CN117471100A - Application of Th40 cell subpopulations in the preparation of kits for auxiliary diagnosis and assessment of systemic lupus erythematosus disease activity - Google Patents

Abstract

The invention discloses the application of detecting Th40 cell subpopulations in preparing kits for auxiliary diagnosis and assessment of SLE disease activity. The present invention is based on the inventor's first discovery that the expression of Th40 cell subpopulations is significantly increased in SLE patients with active disease, and the higher the disease activity, the higher the proportion of Th40 cell subpopulations. Therefore, Th40 cell subpopulation can be used as one of the laboratory immune-related detection indicators to assist in the diagnosis of SLE. At the same time, it also provides an important reference for evaluating disease activity in SLE patients.

Description

Th40 cell subpopulations are useful in preparing auxiliary diagnosis and evaluating systemic lupus erythematosus disease activity. Application in Activation Kit

Technical field

The invention belongs to the field of biomedicine, and particularly relates to the application of Th40 cell subpopulations in the preparation of kits for auxiliary diagnosis and evaluation of systemic lupus erythematosus disease activity.

Background technique

Systemic lupus erythematosus (SLE) is an autoimmune disease resulting in the formation of pathogenic autoantibodies. Dysfunction of lymphocyte subsets plays an important role in the pathogenesis of SLE, among which CD8+ T cells and NK cells Dysfunctional, losing the ability to inhibit CD4 ⁺ T cells, allowing them to continue to stimulate B cell activation to produce autoantibodies, resulting in the persistence of autoimmune reactions. Currently, clinical indicators for evaluating SLE disease activity and efficacy are relatively single, and there are no immune markers that can effectively predict the onset of SLE.

Currently, the disease severity of SLE is mainly assessed by SLEDAI-2000. However, due to the obvious heterogeneity of SLE and different organ involvement, the SLEDAI-2000 scores are completely different. Currently, there is only one tool for evaluating SLE disease activity. , there is a lack of relatively comprehensive, accurate, and objective laboratory indicators to evaluate disease activity. There is an imbalance of T cell immunity in SLE patients. Research has found some immune indicators related to T cell activation, especially the detection of immune indicators related to T cell activation. It not only explores the mechanism of abnormal T cell immunity in SLE patients from the research field, but may also be used as an auxiliary Clinical indicators for diagnosing SLE and evaluating the severity of SLE patients. At present, most of the research on T cell activation is to explore the mechanism of abnormal T cell immunity in SLE patients. There are no characteristic changes in immune-related indicators in SLE patients, let alone immune indicators of T cell activation that can be used to diagnose and judge disease activity. Therefore, a comprehensive understanding of the similarities and differences in T cell immune changes in SLE patients, especially in patients with severe SLE and mild SLE, has good application prospects in the auxiliary diagnosis of severe SLE by clinicians in the future.

Contents of the invention

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide the application of Th40 cell subpopulations in the preparation of kits for auxiliary diagnosis and assessment of SLE disease activity.

Another object of the present invention is to provide a kit to assist in the diagnosis and assessment of SLE disease activity.

Another object of the present invention is to provide a method for detecting Th40 cell subpopulations.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The application of Th40 cell subpopulations in the preparation of kits for auxiliary diagnosis and assessment of SLE disease activity is based on the inventor of the present invention's first discovery of Th40 (CD4 ⁺ CD40) in the peripheral blood of SLE patients (SLE patients in this specification include mild and severe patients) The proportion of ⁺ T) cell subsets was significantly increased in SLE patients, especially in patients with severe SLE.

The kit includes components, operating procedures and judgment standards that can be used to detect Th40 cell subpopulations;

The components that can be used to detect Th40 cell subpopulations are different fluorescently labeled monoclonal antibodies: anti-CD3 antibodies, anti-CD4 antibodies and anti-CD40 antibodies;

The operating procedures described include the following steps:

1) Process the peripheral blood sample to be tested to form a single cell suspension;

2) Add monoclonal antibodies labeled with different fluorescence: anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody into the single cell suspension obtained in step 1), mix gently and then incubate in the dark;

3) After washing the cells, add PBS to resuspend the cells, and perform detection on a flow cytometer to obtain data on fluorescently labeled Th40 cell subpopulations;

The judgment criteria are as follows: when the median proportion of the Th40 cell subpopulation in CD4 ⁺ T cells is 13.30%, it is a high possibility of SLE disease activity, in which the Th40 cell subpopulation is more likely to be active in CD4 ⁺ T cells. The median proportion of cells is higher than 13.30%, which means severe SLE is more likely.

A kit to assist in the diagnosis and assessment of SLE disease activity includes components that can be used to detect Th40 cell subsets, such as the following different fluorescently labeled monoclonal antibodies: anti-CD3 antibody, anti-CD4 antibody and anti-CD40 antibody.

The fluorescent label of the anti-CD3 antibody is preferably FITC.

The fluorescent label of the anti-CD4 antibody is preferably APC-H7.

The fluorescent label of the anti-CD40 antibody is preferably PE-cy7.

The kit for auxiliary diagnosis and evaluation of SLE disease activity also includes instructions, which record the operating procedures and judgment criteria of the kit.

The operating procedures described include the following steps:

1) Process the peripheral blood sample to be tested to form a single cell suspension;

2) Add monoclonal antibodies labeled with different fluorescence: anti-CD3 antibody, anti-CD4 antibody, and anti-CD40 antibody into the single cell suspension obtained in step 1), mix gently and then incubate in the dark;

3) After washing the cells, add PBS to resuspend the cells, and perform detection on a flow cytometer to obtain data on fluorescently labeled Th40 cell subpopulations.

The specific steps for processing the peripheral blood sample to be tested as described in step 1) are as follows: The peripheral blood sample to be tested is subjected to whole blood red blood cell lysis processing according to conventional methods, centrifuged to remove the supernatant, washed with PBS, and in the precipitate obtained by centrifugation. Add cell staining buffer and resuspend to form a single cell suspension.

The volume of the peripheral blood sample is preferably 200 μL/tube.

The dosage of the cell staining buffer is preferably 50 μL/tube.

The amount of anti-CD3 antibody added in step 2) is preferably 1 μL of anti-CD3 antibody per 50 μL of single cell suspension.

The amount of anti-CD4 antibody added in step 2) is preferably 1 μL of anti-CD4 antibody per 50 μL of single cell suspension.

The amount of anti-CD40 antibody added in step 2) is preferably 1 μL of anti-CD40 antibody per 50 μL of single cell suspension.

The specific operation of the light-protected incubation described in step 2) is to incubate at room temperature in the dark for 15 to 30 minutes; preferably, the method is room temperature and dark incubation for 20 minutes.

The room temperature is 5-35°C; preferably 20-30°C; more preferably 24-26°C.

The washing described in step 3) is performed using cell staining buffer.

The PBS described in step 3) is preferably PBS with a pH value of 7.2-7.4 and a concentration of 0.01-0.1M; more preferably, it is PBS with a pH value of 7.4 and a concentration of 0.01M.

The judgment criteria are as follows: when the median proportion of the Th40 cell subpopulation in CD4 ⁺ T cells is 13.30%, it is more likely to be SLE, in which the Th40 cell subpopulation accounts for 13.30% of CD4 ⁺ T cells. If the median proportion is higher than 13.30%, it is more likely to be heavy SLE.

The kit also includes red blood cell lysis solution, cell staining buffer and phosphate buffer solution (PBS) for lysing peripheral blood red blood cells.

A method for detecting Th40 cell subpopulations that is not intended for disease diagnosis or treatment. The above kit can be used for detection, including the following steps:

(1) Process the peripheral blood sample to be tested to form a single cell suspension;

(2) Add monoclonal antibodies labeled with different fluorescence into the single cell suspension obtained in step (1): anti-CD3 antibody, anti-CD4 antibody, anti-CD40 antibody, mix gently and then incubate in the dark;

(3) After washing the cells, add PBS to resuspend the cells, and perform detection on a flow cytometer to obtain data on fluorescently labeled Th40 cell subpopulations.

The specific steps for processing the peripheral blood sample to be tested as described in step (1) are as follows: The peripheral blood sample to be tested is subjected to whole blood red blood cell lysis processing according to conventional methods, centrifuged to remove the supernatant, washed with PBS, and the precipitate obtained by centrifugation is Add cell staining buffer and resuspend to form a single cell suspension.

The dosage of the peripheral blood sample is preferably 200 μL/tube.

The dosage of the cell staining buffer is preferably 50 μL/tube.

The amount of anti-CD3 antibody added in step (2) is preferably 1 μL of anti-CD3 antibody per 50 μL of single cell suspension.

The fluorescent label of the anti-CD3 antibody is preferably FITC.

The amount of anti-CD4 antibody added in step (2) is preferably 1 μL of anti-CD4 antibody per 50 μL of single cell suspension.

The fluorescent label of the anti-CD4 antibody is preferably APC-H7.

The amount of anti-CD40 antibody added in step (2) is preferably 1 μL of anti-CD40 antibody per 50 μL of single cell suspension.

The fluorescent label of the anti-CD40 antibody is preferably PE-cy7.

The specific operation of the light-proof incubation described in step (2) is room temperature and light-proof incubation for 15 to 30 minutes; preferably, room temperature light-proof incubation is 20 minutes.

The room temperature is 5-35°C; preferably 20-30°C; more preferably 24-26°C.

The washing described in step (3) is performed using cell staining buffer.

The PBS described in step (3) is preferably PBS with a pH value of 7.2-7.4 and a concentration of 0.01-0.1M; more preferably, it is PBS with a pH value of 7.4 and a concentration of 0.01M.

The described detection method of Th40 cell subpopulations, which is not intended for disease diagnosis or treatment, is used to study the mechanism of abnormal T cell immunity of Th40 cell subpopulations in SLE patients.

The application specifically includes the following steps: performing statistical analysis on the expression ratio of the Th40 cell subpopulation. When the median expression ratio of the Th40 cell subpopulation is detected to be higher than 13.30%, it indicates that the patient is more likely to be an SLE patient. Large, allowing further analysis of SLE disease activity.

The statistical analysis is preferably rank sum test analysis.

Compared with the existing technology, the present invention has the following advantages and effects:

(1) The inventor found for the first time that the proportion of Th40 cells in the peripheral blood of SLE patients was significantly increased in SLE patients, especially in severe SLE. It can be used as one of the laboratory immune-related detection indicators to assist in the diagnosis of SLE. At the same time, it also provides an important reference for evaluating disease activity in SLE patients.

(2) The present invention provides a detection method for Th40 cell subpopulations. Through this detection method, the phenotypes of the above T cell subpopulations can be quantitatively counted, and has a very broad application in assisting the diagnosis of SLE and evaluating the disease activity of SLE patients. prospect.

Description of the drawings

Figure 1 is a flow cytometry analysis of the proportion of peripheral blood Th40 cell functional subsets in SLE patients and healthy controls; A is the healthy control group and B is the SLE patient.

Figure 2 is a diagram showing the proportion of peripheral blood Th40 cell functional subpopulations in SLE patients and healthy controls.

Figure 3 is a graph showing the relationship between the proportion of Th40 cell functional subsets in SLE patients and SLEDAI-2000.

Figure 4 is a diagram showing the relationship between the proportion of Th40 cell functional subsets and complement C3 in SLE patients.

Figure 5 is a graph showing changes in the proportion of Th40 cell functional subpopulations in SLE patients before and after treatment.

Detailed ways

The present invention will be described in further detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

The anti-CD3 antibody is a fluorescently labeled antibody CD3-FITC, that is, FITC-labeled mouse anti-human CD3 (clone number: HIT3a, purchased from Biolegend);

The anti-CD4 antibody is the fluorescently labeled antibody CD4-APC-H7, that is, APC-H7 labeled mouse anti-human CD4 (clone number: RPA-T4, purchased from BD Pharmingen);

The anti-CD40 antibody is a fluorescently labeled antibody CD40-PE-Cy7, that is, PE-cy7 labeled mouse anti-human CD40 (clone number: 5C3, purchased from Biolegend);

Cell staining buffer (cell staining buffer, purchased from BD Biosciences);

Red cell lysing buffer (Red cell lysing buffer, purchased from BD Biosciences).

Example 1

Use flow cytometry (FCM) to detect the proportion of Th40 T cells:

(1) Blood was collected under the premise of signing an informed consent form with the patient. All specimens were taken from fasting intravenous EDTA anticoagulation in the early morning. A total of 24 peripheral blood samples from SLE patients were collected, of which 15 patients’ peripheral blood samples were collected one month after treatment during follow-up. At the same time, 24 healthy human samples were collected. This part of the research plan has been approved by the ethics committee of this unit. The clinical data of SLE patients are shown in Table 1.

(2) Use red blood cell lysis solution to lyse the collected peripheral blood from healthy people and SLE patients. Mix 2 mL of red blood cell lysis solution for every 200 μL of peripheral blood, and lyse at room temperature for 10 min. During the lysis period, use a pipette to gently mix once, then centrifuge at 350 g for 5 min. Discard the supernatant, add 1×PBS to 2 mL, and mix at 350 g. Centrifuge and wash, discard the supernatant, add 50 μL of cell staining buffer and resuspend to form a single cell suspension.

(3) Flow cytometry to detect the proportion of Th40 cell subpopulations.

3.1 For each sample, two flow cytometry tubes need to be prepared, one tube is set as the tube to be tested, and the other tube is set as the same type tube. Each tube is the single cell suspension prepared in step (2).

3.2 Add 1 μL of corresponding surface analysis fluorescent antibodies, including anti-CD3 antibody and anti-CD4 antibody, to the tube to be tested and the same type of tube respectively; add 1 μL of anti-CD40 antibody to the tube to be tested, mix gently, and incubate at room temperature in the dark for 20 minutes.

3.3 Add cell staining buffer and wash the cells at 350g for 5 minutes.

3.4 After centrifugation, remove the supernatant, resuspend the cells in 200 μL PBS and use a flow cytometer (FACSVerse) to analyze and obtain the data. The raw data obtained are analyzed with FlowJo Software. After the analyzed data are summarized, SPSS23.0 is used to calculate the median of each group of data. , and calculate the P value.

The flow cytometry results are shown in Figure 1.

Analysis of the correlation between the proportion of Th40 cells and disease activity and efficacy: Collect SLEDAI-2000 scores and laboratory indicators from patients with first-episode SLE, and analyze the correlation between the proportion of Th40 cells and disease activity and efficacy. Among the 24 patients with initial SLE (Table 2), 15 patients were followed up to 1 month after treatment. Through analysis, it was found that: the median proportion of Th40 cells in the 24 patients with initial SLE was 13.30%. As shown in Figure 2, the proportion of Th40 cells in SLE patients was significantly greater than that in healthy controls, and the difference was statistically significant (P=0.000); as shown in Figure 3, patients with higher SLEDAI-2000 scores had higher Th40 cell proportions. The greater the proportion of cells, the SLE disease has a certain positive correlation with the proportion of Th40 cells; complement C3 is an indicator of SLE disease activity, and the decrease of C3 has a certain correlation with the SLE disease activity. As shown in Figure 4, The proportion of Th40 cells is negatively correlated with C3; as shown in Figure 5, the proportion of Th40 cells after treatment was significantly reduced, and the difference in Th40 cells before and after treatment in the same patient was statistically significant (P=0.005). The method provided by the present invention can detect the proportion of Th40 cells, and the proportion of Th40 cells can be used as one of the laboratory immune-related detection indicators to assist in the diagnosis of SLE. At the same time, it also provides important reference materials for evaluating the disease activity of SLE patients.

Table 1 Clinical data of SLE patients and healthy controls

Table 2 Proportion of Th40 cells in patients with initial SLE

serial number gender age Hb(g/L) PLT(10 ⁹ /L) WBC(10 ⁹ /L) ANA(10 ⁹ /L) Th40(%) SLE1 F twenty three 112 80 2.32 1.38 5.66 SLE2 F 25 100.2 237.6 2.89 1.46 1.62 SLE3 F 25 73 60 1.54 1.12 14.2 SLE4 F 38 106 166 2.93 2.07 2.99 SLE5 F 26 59 163 8.22 5.38 76 SLE6 F 56 79 267 3.73 3.09 twenty three SLE7 M twenty two 118 85 2.19 1.6 27.3 SLE8 F 26 115 208 3.01 2 25.2 SLE9 F 33 60.7 243.2 4.28 3.76 34.3 SLE10 F 26 65 146 4.45 3.3 3.93 SLE11 F 27 107.6 266.4 2.7 1.48 6.95 SLE12 F twenty one 76 172 2.41 1.46 38 SLE13 F twenty four 98 310 2.92 1.8 12.4 SLE14 F 54 77.6 100.2 3.82 2.83 4.41 SLE15 F twenty two 67 149 3.71 2.63 34.9 SLE16 F 20 83.1 512.2 18.25 16.62 8.23 SLE17 F twenty three 112.1 73.6 10.34 7.93 33.1 SLE18 M 31 127.2 89.6 3.89 2.72 15.7 SLE19 F 35 131 255 6.51 3.22 39.8 SLE20 F twenty four 81.1 269 6.49 4.98 9.47 SLE21 F twenty two 92 122 3.1 1.87 10.5 SLE22 F 28 91 175 3.27 1.68 6.99 SLE23 M 20 105.4 29.3 0.71 16.21 3.08 SLE24 F 53 79 106 5.4 85.4 27.07

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (10)

1. Application of the Th40 cell subgroup in preparing a kit for assisting in diagnosing and evaluating systemic lupus erythematosus disease activity.
2. 2. The use according to claim 1, characterized in that:

   the kit comprises components, operation rules and judgment standards which can be used for detecting the Th40 cell subgroup;

   the components of the monoclonal antibodies which can be used for detecting the Th40 cell subgroup are different fluorescent markers: anti-CD 3 antibodies, anti-CD 4 antibodies, and anti-CD 40 antibodies;

   the operation procedure comprises the following steps:

   1) Treating a peripheral blood sample to be detected to form single cell suspension;

   2) Adding monoclonal antibodies with different fluorescence labels into the single cell suspension obtained in the step 1): anti-CD 3 antibody, anti-CD 4 antibody and anti-CD 40 antibody, and incubating in dark after light mixing;

   3) Washing cells, adding PBS (phosphate buffer solution) to resuspend the cells, and performing on-machine detection by a flow cytometer to obtain data of the fluorescent-labeled Th40 cell subpopulation;

   the judgment criteria are as follows: when the Th40 cell subgroup is in CD4 ⁺ The median of the proportion of T cells was 13.30% and there was a greater likelihood of SLE, a Th40 cell subsetIn CD4 ⁺ The median of the proportion of T cells is higher than 13.30% and the probability of heavy SLE is high.
3. 3. A kit for aiding in the diagnosis and assessment of SLE disease activity, comprising: including components, protocols, and judgment criteria that can be used to detect Th40 cell subsets;

   the components of the monoclonal antibodies which can be used for detecting the Th40 cell subgroup are different fluorescent markers: anti-CD 3 antibodies, anti-CD 4 antibodies, and anti-CD 40 antibodies;

   the operation procedure comprises the following steps:

   1) Treating a peripheral blood sample to be detected to form single cell suspension;

   2) Adding monoclonal antibodies with different fluorescence labels into the single cell suspension obtained in the step 1): anti-CD 3 antibody, anti-CD 4 antibody and anti-CD 40 antibody, and incubating in dark after light mixing;

   3) Washing cells, adding PBS (phosphate buffer solution) to resuspend the cells, and performing on-machine detection by a flow cytometer to obtain data of the fluorescent-labeled Th40 cell subpopulation;

   the judgment criteria are as follows: when the Th40 cell subgroup is in CD4 ⁺ The median of the proportion of T cells is 13.30%, and the activity of SLE disease is more likely, wherein the Th40 cell subgroup is CD4 ⁺ The median of the proportion of T cells is higher than 13.30% and the probability of heavy SLE is high.
4. 4. A kit for aiding in the diagnosis and assessment of SLE disease activity according to claim 3, wherein:

   the kit also comprises at least one of a red blood cell lysate, a cell staining buffer solution and a phosphate buffer solution for lysing peripheral red blood cells.
5. 5. The kit for aiding in the diagnosis and assessment of SLE disease activity according to claim 3 or 4, wherein:

   the fluorescent label of the anti-CD 3 antibody is FITC;

   the fluorescence label APC-H7 of the anti-CD 4 antibody;

   the fluorescent label PE-cy7 of the anti-CD 40 antibody.
6. 6. The kit for aiding in the diagnosis and assessment of SLE disease activity according to claim 3 or 4, wherein:

   the specific steps for treating the peripheral blood sample to be detected in the step 1) are as follows: and (3) performing whole blood cell lysis treatment on the peripheral blood sample to be tested according to a conventional method, centrifuging to remove supernatant, washing with PBS, and re-suspending with a cell staining buffer solution to obtain single cell suspension.
7. 7. The kit for aiding in the diagnosis and assessment of SLE disease activity according to claim 3 or 4, wherein:

   the volume of the peripheral blood sample is 200 mu L/tube;

   the dosage of the cell staining buffer is 50 mu L/tube;

   the addition amount of the anti-CD 3 antibody in the step 2) is 1 mu L of the anti-CD 3 antibody according to the proportion of each 50 mu L of single cell suspension;

   the addition amount of the anti-CD 4 antibody in the step 2) is 1 mu L of the anti-CD 4 antibody according to the proportion of each 50 mu L of single cell suspension;

   the amount of anti-CD 40 antibody added in step 2) is 1 mu L of anti-CD 40 antibody per 50 mu L of single cell suspension;

   the specific operation of the light-shielding incubation in the step 2) is that the incubation is carried out for 15-30 minutes at room temperature in a light-shielding way;

   the washing in step 3) is performed using a cell staining buffer;

   the PBS in the step 3) is PBS with the pH value of 7.2-7.4 and the concentration of 0.01-0.1M.
8. 8. Use of a method for detecting a subpopulation of Th40 cells for non-disease diagnosis or treatment purposes, characterized in that: the detection method of the Th40 cell subgroup for non-disease diagnosis or treatment is used for researching the mechanism of T cell immune abnormality of the Th40 cell subgroup in SLE patients;

   the method comprises the following steps:

   (1) Treating a peripheral blood sample to be detected to form single cell suspension;

   (2) Adding monoclonal antibodies with different fluorescence labels into the single cell suspension obtained in the step (1): anti-CD 3 antibody, anti-CD 4 antibody and anti-CD 40 antibody, and incubating in dark after light mixing;

   (3) And (3) after washing the cells, adding PBS to resuspend the cells, and performing on-machine detection by a flow cytometer to obtain data of the fluorescent-labeled Th40 cell subpopulation.
9. 9. The use according to claim 8, characterized in that:

   the specific steps for treating the peripheral blood sample to be detected in the step (1) are as follows: performing whole blood erythrocyte lysis treatment on a peripheral blood sample to be tested according to a conventional method, centrifuging to remove supernatant, washing with PBS (phosphate buffer solution), and respectively adding 50 mu L of cell staining buffer solution into precipitates obtained by centrifugation to resuspend to form single cell suspension;

   the volume of the peripheral blood sample is 200 mu L/tube;

   the dosage of the cell staining buffer is 50 mu L/tube;

   the addition amount of the anti-CD 3 antibody in the step (2) is 1 mu L of the anti-CD 3 antibody according to the proportion of each 50 mu L of single cell suspension;

   the addition amount of the anti-CD 4 antibody in the step (2) is 1 mu L of the anti-CD 4 antibody according to the proportion of each 50 mu L of single cell suspension;

   the amount of anti-CD 40 antibody added in step (2) was 1. Mu.L of anti-CD 40 antibody per 50. Mu.L of single cell suspension.
10. 10. The use according to claim 8, characterized in that:

    the specific operation of the light-shielding incubation in the step (2) is that the incubation is carried out for 15-30 minutes at room temperature in a light-shielding way;

    the washing in step (3) is performed using a cell staining buffer;

    the PBS in the step (3) is PBS with the pH value of 7.2-7.4 and the concentration of 0.01-0.1M.