Application of reagent for detecting expression quantity of NKG2D protein on CD3+CD8 + cell surface in aGVHD diagnosis

Abstract

The invention provides application of a reagent for detecting the expression quantity of the NKG2D protein on the CD3 + CD8 + cell surface in aGVHD diagnosis. According to the application disclosed by the invention, the research finds that compared with a Non-aGVHD patient, the expression quantity of the NKG2D protein on the CD3 + CD8 + cell surface in a peripheral blood sample of the aGVHD patient is obviously increased for the first time. The further ROC curve analysis result shows that when the CD3 + CD8 + cell surface NKG2D protein is used for aGVHD diagnosis, the AUC is 0.76, and the diagnosis accuracy is high. Therefore, the reagent for detecting the expression quantity of the NKG2D protein on the CD3 + CD8 + cell surface can be used as an aGVHD diagnostic reagent for auxiliary diagnosis of aGVHD, a result can be obtained on the same day, and the reagent is convenient, rapid and high in generalizability.

Description

Technical Field

[0001] The present invention belongs to the field of molecular diagnosis technology, and in particular relates to the detection of CD3 + CD8 + Application of reagents for measuring cell surface NKG2D protein expression in the diagnosis of aGVHD. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important means to cure blood system diseases. At the same time, acute graft-vs-host disease (aGVHD) is the main cause of transplant-related mortality after transplantation. At present, the diagnosis of aGVHD mainly relies on clinical manifestations and biopsy of target organ tissues. It should be differentiated from other causes of corresponding symptoms, such as infection, adverse drug reactions, pretreatment toxicity, transplantation-associated thrombotic microangiopathy (TA-TMA), etc., and the clinical symptoms are atypical. In recent years, new discoveries have been made in the diagnostic biological markers of aGVHD at the cellular level, protein level, and gene level, which help the early diagnosis and later treatment of aGVHD and the prognosis judgment. The research on miRNA is limited to animal experiments, or the number of patients enrolled is small, and a large number of high-quality clinical studies are needed to confirm.

[0003] The six factors predicting GVHD can be used as potential diagnostic markers and can also indicate whether the treatment is refractory. However, due to the lack of specificity, multiple cytokines are often required for combined evaluation. According to the current guidelines in my country, the efficacy is mainly determined by clinical symptoms of multiple target organs, which is highly subjective and difficult to quantify. Clinically, clinical symptoms, liver damage indicators, LDH, and the six factors predicting GVHD are often combined. When these factors appear, severe tissue damage has often occurred. Failure to accurately and timely intervene will cause irreversible damage to the patient's tissues and increase the economic burden on patients.

[0004] Therefore, there is a need in the art for a specific diagnostic marker for aGVHD that can simply and conveniently realize auxiliary diagnosis of aGVHD, achieve early diagnosis and treatment, and reduce the physical and economic burden on patients. Summary of the invention

[0005] Based on this, the object of the present invention is to provide a method for detecting CD3 + CD8 + Application of reagents for measuring cell surface NKG2D protein expression in the diagnosis of aGVHD.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] In a first aspect of the present invention, there is provided a method for detecting CD3 + CD8 + Application of a reagent for measuring the expression of NKG2D protein on the cell surface in the preparation of a detection kit for acute graft-versus-host disease.

[0008] In some embodiments, the reagents include reagents for flow cytometry detection.

[0009] In some embodiments, the reagent comprises a method for detecting CD3 + CD8 + Flow cytometry was used to detect the expression of NKG2D protein on the cell surface using fluorescent antibodies.

[0010] In some embodiments, the antibodies include anti-human CD3 antibodies, anti-human CD8 antibodies, and anti-human NKG2D antibodies modified with different fluorescent groups.

[0011] In some embodiments, the CD3 + CD8 + Cells were obtained from peripheral blood samples.

[0012] In some embodiments, the CD3 + CD8 + Cells were derived from peripheral blood mononuclear cell samples.

[0013] In a second aspect of the present invention, a diagnostic kit for acute graft-versus-host disease is provided, the kit comprising a method for detecting CD3 + CD8 + A reagent for measuring the expression of NKG2D protein on the cell surface.

[0014] In some embodiments, the kit includes an anti-human CD3 antibody, an anti-human CD8 antibody, and an anti-human NKG2D antibody modified with different fluorescent groups.

[0015] In some embodiments, the biological sample comprises a peripheral blood sample.

[0016] In some embodiments, the kit further comprises a lymphocyte separation solution and a red blood cell lysis solution.

[0017] The present invention provides a method for detecting CD3 + CD8 + Application of reagents for the expression of NKG2D protein on the cell surface in the diagnosis of aGVHD. The present invention has found for the first time that compared with patients without graft-versus-host disease (Non-aGVHD) after transplantation, the CD3 + CD8 + The expression of NKG2D protein on the cell surface was significantly increased. Further ROC curve analysis results showed that CD3+ CD8 + The AUC of NKG2D protein on the cell surface was 0.76 when used for the diagnosis of aGVHD, and the diagnostic accuracy was high. + CD8 + The reagent for measuring the expression of NKG2D protein on the cell surface can be used as an aGVHD diagnostic reagent for auxiliary diagnosis of aGVHD.

[0018] CD3 + CD8 + The cell surface NKG2D protein can be used for auxiliary diagnosis in the evaluation of aGVHD efficacy conveniently and quickly, and the results can be obtained on the same day, which is convenient and fast. The operator does not need to undergo lengthy and professional training, but only needs to master basic flow cytometry analysis technology, which is highly scalable. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 CD3 in peripheral blood biospecimens of patients in Non-aGVHD and aGVHD groups + CD8 + Flow cytometry detection results of NKG2D protein expression on the cell surface.

[0020] Figure 2 It is the ROC curve diagram. DETAILED DESCRIPTION

[0021] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0023] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0024] The following describes the invention in conjunction with specific embodiments.

[0025] Example 1

[0026] Fresh peripheral blood samples were collected from patients with confirmed acute graft-versus-host disease (aGVHD) and patients without graft-versus-host disease (Non-aGVHD) after transplantation, and CD3 + CD8 + The expression of NKG2D protein on the cell surface (mean fluorescence intensity).

[0027] 1. Experimental Methods

[0028] 1. Sample source

[0029] (1) aGVHD group

[0030] Inclusion criteria:

[0031] A. Received hematopoietic stem cell transplantation (HSCT) from any donor source (including HLA-matched related and unrelated, monoidentical and umbilical cord donors) and received any conditioning regimen;

[0032] B. Mainly based on clinical symptoms, bilirubin, transaminase and cytomegalovirus tests, exclude other causes of intestinal diarrhea and identify diseases that cause hyperbilirubinemia. If necessary, evaluate the patient and perform a pathological biopsy. According to the IBMTR grading system, the clinical diagnosis is II to IV acute GVHD;

[0033] C. Informed and written consent signed by the subject or the subject's legally acceptable representative.

[0034] (2) Non-aGVHD group

[0035] A. Inclusion criteria: No systemic disease, including:

[0036] Uncontrolled hypertension, unstable angina, angina that started within the last 3 months, congestive heart failure (≥New York Heart Association [NYHA] class I), myocardial infarction within the past six months, severe arrhythmia requiring medication, and no liver, kidney, or metabolic disease;

[0037] B. No infection or mental illness

[0038] C. Informed and written consent signed by the subject or the subject's legally acceptable representative

[0039] Exclusion criteria: All enrolled patients must be excluded from severe systemic diseases or mental illnesses.

[0040] According to the above criteria, peripheral blood samples were collected from 20 patients with aGVHD and 20 patients with Non-aGVHD. There was no statistical difference in age and gender between the two groups.

[0041] 2. Peripheral Blood Mononuclear Cell (PBMC) Isolation

[0042] (1) Add PBS solution 3 times the volume of the blood to the collected peripheral blood sample and mix well to obtain a diluted blood sample;

[0043] (2) Add an equal volume of lymphocyte separation solution to the diluted blood sample into a centrifuge tube, then use a pipette to slowly add the diluted blood sample to the upper layer of the lymphocyte separation solution along the wall of the centrifuge tube, and centrifuge at 18°C ​​and 600g (5 for ascending, 0 for descending) for 25 min;

[0044] (3) After centrifugation, a white film layer can be seen. Carefully aspirate the middle white film layer and place it in a clean centrifuge tube. Add 10 ml of pre-cooled PBS solution and mix well. Centrifuge at room temperature at 2000 rpm / min for 5 min, discard the supernatant, and collect the cell pellet.

[0045] (4) Add 3 ml of red blood cell lysis buffer (ACK) to the cell pellet, lyse at room temperature for 5 min, then add 10 ml of pre-cooled PBS to terminate the lysis; then centrifuge at room temperature at 2000 rpm / min for 5 min, discard the supernatant, and collect the cell pellet;

[0046] (5) Resuspend the cell pellet in PBS and count the cells for later use.

[0047] 3. Flow cytometry

[0048] (1) Take 1 million PBMC single cell suspension from each sample and add it to the flow cytometry tube, then add 5 ml PBS and mix well, centrifuge at 2000 rpm / min for 5 min, discard the supernatant, and collect the cell pellet;

[0049] (2) Taking flow cytometry antibodies for analyzing human activated T cells: human anti-CD3-buv650 antibody, human anti-CD8-PE-Cy5 antibody, and human anti-NKG2D-Percp5.5 antibody, diluting the antibodies with PBS buffer at a ratio of 1:50 to obtain an antibody dilution solution (the above three antibodies are added to the same centrifuge tube for dilution at the same time to obtain an antibody dilution solution);

[0050] (3) Take 100 μl of antibody dilution solution and add it to the flow tube corresponding to each sample. Place the flow tube on a vortex oscillator to oscillate thoroughly, and then stain in a 4°C refrigerator away from light for 30 min.

[0051] (4) Add 4 ml of pre-cooled PBS buffer to the flow tube, then centrifuge at 2000 rpm / min for 5 min and discard the supernatant; add 300 μl of pre-cooled PBS to resuspend the cells, and then use a flow cytometer (BD Canto II, USA) for on-machine detection, acquire data, and analyze with FlowJo10 software.

[0052] 4. Statistical Analysis

[0053] GraphPad 9.50 was used to compare the means of the two groups, and the independent sample t-test method was used to calculate the median of each group and the P value. P < 0.05 was considered to be statistically significant. The sample size of the enrolled patients was calculated using the online sample size estimation website https: / / www.cnstat.org / samplesize / : The standard deviation of the experimental group is 2917, the standard deviation of the control group is 820, the significance level is 0.05, the statistical power is 0.80, and when the sample size ratio of the experimental group and the control group is 1:1, the minimum sample size of each group is 17.

[0054] 2. Experimental results

[0055] Twenty samples were collected from each of the Non-aGVHD group and the aGVHD group. The statistical analysis results showed that the minimum sample size for each group was 17, so the sample size met the research requirements. Figure 1 As shown, compared with Non-aGVHD patients, CD3 + CD8 + The mean fluorescence intensity of NKG2D protein on the cell surface increased significantly, and the difference was statistically significant (P < 0.0001). + CD8 + The expression of NKG2D protein on the cell surface was significantly increased, indicating that CD3 + CD8 + Cell surface NKG2D protein may be a specific diagnostic marker for aGVHD.

[0056] To this end, we further performed ROC curve analysis, and the results ( Figure 2 ) shows that CD3 + CD8 + The AUC of NKG2D protein on the cell surface was 0.76 when used for the diagnosis of aGVHD, with a high diagnostic accuracy. + CD8 + NKG2D protein on the cell surface can be used as a specific diagnostic marker for aGVHD. + CD8 + The reagent for measuring the expression of NKG2D protein on the cell surface can be used as an aGVHD diagnostic reagent for auxiliary diagnosis of aGVHD.

[0057] In summary, the present invention provides a method for detecting CD3 + CD8 + The application of reagents for measuring the expression of NKG2D protein on the cell surface in the diagnosis of aGVHD can quickly and conveniently assist clinicians in diagnosing aGVHD, formulating reasonable treatment methods, achieving early diagnosis and early treatment, and avoiding serious disease progression.

[0058] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0059] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Detection of CD3 + CD8 + Application of a reagent for measuring the expression of NKG2D protein on the cell surface in the preparation of a detection kit for acute graft-versus-host disease.

2. The use according to claim 1, characterized in that The reagents include reagents for flow cytometry detection.

3. The use according to claim 2, characterized in that The reagent includes detection of CD3 + CD8 + Flow cytometry was used to detect the expression of NKG2D protein on the cell surface using fluorescent antibodies.

4. The use according to claim 3, characterized in that The antibodies include anti-human CD3 antibodies, anti-human CD8 antibodies and anti-human NKG2D antibodies modified with different fluorescent groups.

5. The use according to any one of claims 1 to 4, characterized in that: The CD3 + CD8 + Cells were obtained from peripheral blood samples.

6. The use according to claim 5, characterized in that The CD3 + CD8 + Cells were derived from peripheral blood mononuclear cell samples.

7. An acute graft-versus-host disease auxiliary diagnosis kit, characterized in that: The kit includes a detection kit for CD3 + CD8 + A reagent for measuring the expression of NKG2D protein on the cell surface.

8. The acute graft-versus-host disease auxiliary diagnosis kit according to claim 7, characterized in that: The kit comprises anti-human CD3 antibody, anti-human CD8 antibody and anti-human NKG2D antibody modified with different fluorescent groups.

9. The acute graft-versus-host disease auxiliary diagnosis kit according to claim 7, characterized in that: The CD3 + CD8 + Cells were obtained from peripheral blood samples.

10. The acute graft-versus-host disease auxiliary diagnosis kit according to any one of claims 7 to 9, characterized in that: The kit also includes lymphocyte separation solution and red blood cell lysis solution.

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