Application of reagent for detecting follicular regulatory T cells in preparation of diagnostic reagent for prognosis of immunotherapy of allergic rhinitis
阅读说明：本技术 检测滤泡调节性t细胞试剂在制备变应性鼻炎免疫治疗预后诊断试剂中的应用 (Application of reagent for detecting follicular regulatory T cells in preparation of diagnostic reagent for prognosis of immunotherapy of allergic rhinitis ) 是由 刘争 姚银 王志超 刘阳 陆翔 王恒 曾明 于 2021-07-16 设计创作，主要内容包括：本发明公开了一种检测滤泡调节性T细胞试剂在制备变应性鼻炎免疫治疗预后诊断试剂中的应用,属于变应性鼻炎检测技术领域。具体的,包括如下步骤：1)制备外周血单个核细胞；2)采用流式细胞术检测步骤1)中单个核细胞中滤泡调节性T细胞表达水平。本发明通过检测正常样本和变应性鼻炎患者的外周血单个核细胞中滤泡调节性T细胞水平,判断变应性鼻炎严重程度,为变应性鼻炎患者的疾病风险及治疗方案提供重要依据；同时,通过监控变应性鼻炎患者在接受免疫治疗前后外周血单个核细胞中滤泡调节性T细胞水平,以此判断变应性鼻炎免疫治疗预后指标。(The invention discloses an application of a reagent for detecting follicular regulatory T cells in preparation of an allergic rhinitis immunotherapy prognosis diagnosis reagent, and belongs to the technical field of allergic rhinitis detection. Specifically, the method comprises the following steps: 1) preparing peripheral blood mononuclear cells; 2) measuring the expression level of the follicular regulatory T cells in the mononuclear cells of step 1) by flow cytometry. The invention judges the severity of the allergic rhinitis by detecting the level of follicular regulatory T cells in the peripheral blood mononuclear cells of a normal sample and a patient with the allergic rhinitis, and provides an important basis for the disease risk and treatment scheme of the patient with the allergic rhinitis; meanwhile, the level of follicular regulatory T cells in peripheral blood mononuclear cells of a patient with allergic rhinitis before and after receiving immunotherapy is monitored, so that the prognosis index of the allergic rhinitis immunotherapy is judged.)
1. An application of a reagent for detecting follicular regulatory T cells in preparing an immune therapy prognosis diagnostic reagent for allergic rhinitis.
2. The use of the follicular regulatory T cell reagent of claim 1 for the preparation of a prognostic diagnostic reagent for immunotherapy of allergic rhinitis, comprising the step of measuring the level of follicular regulatory T cells in a normal in vitro sample and peripheral blood mononuclear cells of a patient with allergic rhinitis.
3. The use of the follicular regulatory T cell reagent of claim 2 for the preparation of a prognostic diagnostic reagent for immunotherapy of allergic rhinitis, further comprising monitoring the level of follicular regulatory T cells in peripheral blood mononuclear cells of a patient with allergic rhinitis before and after receiving immunotherapy.
4. Use of the follicular regulatory T cell reagent of claim 3 for the preparation of a prognostic diagnostic reagent for immunotherapy of allergic rhinitis, wherein the number of follicular regulatory T cells in said peripheral blood mononuclear cells is increased compared to the number before treatment by monitoring changes and correlations in the expression level of follicular regulatory T cells.
5. The use of the reagent for detecting follicular regulatory T cells according to any one of claims 1 to 4, in the preparation of a prognostic diagnostic reagent for immunotherapy of allergic rhinitis, wherein the expression level of follicular regulatory T cells in peripheral blood mononuclear cells of patients with allergic rhinitis is significantly correlated with the severity of allergic rhinitis, and the correlation coefficient r is-0.206.
6. The use of the reagent for detecting follicular regulatory T cells according to any one of claims 1 to 4 for the preparation of a prognostic diagnostic reagent for immunotherapy of allergic rhinitis, comprising the following detection steps:
1) preparing peripheral blood mononuclear cells;
2) detecting the expression level of follicular regulatory T cells in said mononuclear cells of step 1) by flow cytometry.
7. The use of the reagent for detecting follicular regulatory T cells as defined in claim 6 for the preparation of a prognostic diagnostic reagent for immunotherapy of allergic rhinitis, wherein the specific process of step 1) is as follows:
1.1) taking a peripheral blood lymphocyte separation solution and placing the separation solution at the bottom of a test tube;
1.2) preparing an anticoagulant diluent: placing the vein anticoagulation blood at the bottom of another test tube, and adding PBS with the same volume to dilute;
1.3) premixing: slowly adding the anticoagulation diluent in the step 1.2) into the peripheral blood lymphocyte separation solution in the step 1.1);
1.4) centrifugal treatment;
1.5) preparation of suspension cells: and (3) taking out the interface white cell layer of the solution formed after the centrifugal treatment in the step 1.4), and adding PBS (phosphate buffer solution) for dilution to obtain the suspension cells.
8. The use of the reagent for detecting follicular regulatory T cells according to claim 7 for the preparation of a prognostic diagnostic reagent for immunotherapy of allergic rhinitis, wherein in step 1.2), said venous anticoagulation is 15-25 units per ml of blood heparin.
9. The use of the reagent for detecting follicular regulatory T cells as defined in claim 7 for the preparation of a prognostic diagnostic reagent for immunotherapy of allergic rhinitis, wherein in step 1.4), the centrifugation conditions are: at room temperature, the centrifugal speed is 1800-2200 r/min, and the centrifugal treatment is 15-25 min.
10. An allergic rhinitis immunotherapy prognosis diagnosis kit, characterized in that it comprises an antibody composition for detecting follicular regulatory T cells in peripheral blood mononuclear cells, said antibody composition comprising any one or two or more of a CD3 antibody, a CD4 antibody, a CD45RA antibody, a CXCR5 antibody, a CD127 antibody and a CD25 antibody, and all the antibodies are fluorescently labeled antibodies.
Technical Field
The invention relates to application of a follicular regulatory T cell as an allergic rhinitis marker in judging the prognosis of immunotherapy of an allergic rhinitis patient, belongs to the technical field of allergic rhinitis detection, and particularly relates to application of a reagent for detecting follicular regulatory T cells in preparing an allergic rhinitis immunotherapy prognosis diagnosis reagent.
Background
Allergic Rhinitis (AR) refers to a nasal mucosa non-infectious chronic inflammatory disease mainly mediated by IgE after an organism is exposed to an allergen, and the prevalence rate of AR in continental areas of China is 4-38%. The main symptoms of AR include nasal itching, sneezing, watery nasal discharge, itchy eyes, nasal congestion, etc., which affect the life, sleep, and work quality of patients and cause serious social and economic burden. Current treatment regimens for AR include allergen avoidance, medication, AIT (allergen-specific immunotherapy) and patient education. Unlike temporary relief of symptoms by avoiding exposure to allergens and medications, the allergen extract (vaccine) in AIT induces immune tolerance in the body by repeated exposure to the patient and increasing the dose gradually, thereby controlling or alleviating allergic symptoms. However, AIT also has the disadvantages of long treatment period, high cost, poor patient compliance, and susceptibility to serious complications. More importantly, a considerable part of patients with allergic rhinitis do not respond well to AIT, and a great deal of waste of social and medical resources and individual economic loss are caused. Therefore, the search for biomarkers that can monitor and predict the efficacy of AIT, and the search for new methods that can enhance the efficacy of AIT, are the key and difficult points of research on allergic rhinitis and AIT.
The document "application of biomarkers in the evaluation of the efficacy of specific immunotherapy for allergic rhinitis" mentions that some potential indexes related to the occurrence and diagnosis and treatment of allergic rhinitis, such as specific IgE, interleukin 2(IL-2), IL-4, IL-5, eosinophil cationic protein, eosinophil, specific IgG4, interferon gamma, regulatory T cells, sIgE/tIgE ratio, etc., have been found at home and abroad at present. As reported in the literature, "expression and significance of interleukin 23 in nasal mucosa of patients with allergic rhinitis", the expression of 1L-23 in the lower nasal mucosa of 12 cases of AR patients (AR group) and 11 cases of simple nasal septum deflection patients (control group) were collected and detected by immunohistochemistry and quantitative RT-PCR. The results show that the expression level of 1L-23P19mRNA in the AR group is obviously improved compared with the control group (P is less than 0.01). Immunohistochemistry shows that the 1L-23 protein is mainly expressed in inflammatory infiltration cells of a mucous membrane lamina propria, and the number of 1L-23 positive cells in the nasal mucosa of the AR group is obviously higher than that of a control group (P is less than 0.01). The expression levels of 1L-23P19mRNA and protein are obviously and positively correlated with the infiltration degree of total inflammatory cells of the mucous lamina propria and the deposition degree of collagen fibers of the lamina propria (the correlation coefficients are 0.678 and 0.834 respectively for the expression level of mRNA, and 0.644 and 0.721 respectively for the expression level of protein, and the P is less than 0.01). And the literature, "expression and significance of high-mobility group protein B1 in allergic rhinitis" describes that the expression of HMGB1 in the inferior turbinate mucosa of 12 patients with allergic rhinitis and the expression of HMGB1 in the inferior turbinate mucosa of 11 patients with simple nasal septum deflection (a control group) are collected and detected by adopting immunohistochemistry and RT-PCR (reverse transcription-polymerase chain reaction) methods, and the result shows that the expression level of the HMGB1 in the inferior turbinate mucosa of the patients with allergic rhinitis is obviously higher than that in the control group (P is less than 0.01). Immunohistochemistry shows that positive expression is mainly located in the mesentery of the mucosa, the fluorescence intensity in the mucosa of the turbinate of patients with allergic rhinitis is obviously higher than that of a control group (P < 0.01), the HMGB1mRNA level (5.0 +/-1.2) and the protein level are both positively correlated with the number of mature dendritic cells in the mucosa of the turbinate (r ═ 0.695, r ═ 0.578, P < 0.01), and the HMGB1 protein level and the eosinophil cell number (40.0 +/-3.1) in the mucosa of the turbinate are obviously negatively correlated (r ═ 0.547, P < 0.01).
The search for new markers of allergic rhinitis and the possibility of specific assessment of clinical efficacy is the current focus of research.
Disclosure of Invention
In order to solve the technical problems, the invention discloses an application of a reagent for detecting follicular regulatory T cells in preparation of an allergic rhinitis immunotherapy prognosis diagnostic reagent. The follicular regulatory T cells can be used as an effective marker of allergic rhinitis, can be used for judging the severity of diseases, and can provide basis and related guidance for the disease risk and treatment scheme of patients.
In order to achieve the purpose, the invention discloses an application of a reagent for detecting follicular regulatory T cells in preparing an allergic rhinitis immunotherapy prognosis diagnostic reagent.
Further, it comprises measuring the level of follicular regulatory T cells in peripheral blood mononuclear cells from normal samples and patients with allergic rhinitis in vitro.
Further, it comprises monitoring the level of follicular regulatory T cells in peripheral blood mononuclear cells of patients with allergic rhinitis before and after receiving immunotherapy.
Further, by monitoring changes in and correlation of follicular regulatory T cell expression levels, the number of follicular regulatory T cells in the peripheral blood mononuclear cells is increased compared to before treatment.
Further, the expression level of the follicular regulatory T cells in peripheral blood mononuclear cells of patients with allergic rhinitis is clearly correlated with the severity of allergic rhinitis, and the correlation coefficient r is-0.206, which indicates that the severity of allergic rhinitis is closely related to the expression level of the follicular regulatory T cells in peripheral blood mononuclear cells.
Further, it comprises the following detection steps:
1) preparing peripheral blood mononuclear cells;
2) detecting the expression level of follicular regulatory T cells in said mononuclear cells of step 1) by flow cytometry.
Further, the specific process of step 1) is as follows:
1.1) taking a peripheral blood lymphocyte separation solution and placing the separation solution at the bottom of a test tube;
1.2) preparing an anticoagulant diluent: placing the vein anticoagulation blood at the bottom of another test tube, and adding PBS with the same volume to dilute;
1.3) premixing: slowly adding the anticoagulation diluent in the step 1.2) into the peripheral blood lymphocyte separation solution in the step 1.1);
1.4) centrifugal treatment;
1.5) preparation of suspension cells: and (3) taking out an interface white cell layer of the solution formed after the centrifugal treatment in the step 1.4), adding PBS for dilution, and suspending the cells.
Further, in the step 1.2), the venous anticoagulation is that 15-25 units of heparin is added into each milliliter of blood.
Further, in step 1.4), the centrifugal processing conditions are as follows: at room temperature, the centrifugal speed is 1800-2200 r/min, and the centrifugal treatment is 15-25 min.
In addition, the invention also discloses a diagnostic kit for the prognosis of allergic rhinitis, which comprises the application. Meanwhile, the diagnostic kit comprises a substance for detecting the level of follicular regulatory T cells in peripheral blood mononuclear cells, as shown in any one of the following tables. Namely, any one or two or more of CD3 antibody, CD4 antibody, CD45RA antibody, CXCR5 antibody, CD127 antibody and CD25 antibody, and all antibodies are fluorescently labeled antibodies.
Has the advantages that:
1. the invention judges the severity of the allergic rhinitis by detecting the level of follicular regulatory T cells in the normal sample and peripheral blood mononuclear cells of the patient with the allergic rhinitis, and provides an important basis for the disease risk and treatment scheme of the patient with the allergic rhinitis.
2. The invention judges the allergic rhinitis immunotherapy prognosis index by monitoring the level of follicular regulatory T cells in peripheral blood mononuclear cells of a patient with allergic rhinitis before and after receiving immunotherapy.
3. The invention adopts the follicular regulatory T cells in the peripheral blood mononuclear cells as the allergic rhinitis prognosis diagnostic reagent, and the detection process is convenient and rapid.
Drawings
FIG. 1 is a schematic representation of a study of the correlation of follicular regulatory T cells with allergic rhinitis;
FIG. 2 is a schematic representation of the study of the correlation of follicular regulatory T cells with allergic rhinitis;
FIG. 3 is a schematic representation of a study of follicular regulatory T cell levels in peripheral blood mononuclear cells of patients with allergic rhinitis before and after receiving immunotherapy;
FIG. 4 is a schematic representation of the study of follicular regulatory T cells levels in peripheral blood mononuclear cells before and after receiving immunotherapy in patients with allergic rhinitis.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Definition and use of terms
As used herein, the term "and/or" means any one of the options or two or more of the options.
The term "marker" or "biomarker" is an organic biomolecule that is differentially present in a sample taken from a subject of one phenotypic state (e.g., having a disease) as compared to another phenotypic state (e.g., not having a disease). Biomarkers differ in different phenotypic states if the average or median level of the biomarker, e.g., expression level, in different groups is calculated to be statistically significant.
The term "sample" as used herein refers to a collection of similar cells or tissues isolated from a subject, as well as tissues, cells, and fluids present within a subject. The term "sample" includes any bodily fluid (e.g., blood, lymph, gynecological fluid, cystic fluid, urine, tears, and fluid collected by bronchial lavage and/or peritoneal irrigation) or cell from a subject. In one embodiment, the tissue or cell is removed from the subject. In another embodiment, the cell is present in the subject. Other subject samples include tear drops, serum, cerebrospinal fluid, feces, sputum, and cell extracts. In one embodiment, the biological sample comprises a protein molecule from a test subject. In another embodiment, the biological sample may comprise mRNA molecules from a test subject or genomic DNA molecules from a test subject.
The term "determining" is intended to include methods of detecting the presence or absence of a marker in a sample, quantitatively determining the amount of a marker in a sample, and/or determining the type of biomarker.
The term "monitoring" means a method that is capable of knowing in real time the presence or absence of a marker in a test sample, quantitatively determining the amount of a marker in a sample, and/or determining the type of biomarker.
The term "kit" is any article of manufacture (e.g., package or container) comprising at least one reagent, e.g., probe, primer or antibody, that specifically detects a marker of the invention, which article of manufacture is promoted, distributed or sold as one unit for performing a method of the invention. In certain embodiments, a kit can include a substrate, e.g., a substrate comprising a capture reagent of one or more markers of the invention and/or a capture reagent bound to one or more markers of the invention. In some embodiments, such kits comprise instructions for determining the level of the marker using mass spectrometry.
Subjects or patients may include, but are not limited to, mammals, such as cows, birds, dogs, horses, cats, sheep, pigs, or primates (including humans and non-primates).
Example 1: judging the severity of allergic rhinitis of a patient by detecting the expression level of follicular regulatory T cells
Experimental subject and materials:
1. subject:
peripheral blood samples from allergic rhinitis patients and normal controls were obtained from multi-market hospitals in Wuhan City. Morning fasting anticoagulation.
2. Experimental materials:
1. antibody: as in table 1 below;
TABLE 1 list of antibody compositions
2. Other materials:
(1) FACS buffer: 3% calf serum in PBS;
(2) conical tubes (BD Labware);
(3)PBS；
3. instrument for measuring the position of a moving object
BD LSRFortessaTMX-20 flow cytometry;
3. specific experiments are as follows:
(1) and (5) harvesting the single cell suspension of the peripheral blood.
(2) Cells were transferred to 50mL conical tubes and centrifuged at 300RCF for 5min at 4 ℃.
(3) The supernatant was discarded and the precipitate was retained.
(4) Cells were resuspended in PBS and kept on ice for 5 min.
(5) Then, 15mL of PBS and 300RCF were added, and the mixture was centrifuged at 4 ℃ for 5 min.
(6) The supernatant was discarded, 20mL of PBS was added to resuspend the cells, and the cells were counted.
(7) 5% calf serum was added to 50uL of cell suspension for blocking and incubation on ice for 10 min.
(8) Cells were washed with 200mL PBS, centrifuged at 300RCF at 4 ℃ for 5 min.
(9) The supernatant was discarded, and the cells were resuspended with 30uL each of fluorescently labeled CD3, CD4, CD45RA, CXCR5, CD127 and CD25 antibodies or isotype (1: 50in FACS buffer), and incubated on ice for 30 min.
(10) Cells were washed 3 times with 200mL PBS, centrifuged at 300RCF at 4 ℃ for 5 min.
(11) The cells were resuspended at 400uLPBS and analyzed by BD LSRFortessaTmX-20 flow cytometry, and the follicular regulatory T cells were labeled CD3+ CD4+ CD45RAlowCXCR5+ CD25highCD127 low.
4. The experimental results are as follows:
as can be seen from fig. 1 and 2, the level of follicular regulatory T cells in patients with allergic rhinitis was significantly lower than in normal persons. By correlation, the severity of allergic rhinitis can be judged by detecting the level of follicular regulatory T cells.
Example 2: judging allergic rhinitis immunotherapy prognostic index by monitoring the change of expression level of follicular regulatory T cells in peripheral blood mononuclear cells of patients with allergic rhinitis before and after receiving immunotherapy
Experimental subject and materials:
1. subject:
peripheral blood samples from patients with allergic rhinitis and from multi-market hospitals in Wuhan City were obtained from the same patients after receiving immunotherapy. The immunotherapy method for allergic rhinitis patients of the present application is subcutaneous injection.
2. Experimental materials:
the same as in example 1.
3. The experimental method comprises the following steps:
the same as in example 1.
4. The experimental results are as follows:
as can be seen from fig. 3, when the patients with allergic rhinitis received specific immunotherapy, the level of follicular regulatory T cells was significantly increased, and as can be seen from fig. 4, the clinical score of the patients was also significantly decreased, which was clearly inversely correlated with that of follicular regulatory T cells.
Example 3
The application also discloses a diagnostic reagent for the prognosis of allergic rhinitis, which comprises a substance for detecting the level of follicular regulatory T cells in peripheral blood mononuclear cells, and comprises an antibody composition for detecting follicular regulatory T cells in peripheral blood mononuclear cells, wherein the antibody composition comprises any one or two or more of a CD3 antibody, a CD4 antibody, a CD45RA antibody, a CXCR5 antibody, a CD127 antibody and a CD25 antibody, and all the antibodies are fluorescently labeled antibodies. Specifically, as shown in table 1 above.
Therefore, the diagnostic kit designed by the invention can effectively monitor the expression level of follicular regulatory T cells in peripheral blood mononuclear cells of patients with allergic rhinitis in the prognosis and the prognosis, and provides important guiding significance for predicting the AIT curative effect and searching a new AIT treatment method.
In addition, the diagnostic kit designed by the invention can also cooperate with other detection indexes.
The present invention is not limited to the above-described alternative embodiments, and various other forms of products can be obtained by anyone in light of the present invention. The above detailed description should not be taken as limiting the scope of the invention, which is defined in the claims, and which the description is intended to be interpreted accordingly.