Application of CD3+CD4+CD8+ cells in the diagnosis and therapeutic effect evaluation of myasthenia gravis

Abstract

The present invention provides the application of CD3+CD4+CD8+ cells in the diagnosis and efficacy evaluation of myasthenia gravis. The inventors have found through research that compared with healthy volunteers, the proportion of CD3+CD4+CD8+ cells in the peripheral blood of myasthenia gravis patients increased significantly, and the proportion of CD3+CD4+CD8+ cells decreased accordingly after treatment. Further ROC curve analysis found that the AUC value of CD3+CD4+CD8+ cells was 0.9556, P=0.0008, the sensitivity was 90%, and the specificity was 100% when used for the diagnosis of myasthenia gravis, indicating that the diagnostic accuracy was high. The present invention provides a specific diagnostic marker for myasthenia gravis, the detection of which has the advantages of non-invasive and convenient sampling, simple operation, intuitive and clear results, easy promotion, and is very suitable for early diagnosis and treatment of myasthenia gravis, as well as evaluation of the efficacy of treatment plans.

Description

Technical Field

[0001] The present invention belongs to the field of molecular diagnosis technology, and particularly relates to CD3 + CD4 + CD8 + Application of cells in the diagnosis and efficacy evaluation of myasthenia gravis. Background Art

[0002] Myasthenia gravis (MG) is an autoimmune disease caused by dysfunction of neuromuscular junction transmission. Acetylcholine receptor antibodies are the most common pathogenic antibodies. Its main clinical manifestations are partial or systemic skeletal muscle weakness and fatigue. Symptoms worsen after activity and are relieved after treatment with cholinesterase inhibitors or rest. The global prevalence of MG is 12.4 / 100,000, and the annual incidence is 4-12 / 1 million. The incidence of myasthenia gravis in my country is about 0.68 / 100,000, with a slightly higher incidence in women. It occurs in all age groups, with two peak incidences at 30 and 50 years old. The cause of MG is still unclear, and it is generally believed to be related to infection, drugs and environmental factors. Clinically, 10% to 15% of MG patients have thymoma, and 70% of MG patients have hyperplastic thymus changes. At present, the treatment options for myasthenia gravis mainly include plasma exchange, immunosuppressive therapy, targeted biological agents (complement inhibitors and targeted B cell therapy), thymectomy and autologous hematopoietic stem cell transplantation.

[0003] Currently, there is a lack of specific myasthenia gravis detection markers to assist in diagnosis and achieve early diagnosis and treatment. Summary of the invention

[0004] Based on this, the object of the present invention is to provide CD3 + CD4 + CD8 + Application of cells in the diagnosis and efficacy evaluation of myasthenia gravis.

[0005] To achieve the above objectives, the present invention adopts the following technical solutions.

[0006] In a first aspect of the present invention, there is provided a CD3 + CD4 + CD8 + Application of cells in the preparation of reagents for myasthenia gravis detection and / or efficacy evaluation.

[0007] In a second aspect of the present invention, there is provided a CD3 + CD4 + CD8 + Application of cells in screening reagents for myasthenia gravis detection and / or efficacy evaluation.

[0008] In a third aspect of the present invention, there is provided a method for detecting CD3 +CD4 + CD8 + The invention relates to the use of a reagent for measuring the content of cells in a biological sample in the preparation of a kit for detecting and / or evaluating the efficacy of myasthenia gravis.

[0009] In some embodiments, the reagents include reagents for flow cytometry detection.

[0010] In some embodiments, the reagents include anti-human CD3 antibodies, anti-human CD4 antibodies, and anti-human CD8 antibodies modified with different fluorescent groups.

[0011] In some embodiments, the biological sample comprises a peripheral blood sample.

[0012] In some embodiments, the biological sample comprises a peripheral blood mononuclear cell sample.

[0013] In a fourth aspect of the present invention, a method for in vitro screening of myasthenia gravis detection and / or efficacy evaluation reagents is provided, comprising the following steps: using a candidate reagent to detect CD3 + CD4 + CD8 + If the content of the cells can be detected, the candidate reagent can be used as a myasthenia gravis detection and / or efficacy evaluation reagent.

[0014] A fifth aspect of the present invention provides a myasthenia gravis detection and / or efficacy evaluation kit, the kit comprising a method for detecting CD3 + CD4 + CD8 + Reagents for detecting the presence of cells in biological samples.

[0015] In some embodiments, the kit includes reagents for flow cytometry detection.

[0016] In some embodiments, the kit includes anti-human CD3 antibodies, anti-human CD4 antibodies, and anti-human CD8 antibodies modified with different fluorescent groups.

[0017] In some embodiments, the biological sample comprises a peripheral blood sample.

[0018] In some embodiments, the kit further comprises lymphocyte separation solution, red blood cell lysis solution, and PBS buffer.

[0019] The present invention provides CD3 + CD4 + CD8 + The inventors found that compared with healthy volunteers, the peripheral blood of myasthenia gravis patients had a higher CD3 + CD4 +CD8 + Further ROC curve analysis revealed that CD3 + CD4 + CD8 + When CD3 cells were used to diagnose myasthenia gravis, the AUC value was 0.9556, P = 0.0008, the sensitivity was 90%, and the specificity was 100%, indicating that the diagnostic accuracy was high. + CD4 + CD8 + The reagent for measuring the content of cells in a biological sample can be used as a myasthenia gravis detection reagent and for preparing a myasthenia gravis detection kit.

[0020] In addition, after treatment, the number of CD3 + CD4 + CD8 + The proportion of cells showed a downward trend, suggesting that CD3 + CD4 + CD8 + The cells can be used as an efficacy evaluation indicator to evaluate the therapeutic effect of the treatment plan on patients with myasthenia gravis.

[0021] The present invention provides a myasthenia gravis-specific diagnostic and / or therapeutic efficacy evaluation marker, which has the advantages of non-invasive and convenient sampling, simple operation, intuitive and clear results, and easy promotion. It is very suitable for early diagnosis and treatment of myasthenia gravis, as well as efficacy evaluation of treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 CD3 in peripheral blood of myasthenia gravis patients and healthy controls + CD4 + CD8 + Flow cytometry results of cells.

[0023] Figure 2 For CD4 + CD8 + CD3 + Detection results in T cells.

[0024] Figure 3 CD3 after treatment of myasthenia gravis patients + CD4 + CD8 + cells and CD4 + CD8 + CD3 + T cell detection results.

[0025] Figure 4 This is the ROC curve analysis diagram. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The following describes the invention in conjunction with specific embodiments.

[0030] Example 1

[0031] In this example, flow cytometry was used to detect CD3 + CD4 + CD8 + The proportion of cells.

[0032] 1. Myasthenia gravis patients

[0033] Inclusion criteria:

[0034] ① Patients aged 18 years and above, with medical history and medical records supporting and diagnosed with myasthenia gravis;

[0035] ② No tumor diseases other than thymic tumors exist;

[0036] Exclusion criteria:

[0037] ① Patients with any acute, chronic illness or mental illness that may endanger or impair the subject's ability to participate in this study; ② Myasthenic syndrome and related muscle diseases.

[0038] 2. Healthy Controls

[0039] Inclusion criteria: over 18 years old, no family history of muscle disease or immune disease.

[0040] Exclusion criteria: other diseases.

[0041] According to the above criteria, peripheral blood samples were collected from 9 healthy controls and 10 myasthenia gravis patients. There was no statistical difference in age and gender between the two groups.

[0042] In addition, peripheral blood samples were collected from three myasthenia gravis patients before and after treatment to compare and analyze CD3 + CD4 + CD8 + The treatment options are:

[0043] MG1: pyridostigmine bromide 60 mg Q6h; tacrolimus 2 mg bid; prednisone 20 mg qd; plasma exchange 7 times, ventilator-assisted ventilation.

[0044] MG2: pyridostigmine bromide 60 mg tid; tacrolimus 2 mg bid; methylprednisolone 80 mg qd.

[0045] MG3: pyridostigmine bromide 90 mg Q6h; mycophenolate mofetil 0.5 g bid; methylprednisolone 10 mg qd.

[0046] All samples were isolated from peripheral blood mononuclear cells and then analyzed by flow cytometry.

[0047] 1. Experimental Methods

[0048] 1. Flow cytometry

[0049] (1) Collect 3 ml of whole blood in an EDTA anticoagulant tube, centrifuge at 2500 rpm for 15 min at 4°C, and keep the supernatant. Dilute with PBS at a ratio of 1:1.

[0050] (2) Slowly inject onto 4 ml Ficoll, centrifuge at 18°C, 600 g for 30 min, increase by 5 and decrease by 1.

[0051] (3) Aspirate the middle white film layer, add an equal volume of PBS for washing, centrifuge at 500 g for 5 min at 4°C, increase by 9, decrease by 9, and discard the supernatant.

[0052] (4) To lyse red blood cells, add 5 ml of 1× red blood cell lysis buffer and mix by pipetting; let stand at room temperature for 5 min; add 3-5 times the volume of PBS to stop lysis of red blood cells; centrifuge at 4°C, 500 g for 5 min and discard the supernatant.

[0053] (5) Add 50 μl of flow cytometry antibodies (0.5 μl each of CD3, CD4, and CD8, diluted with 48.5 μl of PBS) to label the cells. Incubate in a 4°C refrigerator away from light for 30 min. Add 1 ml of PBS to terminate the staining, centrifuge at 500 g, 4°C, 5 min, and discard the supernatant.

[0054] (6) Resuspend in 200-400 μl PBS and sieve.

[0055] (7) The samples were tested using a flow cytometer (BD Canto II, USA), and the data were acquired and analyzed using FlowJo10 software.

[0056] 2. Statistical Analysis

[0057] Data were processed using GraphPad Prism 8. Statistical values ​​were expressed as mean ± SD. The mean t test was used. p < 0.05 indicated statistical significance.

[0058] 2. Experimental Results

[0059] like Figure 1 As shown, compared with healthy controls (Con), peripheral blood CD3 + CD4 + CD8 + The proportion of cells increased significantly, and the difference was statistically significant (P = 0.0002). + CD4 + CD8 + The proportion of cells decreased. + CD8 + CD3 + The trend of the proportion of T cells and CD3 + CD4 + CD8 + The trend of cells in PBMC is consistent ( Figure 2 In addition, compared with before treatment, the CD3 + CD4 + CD8 + The proportion of cells in PBMCs, and CD4 + CD8 + CD3 + The proportion of T cells showed a downward trend ( Figure 3 ). The results suggest that CD3 + CD4 + CD8 + The cells may be potential biomarkers for diagnosis and therapeutic efficacy evaluation of myasthenia gravis patients.

[0060] To this end, we further conducted ROC curve analysis, and the results showed that ( Figure 4 ), CD3 + CD4 + CD8 +When CD3 cells were used to diagnose myasthenia gravis, the AUC value was 0.9556, P = 0.0008, the sensitivity was 90%, and the specificity was 100%, indicating that CD3 + CD4 + CD8 + The cells can be used as specific diagnostic markers for myasthenia gravis patients with high diagnostic accuracy.

[0061] In summary, the present invention provides a myasthenia gravis-specific diagnostic and / or therapeutic efficacy evaluation marker, which has high diagnostic accuracy and is very suitable for early diagnosis and treatment of myasthenia gravis, as well as evaluation of the efficacy of treatment regimens.

[0062] 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.

[0063] 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 + CD4 + CD8 + The invention relates to the use of a reagent for measuring the content of cells in a peripheral blood sample in the preparation of a kit for detecting and / or evaluating the efficacy of myasthenia gravis.

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 reagents include anti-human CD3 antibodies, anti-human CD4 antibodies and anti-human CD8 antibodies modified with different fluorescent groups.

4. A method for in vitro screening of myasthenia gravis detection and / or efficacy evaluation reagents, characterized in that: The following steps are involved: Detection of CD3 in peripheral blood samples using the candidate reagent + CD4 + CD8 + If the content of the cells can be detected, the candidate reagent can be used as a myasthenia gravis detection and / or efficacy evaluation reagent.

5. A kit for detecting and / or evaluating the therapeutic effect of myasthenia gravis, characterized in that: The kit includes a detection kit for CD3 + CD4 + CD8 + A reagent for measuring the content of cells in a peripheral blood sample, wherein the reagent is an anti-human CD3 antibody, an anti-human CD4 antibody, and an anti-human CD8 antibody modified with different fluorescent groups.

6. The myasthenia gravis detection kit according to claim 5, characterized in that: The kit includes reagents for flow cytometry detection.

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