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

Abstract

The present invention provides an application of CD3+CD4+CCR1+T cells in the diagnosis of allergic respiratory diseases, relating to the field of biotechnology. Based on the discovery that the proportion of CD3+CD4+CCR1+T cells in the peripheral blood PBMCs of patients with allergic respiratory diseases is higher than that of healthy controls, the application involves applying a substance for detecting CD3+CD4+CCR1+T cells to the preparation of a diagnostic product for allergic respiratory diseases, thereby alleviating the shortcomings of existing diagnostic products for allergic respiratory diseases.

Description

Technical Field

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

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

[0003] Allergic airway disease is a type of inflammatory disease of the respiratory tract caused by an abnormal immune system response, primarily due to oversensitivity to environmental allergens (such as dust mites, pollen, and pet dander). It is often overlooked, underdiagnosed, misdiagnosed, and neglected, which not only harms health but also increases social costs. Allergic airway disease is of significant clinical significance as it underlies numerous complications, is a major risk factor for poor asthma control, and impacts quality of life and productivity at work or school.

[0004] Currently, allergic respiratory diseases are difficult to diagnose. Symptoms are similar to those of colds and COPD (Chronic Obstructive Pulmonary Disease), making differentiation difficult, especially in elderly patients, and prone to misdiagnosis or missed diagnosis. For example, vasomotor rhinitis may be mistaken for allergic rhinitis. Existing diagnostic techniques for allergic respiratory diseases include: allergen skin testing, which is an in vivo allergen test, primarily consisting of SPT and intradermal testing. If a patient experiences a hypersensitivity reaction to an allergen, a wheal and erythema will appear at the skin prick site within 20 minutes. A positive SPT is determined if the average diameter of the wheal is greater than 3 mm compared to a negative control. Serum-specific IgE testing is an in vitro allergen test. Typically, the cutoff value for serum-specific IgE levels is 0.35 kU / L; a value greater than or equal to this is considered positive, indicating sensitization. Nasal provocation testing involves directly applying an allergen to the nasal mucosa, simulating a natural attack, to observe whether symptoms are induced.

[0005] Existing diagnostic technologies for allergic respiratory diseases have the following major drawbacks: skin prick tests are affected by antihistamines and skin conditions (such as scratching), resulting in a high false-positive rate; serum IgE testing lacks specificity, and elevated total IgE levels do not necessarily indicate allergens; the detection range is limited, and due to varying thresholds, some patients may be IgE-positive but asymptomatic, making treatment decisions difficult; non-IgE-mediated allergies, such as some food allergies, are not covered, and desensitization treatment requires reassessment; provocative tests may induce severe reactions, limiting their clinical application; and genetic polymorphisms influence the intensity of allergic reactions, meaning that the same test result may have different meanings in different individuals. Consequently, new diagnostic methods and products are needed.

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

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

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides 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 a second aspect, a diagnostic kit for allergic respiratory diseases is provided, wherein the diagnostic kit for allergic respiratory diseases comprises the substance for detecting CD3+CD4+CCR1+T cells described in the first aspect.

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

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

[0013] The prediction and judgment module includes a computer-readable medium that records judgment rules. When the computer-readable medium is processed and executed, the CD3+CD4+CCR1+T cell content is compared with a threshold value. When the CD3+CD4+CCR1+T cell content is within the range defined by the threshold value, it is determined that the subject suffers from an allergic respiratory disease.

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

[0015] The present invention found that the proportion of CD3+CD4+CCR1+T cells in the peripheral blood PBMCs of patients with allergic respiratory diseases was higher than that of healthy controls. In a mouse model of allergic respiratory disease, the proportion of CD3+CD4+CCR1+T cells in the respiratory mucosa of the model group was also higher than that of the control group. Furthermore, it was found that CD3+CD4+CCR1+T cells expressed elevated Th2-specific cytokines, indicating that CCR1 can serve as a Th2 cell phenotypic marker. A receiver operating characteristic (ROC) curve was constructed to validate the use of CD3+CD4+CCR1+T cells as a diagnostic marker for allergic respiratory diseases.

[0016] Using CD3+CD4+CCR1+ T cells as a diagnostic marker for allergic respiratory diseases eliminates the need for allergen stimulation of subjects, reducing adverse reactions during the diagnostic process. It can also improve the accuracy of diagnostic products, guide individualized treatment, dynamically monitor disease activity, and reduce disease risk. Immune cell testing provides multi-dimensional information from the mechanism to the clinical context for the diagnosis of allergic respiratory diseases, facilitating the subsequent development of precision medicine products, improving disease stratification management, and predicting treatment responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 is the ratio of CD3+CD4+CCR1+T cells in PBMC of healthy subjects in Example 1;

[0019] Figure 2 is the ratio of CD3+CD4+CCR1+T cells in PBMCs of patients with allergic respiratory diseases in Example 1;

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

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

[0022] Figure 5 The ratio of CD3+CD4+CCR1+T cells in the spleen cells of the model group mice after APC cell stimulation in Example 2;

[0023] Figure 6 The IL-4 expression levels of CD3+CD4+CCR1+T cells and CD3+CD4+CCR1-T cells in the model group mice after stimulation with antigen-presenting APC cells in Example 2;

[0024] Figure 7 The IL-5 expression levels of CD3+CD4+CCR1+T cells and CD3+CD4+CCR1-T cells in the model group mice after stimulation with antigen-presenting APC cells in Example 2;

[0025] Figure 8The IL-13 expression levels of CD3+CD4+CCR1+T cells and CD3+CD4+CCR1-T cells in the model group mice after stimulation with antigen-presenting APC cells in Example 2;

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

[0027] Figure 10 This is the ROC curve constructed in Example 3 with the CD3+CD4+CCR1+T cell ratio of 0.855% in PBMC as the cut-off value. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] Immune cells play a central role in the pathogenesis and diagnosis of allergic respiratory diseases (such as allergic rhinitis and asthma). They are not only key players in the occurrence of the disease, but also serve as diagnostic markers and therapeutic targets. Th2 cells (T helper type 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 assess the type and severity of allergic reactions. The present invention found that CD3+CD4+CCR1+T cells expressed elevated Th2 characteristic cytokines, and CCR1 can be used as a Th2 cell phenotypic marker. It was also found that the proportion of CD3+CD4+CCR1+T cells in PBMCs of patients with allergic respiratory diseases was higher than that in healthy people; in a mouse model of allergic respiratory disease with OVA as an allergen, the proportion of CD3+CD4+CCR1+T cells in the respiratory mucosa of the model group mice was 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 respiratory diseases, and finally, the ROC curve was constructed to verify that CD3+CD4+CCR1+T cells can be used as a diagnostic marker for allergic respiratory diseases.

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

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

[0032] In an optional embodiment, the expression of Th2 characteristic cytokines of the CD3+CD4+CCR1+T cells is elevated.

[0033] In an optional embodiment, the expression of one or more of the CD3+CD4+CCR1+ T cell Th2 characteristic cytokines IL-4, IL-5 and IL-13 is increased.

[0034] In an optional embodiment, the substance for detecting CD3+CD4+CCR1+T cells includes a substance for detecting Th2 characteristic 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 optional embodiment, the substance for detecting CD3+CD4+CCR1+T cells includes substances 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 spleen cells.

[0036] In an optional 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 optional embodiment, detecting CD3+CD4+CCR1+T cells includes detecting the proportion of CD3+CD4+CCR1+T cells in PBMCs.

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

[0039] As used herein, the term "antibody" includes any immunoglobulin that can bind to a specific antigen. The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, full-length antibodies, nanobodies, and antigen-binding fragments, as long as 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, lacking at least some of the amino acids present in the full-length chain but still capable of specific binding to the 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 diabodies), single-chain antibody molecules (scFv), and scFv dimers (bivalent bifunctional antibodies).

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

[0041] In an optional embodiment, the allergic respiratory disease diagnostic kit comprises CD3 antibodies, CD4 antibodies and CCR1 antibodies.

[0042] In an optional embodiment, the allergic respiratory disease diagnostic kit comprises a reagent for separating blood, a reagent for separating PBMC, a reagent for separating respiratory mucosa, and a reagent for separating spleen cells.

[0043] In an optional embodiment, the allergic respiratory disease diagnostic kit contains reagents for immunoassay, which includes but is not limited to reagents for flow cytometry, chemiluminescence, enzyme-linked immunosorbent assay, immunomagnetic microparticle detection, immunofluorescence or immunoblotting, and preferably contains reagents for flow cytometry.

[0044] In an optional embodiment, the kit comprises reagents and / or consumables for detection, including but not limited to one or more of primers, probes, buffers, dyes, diluents, washing solutions, developer solutions, lysates, secondary antibodies, signaling agents, negative controls, positive controls, and blank controls. Those skilled in the art can select the reagent composition of the kit based on the specific detection method, and the present invention is not limited thereto.

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

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

[0047] The prediction and judgment module includes a computer-readable medium that records judgment rules. When the computer-readable medium is processed and executed, the CD3+CD4+CCR1+T cell content is compared with a threshold value. When the CD3+CD4+CCR1+T cell content is within the range defined by the threshold value, it is determined that the subject suffers from an allergic respiratory disease.

[0048] In an optional embodiment, obtaining the CD3+CD4+CCR1+T cell content includes obtaining the proportion of the subject's CD3+CD4+CCR1+T cells in PBMC; when the value of the obtained proportion of the subject's CD3+CD4+CCR1+T cells in PBMC is ≥ a threshold value, the subject is determined to be suffering from an allergic respiratory disease state.

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

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

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

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

[0053] In an optional embodiment, the subject of the first, second, or third aspect above comprises a mammal, exemplary subjects including but not limited to humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, alpacas, birds, goats, and sheep. In some embodiments, the subject is a human. In some embodiments, the subject is an animal model constructed with allergic respiratory symptoms.

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

[0055] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0056] Example 1

[0057] Flow cytometry was used to detect the proportion of CD3+CD4+CCR1+T cells in the peripheral blood of healthy subjects and patients with allergic respiratory diseases.

[0058] Peripheral blood is drawn by an experienced nurse, and an anticoagulant (heparin) is added to prevent coagulation. To detect lymphocyte subsets: 1 mL of whole blood is taken from each sample, and the peripheral blood is diluted with sterile PBS at a ratio of 1:1 and mixed. An appropriate amount of Ficoll-Paque PLUS is added to the centrifuge tube, and the diluted blood is slowly added to the upper layer of Ficoll along the wall of the tube to avoid 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 layer, the lower layer is Ficoll, and the bottom layer is red blood cells and granulocytes. Carefully aspirate the PBMC buffy coat layer 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 wash once. The extracted PBMC is used for flow cytometry. Take 1μL zombie to 50μL PBS to form a 50μL system, take 50μL mixed system to resuspend the washed cells, and stain at room temperature for 20 minutes. Add 2mL PBS to wash the cells twice, centrifuge at 2000 rpm at room temperature for 5 minutes to obtain a cell pellet. Add 1μL each of CD3\CD4\CCR1 flow cytometer fluorescent antibodies to 50μL PBS to form a 50μL system. Take 50μL mixed system to resuspend the washed cells, and stain at room temperature for 20 minutes. Add 2mL PBS to wash the cells twice, centrifuge at 2000 rpm at room temperature for 5 minutes to obtain a cell pellet. Resuspend the cells in 180μL PBS and detect on the machine. Quantitatively collect 100μL of resuspended cells and analyze the data. The results are as follows Figure 1 and Figure 2 The results showed that the proportion of CD3+CD4+CCR1+T cells in the peripheral blood of healthy people was 0.55%, while the proportion of CD3+CD4+CCR1+T cells in the peripheral blood of patients with allergic respiratory diseases was 1.43%, indicating that the proportion of CD3+CD4+CCR1+T cells in the peripheral blood of patients with allergic respiratory diseases was higher than that of healthy people.

[0059] Example 2

[0060] The mouse allergic respiratory disease model was established to detect the proportion of CD3+CD4+CCR1+T cells in the respiratory mucosa, and the antigen-specific spleen cell culture was used to detect the response of CD3+CD4+CCR1+T cells to inflammatory factors.

[0061] (1) Experimental purpose: To construct a mouse model of allergic respiratory disease, verify that the CD3+CD4+CCR1+T cells in mice with allergic respiratory disease are different from those in healthy mice, and to study the response of CD3+CD4+CCR1+T cells to inflammatory factors.

[0062] (2) Experimental groups: Group 1 was the control group, and Group 2 was the modeling group.

[0063] (3) Experimental Materials and Modeling Methods: OVA (ovalbumin) (allergen), Al(OH)3 suspension (adjuvant). On days 0, 7, and 14, group 1 received an intraperitoneal injection of 200 μL PBS, and group 2 received an intraperitoneal injection of 200 μL (100 μg OVA, 2 mg Al(OH)3) for sensitization. On days 21 to 27, group 1 received intranasal instillation of 20 μL PBS, and group 2 received intranasal instillation of 20 μL 40 mg / mL OVA.

[0064] (4) Experimental content: Flow cytometry detection of the proportion of CD3+CD4+CCR1+T cells in the respiratory mucosa of mice, and culture and flow cytometry detection of antigen-specific spleen lymphocytes.

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

[0066] On day 28, mice were euthanized, and nasal mucosa and lung respiratory tract tissue were obtained. Single-cell suspensions of mouse respiratory mucosa were prepared by enzymatic dissociation. The respiratory tissue was rinsed with PBS and placed in a 1.5 mL EP tube. The tissue was minced and mechanically dispersed with scissors. 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. The suspension was incubated on a shaker at 37°C for 1 hour. The suspension was gently mixed using a pipette and incubated for a further 30 minutes. The suspension was filtered through a 40 μm filter into a 50 mL Falcon tube and centrifuged at 1500 rpm for 10 minutes, with the supernatant discarded. The suspension was washed with PBS and centrifuged at 1500 rpm for 10 minutes, with the supernatant discarded. This wash was repeated once to obtain single-cell suspensions of mouse respiratory mucosa for flow cytometry analysis. Take 1μL zombie to 50μL PBS to form a 50μL system, take 50μL of the mixed system to resuspend the washed cells, and stain at room temperature for 20 minutes. Add 2mL PBS to wash the cells twice, centrifuge at 2000 rpm at room temperature for 5 minutes to obtain a cell pellet. Add 1μL each of CD3\CD4\CCR1 flow cytometer fluorescent antibodies to 50μL PBS to form a 50μL system. Take 50μL of the mixed system to resuspend the washed cells, and stain at room temperature for 20 minutes. Add 2mL PBS to wash the cells twice, centrifuge at 2000 rpm at room temperature for 5 minutes to obtain a cell pellet. Resuspend the cells in 180μL PBS and detect them on the machine. Quantitatively collect 100μL of the resuspended cells and analyze the data. The results are as follows Figure 3 and Figure 4 The results showed that the proportion of CD3+CD4+CCR1+T cells in the respiratory mucosa of mice in the model group was 6.07%, while the proportion of CD3+CD4+CCR1+T cells in the respiratory mucosa of mice in the control group was 0.23%. The results showed that the proportion of CD3+CD4+CCR1+T cells in the respiratory mucosa of mice with allergic respiratory disease modeling increased.

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

[0068] ① The method for culturing antigen-specific splenic lymphocytes is as follows: On day 28, mice were killed and the spleens were dissected under sterile conditions. A single-cell suspension of spleen cells was obtained by physical grinding. Red blood cells were lysed with red blood cell lysis buffer, washed twice with sterile PBS, and resuspended in complete RPMI-1640 medium. 1×10 cells were added to each well of a 12-well cell culture plate. 7 Splenocytes were cultured in 3 mL of 100 μg / mL L OVA solution and 5 U / mL recombinant mouse IL-2 in an incubator at 37°C, 5% CO2 for 5 days. After culturing, the medium was exchanged halfway with complete RPMI-1640 medium supplemented with 20 U / mL recombinant mouse IL-2. Culture was continued at 37°C, 5% CO2 until day 7, when flow cytometry analysis was performed.

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

[0070] Antigen presenting cells (APCs) were prepared and antigen presentation was performed 10 hours before the test as follows: BALB / c mice without any treatment were euthanized, the fur was disinfected, and the mice were dissected under sterile conditions. The mouse peritoneal cells were collected by flushing with normal saline, and macrophages were obtained by the adherent method to be used as APCs.

[0071] Wash twice with sterile saline and resuspend in complete RPMI-1640 medium. Add 1×10 5 The above APC cells, 100 μg / mL OVA solution or PBS as a control were cultured with OVA in a 37° C., 5% CO 2 incubator overnight to obtain APC cells presenting the antigen.

[0072] After 10 hours, the APC cells that presented the antigen were collected and washed three times with RPMI-1640 medium. 5 APC cell stimulation 1×10 6 Culture the lymphocytes from step ① for 7 days. Add 0.15 μL of Golgi blocker to 200 μL of the culture medium. Continue culturing in a 37°C, 5% CO2 incubator for 5 hours.

[0073] Then collect the cells and wash them twice with PBS. Resuspend the above cells in 100μL PBS, add 1μL each of CD3\CD4\CCR1 flow cytometer fluorescent antibodies, and stain them 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 a cell pellet. After using a membrane permeabilization agent to break the membrane, resuspend in 100μL PBS, add 1μL each of IL-4\IL-5\IL-13\IFN-γ\IL-17A flow cytometer fluorescent antibodies, and stain them 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 a cell pellet. Resuspend the cells in 180μL PBS and detect them on the machine. Quantitatively collect 100μL of resuspended cells and analyze the data. The results are as follows Figure 5 、 Figure 6 、 Figure 7 and Figure 8As shown, the results showed that the expression level of IL-4 in CD3+CD4+CCR1+T cells was higher than that in CD3+CD4+CCR1-T cells; the expression level of IL-5 in CD3+CD4+CCR1+T cells was higher than that in CD3+CD4+CCR1-T cells; and the expression level of IL-13 in CD3+CD4+CCR1+T cells was higher than that in CD3+CD4+CCR1-T cells. The above results indicate that the expression of Th2 characteristic cytokines in spleen antigen-specific CD3+CD4+CCR1+T cells in the modeling group mice is increased.

[0074] Example 3

[0075] The role of human peripheral blood CD3+CD4+CCR1+T cells in the diagnosis of allergic respiratory diseases.

[0076] (1) Experimental purpose: 16 healthy subjects and 18 patients with allergic respiratory diseases were recruited to detect the proportion of PBMCCD3+CD4+CCR1+T cells by flow cytometry.

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

[0078] ① Clear diagnosis: meet the diagnostic criteria for allergic respiratory diseases such as allergic rhinitis and asthma (such as GINA guidelines and ARIA guidelines).

[0079] ②Age range: usually 18 to 65 years old.

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

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

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

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

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

[0085] ② Recent infection or surgery: exclude those who have had respiratory infection or major surgery in the recent period (e.g., within 4 weeks).

[0086] ③Drug use: Patients who used immunosuppressants or systemic glucocorticoids for a long time were excluded.

[0087] ④ Pregnancy or lactation: Pregnant or lactating women are excluded.

[0088] ⑤Other allergic diseases: exclude severe atopic dermatitis, food allergies and other allergic diseases.

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

[0090] (4) Experimental method: The method for obtaining peripheral blood and the method for extracting and separating PBMC are the same as those in Example 1. Take 1 μL zombie to 50 μL 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 PBS to wash the cells twice, and centrifuge at 2000 rpm at room temperature for 5 minutes to obtain a cell pellet. Add 1 μL each of CD3\CD4\CCR1 flow cytometer fluorescent antibodies to 50 μL 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 PBS to wash the cells twice, and centrifuge at 2000 rpm at room temperature for 5 minutes to obtain a cell pellet. Resuspend the cells in 180 μL PBS and detect on the machine. Quantitatively collect 100 μL of the resuspended cells and analyze the data. The results are as follows Figure 9 As shown in the figure, the proportion of CD3+CD4+CCR1+T cells in the allergic respiratory disease group was higher than that in the healthy group, and the statistical test p value was <0.05, indicating that there was a significant difference in the proportion of CD3+CD4+CCR1+T cells between the allergic respiratory disease group and the healthy group. The ROC curve was drawn with the proportion of CD3+CD4+CCR1+T in PBMC as 0.855% as the cut-off value, as shown in the figure. Figure 10 As shown, the AUC was 0.7143, the sensitivity was 61.11%, and the specificity was 75%, indicating that the proportion of CD3+CD4+CCR1+T cells in human PBMCs 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, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Detection of CD3 + CD4 + CCR1 + Application of T cell substances in the preparation of diagnostic products for allergic respiratory diseases.

2. The use according to claim 1, characterized in that The products include detection reagents, kits, antibody chips, antibody probes or detection equipment.

3. The use according to claim 1, characterized in that CD3 + CD4 + CCR1 + The expression of Th2 characteristic cytokines in T cells was elevated.

4. The use according to any one of claims 1 to 3, characterized in that Detection of CD3 + CD4 + CCR1 + T cells including CD3 + CD4 + CCR1 + The proportion of T cells in PBMCs.

5. A diagnostic kit for allergic respiratory diseases, characterized in that: Comprising the detection CD3 according to any one of claims 1 to 4 + CD4 + CCR1 + T cell substances.

6. The allergic respiratory disease diagnostic kit according to claim 5, characterized in that: Contains CD3 antibodies, CD4 antibodies and CCR1 antibodies.

7. The allergic respiratory disease diagnostic kit according to claim 5, characterized in that: Contains reagents for separating blood, reagents for separating PBMC, reagents for separating respiratory mucosa, and reagents for separating spleen cells; and / or, includes reagents for immunoassay.

8. Allergic respiratory disease diagnostic device, characterized in that Contains detection module and prediction and judgment module; The detection module obtains CD3 + CD4 + CCR1 + T cell content; The prediction and judgment module includes a computer readable medium recording judgment rules, and the computer readable medium is used to process and execute the CD3 + CD4 + CCR1 + T cell content was compared with the threshold value, and when CD3 + CD4 + CCR1 + If the T cell content is within the range defined by the threshold, the subject is determined to have an allergic respiratory disease.

9. The diagnostic device according to claim 8, wherein The acquisition of CD3 + CD4 + CCR1 + T cell content includes obtaining the subject's CD3 + CD4 + CCR1 + The proportion of T cells in PBMC; when the subject's CD3 + CD4 + CCR1 + If the proportion of T cells in PBMCs is ≥ the threshold, the subject is determined to have allergic respiratory disease.

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

Images