CN109187941B - Application of CD4+ CD70+ T cell subset in preparation of kit for auxiliary diagnosis of very severe aplastic anemia - Google Patents
Application of CD4+ CD70+ T cell subset in preparation of kit for auxiliary diagnosis of very severe aplastic anemia Download PDFInfo
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Abstract
The invention discloses application of a CD4+ CD70+ T cell subgroup in preparation of a kit for auxiliary diagnosis of very severe aplastic anemia. The inventor of the invention firstly finds that the proportion of CD4+ CD70+ T cells in the peripheral blood of AA patients is obviously reduced in the AA patients, and particularly is most obvious in VSAA. The kit can be used as one of laboratory immunity related detection indexes for assisting in diagnosing VSAA patients, and meanwhile, provides important reference data for clinically selecting treatment strategies for VSAA patients in the future.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of a CD4+ CD70+ T cell subgroup in preparation of a kit for auxiliary diagnosis of very severe aplastic anemia.
Background
Aplastic Anemia (AA) is a syndrome of myeloid hematopoietic failure characterized primarily by decreased myelohematopoietic cell proliferation and pancytopenia. Anemia, bleeding, and infection are the major clinical manifestations of AA. The annual incidence rate of AA in China is higher than that in Europe and America. The pathogenesis of AA is not clear at present, and it is considered that one of the causes of impairment of bone marrow hematopoietic function due to T cell dysfunction in the pathogenesis of AA. Thus, AA is also a T cell mediated autoimmune disease. In the research of T cell immune abnormality of AA patients, abnormal T cell activation plays an important role in the pathogenesis of AA. We have previously found that mRNA is abnormally expressed in AA patients in comparison with signal molecules involved in T cell activation, such as CD3 ζ, CD28 and CTLA-4.
CD70 is a ligand of the T cell activation costimulatory signaling molecule CD27, and belongs to a member of the tumor necrosis factor-tumor necrosis factor receptor superfamily. The human CD70 gene maps to chromosome 19p13, and the mRNA has 913 nucleotides in length. Previous studies have shown that the expression of CD27, a ligand for CD27, is restricted to germinal center B cells and a small fraction of T cells, and regulates T cell proliferation and differentiation by interacting with CD 27. Recent studies have shown that CD70 is also expressed on mature dendritic cells and antigen-induced T and B cells. In other words, activated T cells express CD 70. CD70 expressed on T cells is found to be elevated in some patients with autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, but the mechanism of action differs in different diseases. Recently, CD70 expressed on activated T cells was found to play a negative role in the activation and function of T cells. This suggests that CD70 on activated T cells functions differently under different pathological conditions. The domestic scholars find that the peripheral blood CD8 of patients with severe aplastic anemia (a part of patients with severe aplastic anemia who relapse after being treated by immunosuppressant)+The proportion of CD70 in T cells is increased, but the peripheral blood CD4 of AA patients is not found at present+A related study of CD70 status in T cells was reported.
The current clinical diagnosis standard of AA mainly depends on the routine examination and multiple examinations of peripheral blood of patientsBone marrow aspiration smear analysis, bone marrow biopsy, and other diseases excluding congenital and pancytopenia and myelodysplasia. The evaluation of AA in different disease degrees is mainly based on the degree of myeloproliferation and blood routine examination, and AA is classified into Non-Severe aplastic anemia (NSAA), Severe Aplastic Anemia (SAA) and Very Severe Aplastic Anemia (VSAA), wherein patients with SAA and VSAA are in an acute disease, and if they cannot be effectively treated, the death rate of the patients is extremely high. Determination of AA severity classified by Camitta criteria, SAA patients were evaluated if their absolute neutrophil value was below 0.2X 109VSAA can be diagnosed by/L. With the intensive study on the T cell immune mechanism of AA, the existence of T cell immune imbalance in AA patients is widely accepted by the researchers. Some immune indicators, such as soluble CD30, which are associated with T cell activation, were found to be significantly correlated with the blood routine used to diagnose AA. In addition, there is a difference in signal molecule expression from T cell activation in AA patients with varying degrees of disease. This shows that the detection of T cell immunity, especially T cell activation related immunity index of AA patients not only studies the mechanism of T cell immunity abnormality of AA patients from research field, but also may be used as clinical index for auxiliary judgment of disease degree of AA patients. Therefore, some of the tests related to AA immune related indexes are gradually introduced into the necessary laboratory test items for diagnosing aplastic anemia. The latest edition of 'the diagnosis of aplastic anemia and the consensus of Chinese experts on treatment' particularly emphasizes the detection of the immune indexes of AA patients, such as the detection of indexes such as regulatory T cells, Th1/Th2 and the like, so as to assist clinicians in the diagnosis and differential diagnosis of AA. However, no immune-related indicators of characteristic changes in VSAA patients have been reported. Therefore, the method can comprehensively understand the difference of T cell immune characteristics of AA patients, particularly VSAA patients and SAA patients, and has good application prospect in the aspect of auxiliary diagnosis of VSAA by clinicians in the future.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides application of a CD4+ CD70+ T cell subgroup in preparation of a kit for auxiliary diagnosis of very severe aplastic anemia.
The invention also aims to provide a kit for auxiliary diagnosis of very severe aplastic anemia.
Still another objective of the present invention is to provide a method for detecting CD4+ CD70+ T cell subset and the application thereof.
The purpose of the invention is realized by the following technical scheme: the application of CD4+ CD70+ T cell subset in preparing a kit for the auxiliary diagnosis of very severe aplastic anemia is based on the first discovery by the inventor of the invention that the proportion of CD4+ CD70+ T cell subset in peripheral blood of AA patients (the AA patients in the specification comprise SAA and VSAA patients) is remarkably reduced in the AA patients, especially is most remarkable in the VSAA patients.
When the CD4+ CD70+ T cell subset is in CD4+The median proportion of T cells was 1.9%, and the likelihood of diagnosis of VSAA was high.
A kit for aiding in the diagnosis of extreme aplastic anemia comprising components useful for detecting a subpopulation of CD4+ CD70+ T cells, such as the following different fluorescently labeled monoclonal antibodies: anti-CD 3 antibodies, anti-CD 4 antibodies, and anti-CD 70 antibodies.
The fluorescent label of the anti-CD 3 antibody is preferably FITC.
The fluorescent label of the anti-CD 4 antibody is preferably APC-H7.
The fluorescent label of the anti-CD 70 antibody is preferably PE.
The kit also comprises erythrocyte lysate, cell staining buffer solution and Phosphate Buffer Solution (PBS) for lysing peripheral red blood cells.
A method for detecting CD4+ CD70+ T cell subsets for non-disease diagnosis or treatment purposes can be used for detection by using the kit for the auxiliary diagnosis of the extreme aplastic anemia, and comprises the following steps:
(1) processing a peripheral blood sample to be detected to form a single cell suspension;
(2) adding monoclonal antibodies marked with different fluorescence into the single cell suspension obtained in the step (1): the anti-CD 3 antibody, the anti-CD 4 antibody and the anti-CD 70 antibody are mixed evenly and incubated in dark;
(3) after washing the cells, adding PBS to resuspend the cells, detecting on a flow cytometer to obtain data of the CD4+ CD70+ T cell subset after fluorescent labeling.
The specific steps for processing the peripheral blood sample to be detected in the step (1) are as follows: and (3) performing whole blood erythrocyte lysis treatment on peripheral blood to be detected according to a conventional method, centrifuging to remove supernatant, and resuspending by using cell staining buffer solution to obtain single cell suspension.
The volume of the peripheral blood sample in the step (1) is preferably 200. mu.L.
The addition amount of the anti-CD 3 antibody in the step (2) is preferably calculated by 3-6 μ L of anti-CD 3 antibody (with the concentration of 200 μ g/ml) per 100 μ L of single cell suspension; further preferably, the amount of the anti-CD 3 antibody is 5. mu.L per 100. mu.L of the single cell suspension.
The fluorescent label of the anti-CD 3 antibody is preferably FITC.
The addition amount of the anti-CD 4 antibody in the step (2) is preferably calculated by 3-6 μ L of anti-CD 4 antibody per 100 μ L of single cell suspension; further preferably, the amount of the anti-CD 4 antibody is 5. mu.L per 100. mu.L of the single cell suspension.
The fluorescent label of the anti-CD 4 antibody is preferably APC-H7.
The addition amount of the anti-CD 70 antibody in the step (2) is preferably calculated by 5-25 μ L of anti-CD 70 antibody per 100 μ L of single cell suspension; more preferably, the amount of the anti-CD 70 antibody is 20. mu.L per 100. mu.L of the single cell suspension.
The fluorescent label of the anti-CD 70 antibody is preferably PE.
The specific operation of the lucifugal incubation in the step (2) is the lucifugal incubation at room temperature for 15-30 minutes; preferably, the incubation is carried out for 20 minutes at room temperature in the absence of light.
The room temperature is 5-35 ℃; preferably 20-30 ℃; more preferably 24 to 26 ℃.
The washing in step (3) is carried out using a cell staining buffer.
The PBS in the step (3) is preferably PBS with the pH value of 7.2-7.4 and the concentration of 0.01-0.1M; more preferably, PBS at a pH of 7.4 and a concentration of 0.01M.
The detection method of the CD4+ CD70+ T cell subset for non-disease diagnosis or treatment is used for researching CD4+CD70+The mechanism of T cell immune abnormality of T cell subgroup in patients with severe and extreme aplastic anemia can also be applied to the auxiliary diagnosis of patients with extreme aplastic anemia.
The application specifically comprises the following steps: the statistical analysis of the expression ratio of the CD4+ CD70+ T cell subset shows that the VSAA patient has high possibility when the median expression ratio of the CD4+ CD70+ T cell subset is not higher than 1.9%, and the method can be further used for analyzing the cause of the disturbance of the T cell immune system.
The statistical analysis is preferably a rank sum test analysis.
The current diagnostic criteria for AA are mainly based on the patient's peripheral blood routine, bone marrow smear, bone marrow biopsy and the exclusion of other pancytopenia and myelodysplastic diseases, while the judgment of different degrees of disease is mainly based on bone marrow biopsy and blood routine. With the intensive research on the T cell immune mechanism of AA, some immunity-related index tests related to AA are gradually introduced into necessary laboratory test items for diagnosing AA. However, no immune-related markers have been found for the evaluation of different disease levels of AA. The inventor of the invention analyzes the blank of the research on the proportion of CD70 which plays a role in regulating and controlling the T cell activation in the peripheral blood T cells and different cell subsets of AA patients for the first time so as to provide more comprehensive characteristics of T cell immune abnormality of the AA patients and detection and prediction indexes related to the disease degree. Therefore, the research combines the clinical data of AA patients, innovatively and more thoroughly researches the AA patients and the T cell activation regulator-CD 70 in the peripheral blood CD4 of the AA patients by using flow cytometry+The proportion characteristics of T cells and the relation between the T cells and different disease degrees not only provide the peripheral blood CD4 of AA patients for the first time internationally+CD70+Scientific research data of the relation between the T cell proportion and the disease also provides theoretical support for clinically applying the indexes to assist in judging VSAA.
Compared with the prior art, the invention has the following advantages and effects:
1. the inventor firstly found that the proportion of CD4+ CD70+ T cells in the peripheral blood of AA patients is obviously reduced in the AA patients, and particularly is most obvious in VSAA. The kit can be used as one of laboratory immunity related detection indexes for assisting in diagnosing VSAA patients, and meanwhile, provides important reference data for clinically selecting treatment strategies for VSAA patients in the future.
2. The invention provides a detection method of a CD4+ CD70+ T cell subset, the phenotype of the T cell subset can be quantitatively counted by the detection method, and the method has a very wide application prospect in the aspect of judging auxiliary diagnosis of VSAA patients.
Drawings
FIG. 1 is the peripheral blood CD4 of healthy control group and AA patients+CD70+A flow cytometry result analysis graph of the ratio of the T cell functional subpopulations; wherein panel (A) is a healthy control group and panel (B) is an AA patient.
FIG. 2 is the CD4 peripheral blood of the healthy control group and AA patients+CD70+Analysis chart of proportion of cell functional subgroups; wherein represents P<0.01。
FIG. 3 is the peripheral blood CD4 of AA patients in both VSAA and SAA disease of healthy control group+CD70+A result graph of analysis of the proportion of the cell functional subgroups; wherein represents P<0.01。
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the embodiments of the present invention are not limited thereto.
The reagent information used in the examples is specifically as follows:
FITC-labeled mouse anti-human CD3 (clone number HIT3a, purchased from Biolegend);
APC-H7-labeled mouse anti-human CD4 (clone number: RPA-T4, purchased from BD Pharmingen);
PE-labeled mouse anti-human CD70 (clone number: Ki-24, purchased from BD Pharmingen);
cell staining buffer (cell staining buffer, available from Biolegend);
erythrocyte lysate (Red cell lysis buffer, from BD Pharmingen).
Example 1
(1) Blood was collected with patient informed consent and all specimens were collected from early morning fasting vein EDTA anticoagulation. A total of 23 weekly AA samples were collected, 11 VSAA and 12 SAA. At the same time, 31 samples of healthy persons were collected, and the study protocol was approved by the ethical committee of this unit. Clinical data was also collected from AA patients (as shown in table 1).
TABLE 1
(2) The collected peripheral blood of healthy persons and AA patients was subjected to red blood lysis using a red blood cell lysate. 2mL of erythrocyte lysate is prepared per 200 mu L of peripheral blood, the erythrocyte lysate is lysed for 10min at room temperature, a pipette is used for gently blowing and uniformly mixing once during the lysis, then the erythrocyte lysate is centrifuged for 5min at the rotating speed of 200g, the supernatant is discarded, 1 XPBS (phosphate buffer solution) is added to 2mL, the erythrocyte lysate is centrifugally washed at the rotating speed of 300g, the supernatant is discarded, and 100 mu L of cell staining buffer solution is added to form single cell suspension for resuspension.
(3) Flow cytometry for CD4 detection+CD70+The proportion of cell subsets.
3.1 for each sample, 2 flow tubes are prepared, wherein 1 tube is set as a tube to be detected, 1 tube is set as a same tube, and each tube is the single-cell suspension prepared in the step (2).
3.2 adding corresponding surface analysis fluorescent antibodies of 5 microlitres respectively into the tube to be detected and the homotypic tube, wherein the antibodies comprise mouse anti-human FITC-CD3 and APC-H7-CD 4; adding PE-CD 7020 mu L into the tube to be detected, mixing the mixture evenly and lightly, and incubating the mixture for 20min at room temperature in a dark place.
3.3 Add cell staining buffer (cell staining buffer) to wash cells for 5min at 300g rpm.
3.4 centrifugation was performed, the supernatant was removed, the cells were resuspended in 500. mu.L of PBS, the obtained data were analyzed by a flow analyzer (BD Verse, USA), the obtained raw data were analyzed by FlowJo Software, the analysis data were summarized, the median of each data was calculated by SPSS13.0, and the P value was calculated.
(4) CD4 in peripheral blood of AA patients+CD70+The T cell subpopulation ratio was significantly lower than the healthy controls (fig. 1 and 2); combining the clinical data of AA patients, further dividing the AA patients into SAA and VSAA groups according to different disease degrees, and analyzing each group of CD4+CD70+T cell ratio (FIG. 3), the results suggest that CD4 is present in peripheral blood of VSAA patients+CD70+The T cell subset proportion was significantly lower than that of the healthy controls, with a median expression of CD4+ CD70+ T cell subset of 1.9%. Despite the CD4 in the peripheral blood of SAA patients+CD70+The T cell subset proportion (median 3.05%) was lower than that of healthy humans, but not significantly different from healthy humans and slightly higher than VSAA patients. The above experimental results show that the peripheral blood CD4 of AA patients is detected+CD70+The proportion of T cell subsets is of great importance in the aided diagnosis of VSAA patients.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (5)
1. The application of the composition in preparing a kit for auxiliary diagnosis of the extremely severe aplastic anemia is characterized in that: the composition is used for detecting CD4 by marking different fluorescence+CD70+Monoclonal antibodies of T cell subsets: anti-CD 3, anti-CD 4, and anti-CD 70 antibodies;
the use method of the kit comprises the following steps:
(1) processing a peripheral blood sample to be detected to form a single cell suspension;
(2) adding monoclonal antibodies marked with different fluorescence into the single cell suspension obtained in the step (1): the anti-CD 3 antibody, the anti-CD 4 antibody and the anti-CD 70 antibody are mixed evenly and incubated in dark;
(3) washing the cells, adding PBS to resuspend the cells, detecting on a flow cytometer to obtain the CD4 after fluorescent labeling+CD70+Data for T cell subsets;
CD4 in peripheral blood of patient with severe aplastic anemia+CD70+The expression ratio of the T cell subset is obviously lower than that of healthy people.
2. Use according to claim 1, characterized in that:
the fluorescence label of the anti-CD 3 antibody is FITC;
the fluorescence label of the anti-CD 4 antibody is APC-H7;
the fluorescent label of the anti-CD 70 antibody is PE.
3. Use according to claim 1, characterized in that: the kit also comprises erythrocyte lysate, cell staining buffer solution and phosphate buffer solution which are used for lysing peripheral red blood cells.
4. Use according to claim 1, characterized in that:
the adding amount of the anti-CD 3 antibody in the step (2) is calculated according to the proportion of 3-6 mu L of anti-CD 3 antibody per 100 mu L of single cell suspension;
the adding amount of the anti-CD 4 antibody in the step (2) is calculated according to the proportion of 3-6 mu L of anti-CD 4 antibody per 100 mu L of single cell suspension;
the addition amount of the anti-CD 70 antibody in step (2) is calculated by 5-25. mu.L of anti-CD 70 antibody per 100. mu.L of single cell suspension.
5. Use according to claim 1, characterized in that:
the specific steps for processing the peripheral blood sample to be detected in the step (1) are as follows: performing whole blood erythrocyte lysis treatment on peripheral blood to be detected according to a conventional method, centrifuging to remove supernatant, and carrying out heavy suspension by using a cell staining buffer solution to obtain a single cell suspension;
the specific operation of the lucifugal incubation in the step (2) is the lucifugal incubation at room temperature for 15-30 minutes;
the washing in step (3) is carried out using a cell staining buffer.
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| CN113945722B (en) * | 2021-10-15 | 2022-08-30 | 广东医科大学附属医院 | Aplastic anemia diagnosis marker and application thereof in preparation of kit |
| CN114894698B (en) * | 2022-04-15 | 2023-02-03 | 广东省第二人民医院（广东省卫生应急医院） | Application of MDSCs in diagnosis and/or typing of aplastic anemia |
| CN114739890B (en) * | 2022-04-15 | 2022-12-13 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Application of human MDSCs in CAA and AAA identification and identification kit |
| CN115877017B (en) * | 2023-03-01 | 2023-05-05 | 首都医科大学附属北京地坛医院 | Products and systems for predicting immune reconstitution in HIV/AIDS patients |
| CN116622632A (en) * | 2023-05-29 | 2023-08-22 | 广州市妇女儿童医疗中心 | Application of CD4TEMRA in diagnosis of autism co-morbid epilepsy |
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