratory mucosa of mice, and antigen-specific splenocytes were cultured and detected by flow cytometry.

[0065] (5) Flow cytometry was used to detect the proportion of CD3+CD4+CCR1+ T cells in the respiratory mucosa of mice:

[0066] On the 28th day, euthanized mice were used to collect nasal mucosa to lung respiratory tract tissues, and single-cell suspensions of respiratory tract mucosa of mice were separated and prepared by enzymatic digestion method. The respiratory tract tissues were rinsed with PBS and placed in a 1.5 mL EP tube, and then minced with scissors for mechanical dispersion. 1.2 mL of digestion buffer (100 mg / mL Dispase II, 10 mg / mL collagenase A, 1500 kU / mL DNase I and 0.025 M CaCl2) was added to each specimen; incubated at 37 °C on a shaker for 1 h; the suspension was gently mixed with a pipette and further incubated for 30 min; filtered through a 40 μm filter into a 50 mL Falcon tube; centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded; added PBS for washing, centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. Repeat the washing once to obtain single-cell suspensions of respiratory tract mucosa of mice for flow cytometry detection. Take 1 μL of zombie into 50 μL of PBS to form a 50 μL system, and take 50 μL of the mixed system to resuspend the washed cells, and stain at room temperature for 20 minutes. Add 2 mL of PBS to wash the cells twice, and centrifuge at 2000 rpm at room temperature for 5 minutes to obtain cell pellets. Add 1 μL of each of the fluorescent antibodies for CD3\CD4\CCR1 flow cytometry into 50 μL of PBS to form a 50 μL system. Take 50 μL of the mixed system to resuspend the washed cells, and stain at room temperature for 20 minutes. Add 2 mL of PBS to wash the cells twice, and centrifuge at 2000 rpm at room temperature for 5 minutes to obtain cell pellets. Resuspend the cells with 180 μL of PBS and perform on-machine detection. Quantitatively collect 100 μL of resuspended cells and analyze the data. The results are as Figure 3 and Figure 4 shown. The results showed that the proportion of CD3+CD4+CCR1+ T cells in the respiratory tract mucosa of the model group mice was 6.07%, and the proportion of CD3+CD4+CCR1+ T cells in the respiratory tract mucosa of the control group mice was 0.23%. The results indicated that the proportion of CD3+CD4+CCR1+ T cells in the respiratory tract mucosa of the model group with allergic respiratory diseases in mice increased.

[0067] (6) Antigen-specific splenic lymphocyte culture and detection:

[0068] ① The method for culturing antigen-specific splenic lymphocytes is as follows: After killing the mice on the 28th day, the spleen was dissected under sterile conditions. Splenic single-cell suspensions were obtained by physical grinding method. Erythrocytes were lysed with erythrocyte lysate and washed twice with sterile PBS, and then resuspended in complete RPMI-1640 medium. In a 12-well cell culture plate, 1×10 7 spleen cells, 100 μg / mL OVA solution, 5 U / mL recombinant mouse IL-2 were added to each well, and the culture system was 3 mL. Incubated in an incubator at 37 °C and 5% CO2 for 5 days, and then half of the medium was replaced with complete RPMI-1640 medium containing 20 U / mL recombinant mouse IL-2. Continue to culture in an incubator at 37 °C and 5% CO2 until flow cytometry detection was performed on the 7th day.

[0069] ② The method for detecting antigen-specific CD3+CD4+CCR1+ T cell responses is as follows:

[0070] Prepare antigen-presenting cells (APCs) and perform antigen presentation 10 hours before detection, as follows: Euthanize BALB / c mice without any treatment, disinfect the fur, dissect under sterile conditions, collect peritoneal cells from the mice by saline flushing method, and obtain macrophages by adherent method for use as APCs.

[0071] After washing twice with sterile saline, resuspend in complete RPMI-1640 medium. In a 96-well round-bottom cell culture plate, add 1×10 5 of the above APC cells, 100 μg / mL OVA solution or PBS as a control. Incubate the above APC cells with OVA overnight in an incubator at 37°C and 5% CO2 to obtain APC cells presenting the antigen.

[0072] After 10 hours, collect the APC cells presenting the antigen and wash them 3 times with RPMI-1640 medium. Stimulate 1×10 5 APC cells with 1×10 6 lymphocytes cultured in step ① above for 7 days. The culture system is 200 μL, and add 0.15 μL of Golgi blocker. Continue to culture in an incubator at 37°C and 5% CO2 for 5 hours.

[0073] Then collect the cells and wash them twice with PBS. Resuspend the above cells in 100 μL of PBS, add 1 μL of each of the CD3\CD4\CCR1 flow cytometry fluorescent antibodies, and stain at room temperature in the dark for 20 minutes. Add PBS to wash the cells twice, centrifuge at 2000 rpm at room temperature for 5 minutes to obtain cell pellets. After permeabilization with a permeabilizing agent, resuspend in 100 μL of PBS, add 1 μL of each of the IL-4\IL-5\IL-13\IFN-γ\IL-17A flow cytometry fluorescent antibodies, and stain at room temperature in the dark for 20 minutes. Add PBS to wash the cells twice, centrifuge at 2000 rpm at room temperature for 5 minutes to obtain cell pellets. Resuspend the cells in 180 μL of PBS and load them onto the machine for detection. Quantitatively collect 100 μL of the resuspended cells and analyze the data. The results are as Figure 5 、 Figure 6 、 Figure 7 and Figure 8As shown, the results showed that the expression levels of IL-4, IL-5, and IL-13 in CD3+CD4+CCR1+ T cells were higher than those in CD3+CD4+CCR1- T cells. The above results indicate that the expression of Th2 characteristic cytokines in splenic antigen-specific CD3+CD4+CCR1+ T cells of the model group mice increased.

[0074] Example 3

[0075] Role of human peripheral blood CD3+CD4+CCR1+ T cells in the diagnosis of allergic airway diseases.

[0076] (1) Experimental purpose: Recruit 16 healthy individuals and 18 patients with allergic airway diseases, and detect the proportion of CD3+CD4+CCR1+ T cells in PBMCC by flow cytometry.

[0077] (2) Inclusion criteria for patients with allergic airway diseases:

[0078] ① Definite diagnosis: Meet the diagnostic criteria for allergic airway diseases such as allergic rhinitis and asthma (such as GINA guidelines, ARIA guidelines).

[0079] ② Age range: Usually 18 - 65 years old.

[0080] ③ Duration and severity of symptoms: Symptoms persist for more than a certain period (such as 6 months) and reach a certain severity.

[0081] ④ Positive allergen detection: Skin prick test or serum specific IgE detection shows positive for common allergens (such as dust mites, pollen).

[0082] ⑤ Informed consent: The subjects sign the informed consent form and voluntarily participate in the study.

[0083] (3) Exclusion criteria: Other respiratory diseases: Exclude other respiratory diseases such as chronic obstructive pulmonary disease (COPD), bronchiectasis, etc.

[0084] ① Severe comorbidities: Exclude severe cardiovascular diseases, immune system diseases, malignant tumors, etc.

[0085] ② Recent infection or surgery: Exclude those with recent (such as within 4 weeks) respiratory tract infection or major surgery.

[0086] ③ Drug use: Exclude those who have long-term use of immunosuppressants or systemic glucocorticoids.

[0087] ④ Pregnancy or lactation: Exclude pregnant or lactating women.

[0088] ⑤ Other allergic diseases: Exclude other allergic diseases such as severe atopic dermatitis and food allergy.

[0089] ⑥ Unable to cooperate with the study: Exclude those who are unable to cooperate with follow-up or complete the study requirements.

[0090] (4) Experimental method: The method for obtaining peripheral blood and the separation method for extracting PBMC are the same as in Example 1. Take 1 μL of zombie into 50 μL of PBS to form a 50 μL system. Take 50 μL of the mixed system to resuspend and wash the cells, and stain the cells at room temperature for 20 minutes. Add 2 mL of PBS to wash the cells twice, and centrifuge at 2000 rpm at room temperature for 5 minutes to obtain cell pellets. Add 1 μL of each of the CD3\CD4\CCR1 flow cytometry fluorescent antibodies into 50 μL of PBS to form a 50 μL system. Take 50 μL of the mixed system to resuspend and wash the cells, and stain the cells at room temperature for 20 minutes. Add 2 mL of PBS to wash the cells twice, and centrifuge at 2000 rpm at room temperature for 5 minutes to obtain cell pellets. Resuspend the cells with 180 μL of PBS and perform on-machine detection. Quantitatively collect 100 μL of resuspended cells and analyze the data. The results are as Figure 9 shown. The proportion of CD3+CD4+CCR1+ T cells in the allergic respiratory disease patient group is higher than that in the healthy group, and the p-value of the statistical test < 0.05, indicating that there is a significant difference in the proportion of CD3+CD4+CCR1+ T cells between the allergic respiratory disease patient group and the healthy group. Taking the proportion of CD3+CD4+CCR1+ T in PBMC as 0.855% as the Cut-off value to draw an ROC curve, as Figure 10 shown, the AUC is 0.7143, the sensitivity is 61.11%, and the specificity is 75%. It shows that the proportion of CD3+CD4+CCR1+ T cells in human PBMC has diagnostic significance for allergic respiratory diseases.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a substance for detecting CD3+CD4+CCR1+ T cells in the preparation of a diagnostic product for allergic respiratory diseases.

2. The application according to claim 1, wherein The product includes a detection reagent, a kit, an antibody chip, an antibody probe or a detection device.

3. The application according to claim 1, characterized in that, The expression of Th2 characteristic cytokines in the CD3+CD4+CCR1+ T cells is increased.

4. The application according to any one of claims 1 to 3, characterized in that Detecting CD3+CD4+CCR1+ T cells includes detecting the proportion of CD3+CD4+CCR1+ T cells in PBMC.

5. A diagnostic kit for allergic respiratory diseases, characterized in that, A substance for detecting CD3+CD4+CCR1+ T cells according to any one of claims 1 to 4.

6. The diagnostic kit for allergic respiratory diseases according to claim 5, characterized in that, Including a CD3 antibody, a CD4 antibody and a CCR1 antibody.

7. The diagnostic kit for allergic respiratory diseases according to claim 5, wherein Including a reagent for separating blood, a reagent for separating PBMC, a reagent for separating respiratory mucosa and a reagent for separating splenocytes; and / or, including a reagent for immunoassay.

8. An allergic respiratory disease diagnosis device, characterized in that, Including a detection module and a prediction and judgment module; The detection module obtains the content of CD3+CD4+CCR1+ T cells; The prediction and judgment module includes a computer-readable medium recording judgment rules, and when the computer-readable medium is processed and executed, it realizes comparing the content of CD3+CD4+CCR1+ T cells with a threshold value. When the content of CD3+CD4+CCR1+ T cells is within the range defined by the threshold value, it is determined that the subject has allergic respiratory diseases.

9. The diagnostic device according to claim 8, characterized in that, Obtaining the content of CD3+CD4+CCR1+ T cells includes obtaining the proportion of CD3+CD4+CCR1+ T cells in PBMC of the subject; when the proportion of CD3+CD4+CCR1+ T cells in PBMC of the obtained subject is ≥ the threshold value, it is determined that the subject has allergic respiratory diseases.

10. The diagnostic device according to claim 8 or 9, characterized in that, The detection module includes an antibody composition and an immunoassay device; the antibody composition includes a CD3 antibody, a CD4 antibody and a CCR1 antibody.

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