CN114910652A - Application of follicular helper T lymphocyte 13 as diagnostic marker in preparation of children allergic asthma kit - Google Patents
Application of follicular helper T lymphocyte 13 as diagnostic marker in preparation of children allergic asthma kit Download PDFInfo
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Abstract
According to the invention, the follicular helper T lymphocytes 13 are used as a diagnostic marker in the preparation of the kit for the auxiliary diagnosis of the allergic asthma in children, the follicular helper T lymphocytes 13 are used as the diagnostic marker, the level of the follicular helper T lymphocytes 13 in an external blood sample is used as a diagnostic reference index of the allergic asthma in children, and the data is objective and is not easy to make mistakes; the detection of the level of the follicular helper T lymphocytes 13 in the blood sample is realized by using the detection reagent of the follicular helper T lymphocytes 13 in the preparation of the kit for treating the allergic asthma in children, and the detection process is simple and easy to implement and is suitable for popularization.
Description
Technical Field
The invention relates to the technical field of medical diagnostics, in particular to application of follicular helper T lymphocytes 13 as a diagnostic marker in preparing a kit for treating allergic asthma in children.
Background
Allergic asthma (Allergic asthma) is a kind of chronic Allergic diseases which are triggered by allergens such as pollen, animal dander, dust mite and the like and are mainly characterized by the increase of Immunoglobulin E (IgE). Allergic asthma is one of the most common chronic diseases in childhood. The main diagnostic basis for allergic asthma in children at present is mainly confirmed from three aspects: (1) typical clinical manifestations; (2) evidence of airflow restriction; (3) abnormal immune response levels.
Among the typical clinical symptoms are: wheezing, coughing, chest tightness, dyspnea, etc., and double-lung full-blown expiratory phase wheezing.
Evidence of airflow restriction: the main manifestation during physical examination is full expiratory phase wheezing sound during double-lung auscultation, the detection is objectively based on the lung ventilation function, the method is mainly used for children over 5 years old, the accuracy of the result depends on the matching of the children, the individual difference is large, and experienced clinicians can judge the airway obstruction condition of asthma children by diagnostically applying airway dilators in children under 5 years old and children over 5 years old who cannot be matched. The diagnosis of the younger patients still depends on the clinical diagnosis of the pediatrician due to the incompatibility of the lung ventilation function measurement, and the younger patients have certain subjectivity and inevitably cause certain diagnosis deficiency or over-diagnosis.
Abnormal immune response levels: currently, methods for clinically evaluating individual immune response are mainly divided into two categories, the most common in vivo experiment is skin prick test, which is simple, convenient and accurate, but the test result is easily affected by skin condition, medication history, level of operators and the like of patients, and in addition, the test may cause severe allergic reaction, such as asthma attack, anaphylactic shock and the like, so patients in the acute stage of allergic asthma are not suitable for performing. In vitro experiments include the earliest radiosensitive adsorption experiments, as well as currently used enzyme-linked immunosorbent assays for IgE, immunoblotting and the like. The enzyme-linked immunosorbent assay for detecting IgE can only achieve qualitative/semi-quantitative results, and is complex and low in sensitivity. The immunoblotting method used in clinic at present is complicated to operate, and very accurate results are difficult to obtain.
At present, the diagnosis of allergic asthma in children depends on the clinical diagnosis of pediatricians to a large extent, and the accuracy of the diagnosis result is influenced by the abilities and experience of the clinicians.
Disclosure of Invention
Aiming at solving the problem that the current diagnosis of the allergic asthma in children depends on the clinical diagnosis of pediatricians to a greater extent, the invention provides the application of the follicular helper T lymphocytes 13 as a diagnosis marker in the preparation of a kit for the allergic asthma in children, the follicular helper T lymphocytes 13 can be used as the diagnosis marker, an objective reference index is provided for the diagnosis of the allergic asthma in children, the diagnosis accuracy is improved, meanwhile, the detection process is simple and easy, the detection result is accurate, and the kit is very suitable for clinical popularization.
The technical scheme of the invention is as follows:
the application of the follicular helper T lymphocytes 13 as a diagnostic marker in the preparation of a kit for the treatment of childhood allergic asthma is characterized in that the proportion of the follicular helper T lymphocytes 13 in an extracorporeal blood sample in the follicular helper T lymphocytes is detected to be used as the characterization of childhood allergic asthma.
It is further characterized in that:
the blood sample is peripheral blood.
The detection of the level of the follicular helper T lymphocytes 13 is realized by applying a detection reagent of the follicular helper T lymphocytes 13 in the preparation of a kit for detecting the allergic asthma in children.
The detection reagent comprises a fluorescent antibody which is provided with a label for detection.
The kit comprises: antibody composition, fixed membrane rupture liquid, fixed membrane rupture buffer solution, BFA, PMA and ionomycin.
The antibody composition comprises: one or two or more of CD45RA-AF488, CD3-AF532, CXCR5-APC, CD19-Super Bright 436, TCR gamma delta-BV 480, CD8a-BV570, CD4-BV750, IL-4-PE-Dazle 594 and IL-13-PE.
According to the invention, the follicular helper T lymphocytes 13 are used as a diagnosis marker in the preparation of the kit for assisting the diagnosis of the allergic asthma in children, the follicular helper T lymphocytes 13 are used as the diagnosis marker, the level of the follicular helper T lymphocytes 13 in an external blood sample is used as a diagnosis reference index of the allergic asthma in children, and the data is objective and is not easy to make mistakes; the detection of the level of the follicular helper T lymphocytes 13 in the blood sample is realized by using the detection reagent of the follicular helper T lymphocytes 13 in the preparation of the kit for treating the allergic asthma in children, and the detection process is simple and easy to implement and is suitable for popularization.
Drawings
FIG. 1 is T FH 13 cell circle gating strategy figure 2 is T FH 13 cell count T FH Examples of ratios in cells;
FIG. 3 is T FH Correlation between 13 cells and clinical index serum total ige (tge).
Detailed Description
Follicular helper T lymphocyte 13 (hereinafter referred to as T) FH 13 cells) is a group of newly named cells, and the inventor finds that T is contained in peripheral blood of children allergic asthma patients FH 13 cells occupy follicular helper T lymphocytes (hereinafter referred to as T) FH Cells) was significantly increased (P < 0.0001) as shown in figure 2, and had a strong positive correlation with total IgE levels in serum (r: 0.5162, P: 0.0002), as shown in figure 3 in particular. Indicating T in peripheral blood FH The increased proportion of 13 cells is associated with the onset of allergic asthma and acts through its interaction with IgE, and thus the present invention relates to T FH 13 cells as an immune cell marker to assess the level of aberrant immune responses in allergic asthma children.
In FIG. 3, where P represents the statistical difference, it represents whether the two data sets are statistically significant, and P < 0.05 is a significant statistical difference; r is a correlation coefficient representing the correlation between two sets of data.
In the present invention, T in peripheral blood is detected FH 13 cells in T FH The change of the proportion in the cells is used as an objective index for assisting in evaluating the allergic asthma of children, which has important significance for assisting in diagnosis, guiding treatment, regulating abnormal immune level and evaluating the curative effect of targeted immunotherapy.
In the technical scheme of the invention, T FH 13 the detection reagent for the cells comprises a fluorescent antibody having a detectable label.
Detection reagent-based implementation of T FH 13 a method of detecting a cell, comprising: preparing peripheral blood mononuclear cells, and detecting T in the peripheral blood mononuclear cells by flow cytometry FH Cellular expression level, at T FH Obtaining T from cells FH 13 cells; obtained T FH 13 at T FH The ratio of the cells is the detection result. The method specifically comprises the following steps.
Peripheral blood was collected at about 4mL using an EDTA anticoagulant tube, PBMCs (peripheral blood mononuclear cells) were separated by Ficoll density gradient centrifugation, and the PBMCs were frozen with FBS containing 10% DMSO for use. The liquids required for the detection process were prepared according to table 1 and kept for future use.
Table 1: reagent preparation
Step one, recording spectral data.
S1-1: recovering PBMCs and counting.
S1-2: PBMCs are divided into approximately every 0.6X 10 6 The cells/200. mu.L were resuspended in cIMDM medium, transferred to a 96-well U-plate and placed in an incubator (5% CO2,37 ℃) for 12 hours.
After 12 hours, 100. mu.L of supernatant was removed by pipetting with a pipette, 66. mu.L of medium was added to each well, and 9. mu.L of PMA and 25. mu.L of ionomycin were added to the mixture in Table 1 to achieve the final concentration of PMA: 150ng/mL, ionomycin: 1. mu.g/mL.
The plates were removed after 1 hour by placing in a 5% CO2,37 ℃ incubator, 0.5. mu.L BD-GolgiPlug (containing BFA) was added to each well and the plates were incubated for 7 hours with continued placement in a 5% CO2,37 ℃ incubator.
S1-3: after incubation, 11 EP tubes were taken, numbered: 1-11, transferring the cells incubated in the 96-well plate into an EP tube, adding 1mL FACS into each tube, mixing, centrifuging (4 ℃, 450g, 5 minutes), and discarding the supernatant.
Resuspend tube cells # 1-9 in FACS, add single antibody (total reaction volume 50 μ L) as per table 2, where no reagent was added to EP tube # 1 as blank, incubate for 30 min at 4 ℃ in dark.
FACS washes were added at 1mL per tube, centrifuged (4 ℃, 450g, 5 min) and the supernatant discarded.
The cell pellet was finally resuspended in 200. mu.L FACS.
S1-4: the cell pellet of tubes 10 and 11 was added with 250. mu.L of the fixing/permeating solution, the cells were uniformly resuspended by blowing, incubated at 4 ℃ for 20 minutes, washed with 1mL of 1 XBD Perm/Wash buffer per tube, mixed well, centrifuged (4 ℃, 450g, 5 minutes), and the supernatant was discarded.
The cell pellet was resuspended in 1 XBD Perm/Wash buffer, the fluorescent antibody (total volume 50. mu.L) was added as in Table 3, and incubated at 4 ℃ for 45 minutes in the absence of light.
1mL of 1 XBD Perm/Wash buffer was added to each tube, washed, centrifuged (4 ℃, 450g, 5 min), and the supernatant discarded.
The cell pellet was finally resuspended in 200. mu.L FACS.
S1-5: detecting by using a 38-channel spectral flow cytometer Cytek NL-CLC-3000, collecting 50000-100000 PBMCs for each fluorescent antibody, and recording the fluorescence spectrum data of each fluorescence for subsequent positioning of T FH 13 cells.
Table 2: antibody List 1
| Serial number | Antibody-fluorescence | Antibody amount (μ L/50 μ L) | Incubation time |
| 1 | |
0 | 0 minute |
| 2 | 7-AAD | 0.5 | 5 minutes |
| 3 | CD45RA-AF488 | 0.5 | 30 |
| 4 | CD3-AF532 | 0.25 | 30 minutes |
| 5 | CXCR5-APC | 1 | 30 minutes |
| 6 | CD19-Super Bright 436 | 0.5 | 30 |
| 7 | TCRγδ-BV480 | 0.5 | 30 minutes |
| 8 | CD8a-BV570 | 0.25 | 30 minutes |
| 9 | CD4-BV750 | 0.25 | 30 minutes |
Table 3: antibody list 2
| Serial number | Antibody-fluorescence | Antibody amount (μ L/50 μ L) | Incubation time |
| 10 | IL-4-PE/Dazzle 594 | 2 | 45 minutes |
| 11 | IL-13-PE | 8 | 45 minutes |
Step two, T FH 13 cell detection.
S2-1: steps S1-1 and S1-2 are repeated.
S2-2: corresponding number of EP tubes were taken, numbered, the cells incubated in the 96-well plate were transferred to the EP tubes, 1mL FACS was added to each tube, mixed well, centrifuged (4 ℃, 450g, 5 min), the supernatant was discarded, the cell pellet was resuspended in 100. mu.L FACS, 1. mu.L 7AAD was added to each tube, incubated for 5 min in the dark, 1mL FACS was added to each tube, washed, centrifuged (4 ℃, 450g, 5 min), and the supernatant was discarded.
Add 46.25. mu.L FACS per tube and mix well, add fluorescent antibody Nos. 3-9 according to Table 2. In this example, each reaction volume included 46.25. mu.L of LFACS, 0.5. mu.L of CD45RA-AF488, 0.25. mu.L of CD3-AF532, 1. mu.L of CXCR5-APC, 0.5. mu.L of CD19-Super Bright 436, 0.5. mu.L of TCR γ δ -BV480, 0.25. mu.L of CD8a-BV570, 0.25. mu.L of CD4-BV750, and a total reaction volume of 50. mu.L.
After incubation at 4 ℃ for 30 minutes in the dark, 1mL of FACS was added, washed, centrifuged (4 ℃, 450g, 5 minutes), and the supernatant was discarded.
After the cell pellet was resuspended in 250. mu.L of the fixation/permeation solution and incubated at 4 ℃ for 20 minutes, 1mL of 1 XBD Perm/Wash buffer was added to each tube, centrifuged (4 ℃, 450g, 5 minutes), and the supernatant was discarded.
The cell pellet from each tube was resuspended in 40. mu.L of 1 XBD Perm/Wash buffer and the fluorescent antibody was added as per Table 3. In this example, each reaction volume included 40. mu.L of 1 XBD Perm/Wash buffer, 2. mu.L of IL-4-PE/Dazle 594, and 8. mu.L of IL-13-PE. Incubate at 4 ℃ for 45 minutes in the dark.
1mL of 1 XBD Perm/Wash buffer was added to each tube, washed, centrifuged (4 ℃, 450g, 5 minutes), and the supernatant was discarded.
The cell pellet was finally resuspended in 200. mu.L FACS.
S2-3: detecting on a 38-channel spectral flow cytometer Cytek NL-CLC-3000, collecting 150000-300000 PBMCs, and detecting the number of target cells.
In the present invention, based on T FH 13 the kit for preparing the detection reagent of the cells comprises: antibody composition, fixed rupture membrane and fixed rupture membrane buffer solution, BFA, PMA and ionomycin; the antibody composition comprises: one or two or more of CD45RA-AF488, CD3-AF532, CXCR5-APC, CD19-Super Bright 436, TCR gamma delta-BV 480, CD8a-BV570, CD4-BV750, IL-4-PE-Dazle 594 and IL-13-PE. The kit prepared based on the method comprises the kit for detecting T in PBMCs (peripheral blood mononuclear cells) FH 13 cell substances can provide important basis for the auxiliary diagnosis, treatment effect evaluation, prognosis evaluation and the like of the allergic asthma in children.
Also, other forms of kits for diagnosing allergic asthma, which are prepared based on the prior art using follicular helper T lymphocytes 13 as a diagnostic marker, are also within the scope of the present invention.
s 1: adding PMA and ionomycin in the kit into the culture medium to make the final stimulation concentration of the cells be PMA150ng/ml and Ion
1ug/ml；
s 2: using the surface antibody in the kit, according to T FH 13 the specific steps of the cell detection method, adding the cells into the cell suspension in turn according to the dosage;
s 3: two types of reagents were used in this step:
firstly, fixing and breaking the cell by using a fixing/membrane breaking solution, so that the cytokine antibody can smoothly enter the cell;
then press T FH 13 the specific steps of the method for detecting the cells, sequentially adding the intracellular antibodies into the cell suspension according to the dosage, and finally analyzing by using a flow cytometer to obtain the final detection result.
The specific application process of the kit provided by the invention is as follows.
This example collects basic information and clinical data from 30 Allergic asthmatic children (Allergic athma) and 16 physical examination children (health controls) as controls, including: sex, age, serum total IgE levels, percentage eosinophils (EOS%).
The basic information and clinical examination indices of asthmatic and healthy volunteers are shown in Table 4. The gender and age of the healthy control group and the allergic asthma group were not statistically different (P > 0.05). The serum total IgE and EOS% level of asthma patients is obviously higher than that of healthy controls (P is less than 0.05).
Table 4: examples of basic information and clinical examination indices for asthmatic and healthy volunteers
In the test process, after peripheral blood is extracted, PBMCs need to be extracted and cryopreserved within 4-6 hours, and the kit in the method is applied according to the T realized based on the detection reagent FH 13 method for detecting cells ". The number of cytokine-secreting cells was increased by culturing overnight prior to the stimulating step. Specifically, as shown in table 5, it was found by the inventors that the cytokine secretion effect was most significant when the stimulation concentration was PMA150ng/ml, Ion 1ug/ml and the stimulation time was 8 hours, and the subsequent experiments in this example were performed based on this.
Table 5: stimulation concentration preliminary Experimental example
| 4h | 6h | 8h | 10h | |
| unstimulate | ＜1.5 | ＜1.5 | 17.04 | 18.8 |
| PMA 50ng/ml，Ion 1ug/ml | 30.32 | 46.52 | 65.6 | 65.12 |
| PMA 75ng/ml，Ion 1ug/ml | 27.69 | 42.76 | 69.96 | 61.04 |
| PMA 100ng/ml，Ion 1ug/ml | 29.28 | 47.4 | 65.96 | 58.28 |
| PMA 150ng/ml，Ion 1ug/ml | 28.68 | 50.76 | 88.2 | 78.92 |
As shown in FIG. 1 of the specification, peripheral blood T FH 13 cell gating strategy. First, the lymphocyte subpopulation is circled according to FSC and SSC, then the adhesion signals are removed according to FSC-A and FSC-H to obtain single cells, and then the living cells are circled according to the living cell dye 7-AAD. B cells were removed by gating with CD3 and CD19 to obtain T cells. And setting a gate by CD3 and TCR gamma delta, and excluding gamma delta T cells to obtain alpha beta T cells. The CD4 and CD8 are used for setting a door to exclude CD8 + T cells, obtaining CD4 + T cells. Setting gates on CD45RA and CXCR5 to obtain T FH A cell. Gating IL-4 and IL-13 to obtain T FH 13 cells. The kit and the detection method based on the invention can accurately obtain T FH 13 cells, thereby providing objective and accurate reference data for the auxiliary diagnosis of the allergic asthma.
In this example, T in peripheral blood was detected using a full-spectrum flow cytometer FH 13 cell count T FH Proportion of cells, T in allergic asthmatic children FH 13 ratio significantly higher than healthy children, T FH 13 cell count T FH The proportion of cells in the children with allergic asthma is obviously increased by [ (0.91 +/-0.43)% vs (0.41 +/-0.16)%, and P is less than 0.0001%]. Referring to fig. 2, Health represents healthy persons, astrma represents allergic Asthma, and P < 0.0001. As can be seen from FIG. 2, T FH The ratio of 13 is of great significance in the auxiliary diagnosis of allergic asthma in children.
By applying the kit and the detection method of the invention, T in peripheral blood of children suffering from allergic asthma is detected FH The proportion of 13 cells is combined with the clinical performance of a patient, so that objective data is provided for the diagnosis of the allergic asthma of children, the diagnosis accuracy of the allergic asthma in children is improved, and the harm to children patients and family members caused by untimely diagnosis and treatment and excessive diagnosis is reduced. And the operation process is simple and easy to implement, can be operated in batches, has objective detection results, and is very suitable for clinical popularization.
Claims (6)
1. The application of the follicular helper T lymphocytes 13 as a diagnostic marker in the preparation of a kit for the treatment of childhood allergic asthma is characterized in that the proportion of the follicular helper T lymphocytes 13 in an extracorporeal blood sample in the follicular helper T lymphocytes is detected to be used as the characterization of childhood allergic asthma.
2. Use of follicular helper T lymphocytes 13 as defined in claim 1 as diagnostic marker for the preparation of a kit for the treatment of allergic asthma in children, characterized in that: the blood sample is peripheral blood.
3. The use of the follicular helper T lymphocyte 13 as a diagnostic marker for the manufacture of a kit for childhood allergic asthma according to claim 1, wherein: the detection of the level of the follicular helper T lymphocytes 13 is realized by applying a detection reagent of the follicular helper T lymphocytes 13 in the preparation of a kit for detecting the allergic asthma in children.
4. Use of follicular helper T lymphocytes 13 as a diagnostic marker in the manufacture of a kit for childhood allergic asthma according to claim 3, characterized in that: the detection reagent comprises a fluorescent antibody, and the fluorescent antibody is provided with a detectable label.
5. The use of the follicular helper T lymphocyte 13 as a diagnostic marker for the manufacture of a kit for childhood allergic asthma according to claim 3, wherein: the kit comprises: antibody composition, fixed membrane rupture liquid, fixed membrane rupture buffer solution, BFA, PMA and ionomycin.
6. Use of follicular helper T lymphocytes 13 as a diagnostic marker in the manufacture of a kit for childhood allergic asthma according to claim 5, characterized in that: the antibody composition comprises: one or two or more of CD45RA-AF488, CD3-AF532, CXCR5-APC, CD19-Super Bright 436, TCR gamma delta-BV 480, CD8a-BV570, CD4-BV750, IL-4-PE-Dazle 594 and IL-13-PE.
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