Use of a reagent for detecting 2-NBDG-positive B cells in the preparation of a medicament for diagnosing and / or predicting the prognosis of autoimmune diabetes

Abstract

The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a reagent for detecting 2-NBDG positive B cells in the preparation of a preparation for diagnosing and / or prognosticating autoimmune diabetes. At present, there is still a lack of effective disease markers for autoimmune diabetes, and early screening and treatment face huge challenges. There is an urgent need to find new disease markers to improve the diagnostic efficacy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a reagent for detecting 2-NBDG positive B cells in the preparation of a preparation for diagnosing and / or prognosticating autoimmune diabetes. Background Art

[0002] Autoimmune diabetes (AID) includes classical type 1 diabetes (T1D) and latent autoimmune diabetes in adults (LADA), and is caused by the absolute lack of insulin due to the autoimmune destruction of pancreatic islet beta cells. Patients rely on insulin injection treatment throughout their lives, which seriously endangers their lives and health. The latest research data shows that although the incidence rate of T1DM in China is relatively low compared with Western countries (1.01 / 100,000 / year, including all age groups), due to the large population base and the increasing incidence rate year by year, the number of patients is huge, bringing a huge burden to both families and society, making diabetes one of the major public health problems in China.

[0003] Autoimmune diabetes occurs due to the imbalance of immune tolerance in the body under the combined action of genetic and environmental factors, and is currently considered to be mainly caused by the destruction of pancreatic islet beta cells mediated by T lymphocytes. Recent studies have shown that B cells play an indispensable and important role in the pathogenesis of autoimmune diabetes. B cells are important antibody-producing cells, producing various antibodies including insulin autoantibody (IAA), protein tyrosine phosphatase antibody (1A-2A), glutamic acid decarboxylase antibody (GADA), and zinc transporter-8 antibody (ZnT8A). The detection of these autoantibodies is of great value and significance for predicting and diagnosing autoimmune diabetes. At the same time, as important antigen-presenting cells, B cells express co-stimulatory signal molecules and participate in the activation and amplification of autoreactive CD4+ T cells and CD8+ cytotoxic T cells, thus causing the occurrence of autoimmune diabetes. Studies on humanized NOD mice have confirmed that removing B cells or blocking B cell activation can effectively reduce or reverse the occurrence of T1D in NOD mice. Clinical trials on T1D patients have found that the use of rituximab (anti-CD20 monoclonal antibody) can effectively slow down the decline of pancreatic islet beta cell function and become the hope for the treatment of T1D.

[0004] When immune cells (such as T cells) transition from an initial state to an activated state, a huge energy demand comes along with significant cell growth and clonal expansion. In recent years, studies have shown that the energy metabolism level of immune cells plays a key role in immune function regulation and immune responses. However, most current studies have focused on macrophages and T cells, and relatively few studies have been conducted on B cell metabolism. In vitro experiments have confirmed that upon stimulation with IL-4, BAFF, LPS, or BCR, B cells will rapidly increase glucose uptake and accelerate the process of glycolysis, thereby quickly providing energy for cell proliferation, suggesting that regulating the energy metabolism of B cells is crucial for their development and function. Therefore, in-depth understanding of the characteristics and mechanisms of B cell activation is of great scientific significance and social value for comprehensively clarifying its role in the occurrence and development of autoimmune diabetes, finding new disease prevention, treatment, and intervention targets, and reducing mortality and medical burden.

[0005] Currently, C-peptide and islet autoantibodies such as glutamic acid decarboxylase antibody (GADA), zinc transporter 8 antibody (ZnT8A), and islet cell antibody (ICA) are commonly used clinically to evaluate the degree of islet cell damage and the risk of developing type 1 diabetes. However, using these as disease markers still has many limitations. For example, C-peptide can only reflect the characteristics of the disease in the late stage of islet destruction and lacks specificity; islet autoantibodies often appear successively, with a time interval of several months or even up to a decade. Not all patients have positive antibodies at the time of diagnosis, and many susceptible individuals with positive autoantibodies do not necessarily develop type 1 diabetes, etc. These protein molecule-based detections are cumbersome, expensive, and time-consuming in clinical routine, and their application prospects are not optimistic. Summary of the Invention

[0006] Based on the above problems, the primary object of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a set of markers for the diagnosis of type 1 diabetes with high sensitivity and high specificity; that is, to provide the use of a reagent for detecting 2-NBDG-positive B cells in the preparation of a preparation for diagnosing and / or predicting autoimmune diabetes.

[0007] Further, the autoimmune diabetes includes LADA and T1D.

[0008] Furthermore, the reagent for detecting 2-NBDG-positive B cells includes: a reagent for detecting the proportion of 2-NBDG-positive B cells among B cells and / or a reagent for detecting the 2-NBDG uptake amount in B cells.

[0009] The second object of the present invention is to provide a kit for diagnosing or predicting autoimmune diabetes, comprising the above-mentioned detection reagent.

[0010] The third object of the present invention is to provide the use of the above-mentioned detection reagent in the preparation of a preparation for the fasting C-peptide level of autoimmune diabetes patients.

[0011] The fourth object of the present invention is to provide the use of the above detection reagent in the preparation of a preparation for the postprandial C-peptide level of patients with autoimmune diabetes.

[0012] The fifth object of the present invention is to provide the use of the above detection reagent in the preparation of a preparation for the glycated albumin level of patients with autoimmune diabetes.

[0013] The sixth object of the present invention is to provide the use of the above detection reagent in the preparation of a preparation for the glycated hemoglobin level of patients with autoimmune diabetes.

[0014] The present invention is mainly used for detecting highly metabolic B cells, especially for detecting the intake of 2-NBDG in B cells or the proportion of 2-NBDG-positive B cells in B cells. Highly metabolic B cells are strongly correlated with the decline of islet function in autoimmune diabetes and can be used as a predictive indicator. The present invention has the following advantages: 1) It is applicable to all patients with autoimmune diabetes; 2) The detection is convenient, the detection index is significantly correlated with the prognosis, the detection index is single, and the operation is simple; 3) Most of all reagents are relatively common, and the cost is controlled within a feasible range. Description of the Drawings

[0015] Figure 1 A is a schematic diagram of the flow cytometry gating of highly metabolic B cells;

[0016] Figure 1 B is a result graph showing that the proportion of highly metabolic B cells in peripheral blood of patients with autoimmune diabetes (including T1D and LADA patients) is much higher than that of healthy subjects (HD) and patients with type 2 diabetes (T2D).

[0017] Figure 2 A is a result graph showing that the proportion of highly metabolic B cells is negatively correlated with the fasting C-peptide level of patients;

[0018] Figure 2 B is a result graph showing that the proportion of highly metabolic B cells is negatively correlated with the postprandial C-peptide level of patients;

[0019] Figure 2 C is a result graph showing that highly metabolic B cells are positively correlated with glycated albumin;

[0020] Figure 2 D is a result graph showing that highly metabolic B cells are positively correlated with glycated hemoglobin.

[0021] Figure 3 It is a result graph showing that the speed of islet function failure in patients with autoimmune diabetes with a higher subpopulation of B cells with high glucose uptake (>40%) is faster.

[0022] Figure 4ROC curve for predicting autoimmune diabetes based on the proportion of hypermetabolic B cells in the autoimmune diabetes group. Detailed implementation manners

[0023] The following embodiments are intended to further illustrate the present invention rather than limit the present invention.

[0024] Example 1:

[0025] All participants in the experiment were informed of the research procedures and the uses of the specimens and signed informed consent forms. Demographic information and clinical data of all participants were obtained through questionnaire collection, physical examinations, and blood tests. There were no statistically significant differences in the general information of the patients.

[0026] Peripheral blood of the subjects was collected, including 38 healthy individuals, 61 patients with autoimmune diabetes (AID), among which 37 had type 1 diabetes (T1D), 24 were latent autoimmune diabetes in adults (LADA) patients, and 24 had type 2 diabetes (T2D). Approximately 5 ml of anticoagulated blood from the subjects was collected, and PBMC was separated using density gradient centrifugation, and the cells were adjusted to 3 - 4×10 6 cells / tube for flow cytometry.

[0027] The staining protocol is as follows:

[0028] 1. Prepare 100 μM 2-NBDG staining antibody (using sugar-free 1640 as the base) and preheat it at 37°C.

[0029] 2. Add 1 ml of 2-NBDG (11046, Cayman) to each tube of cells, vortex and mix well, and incubate at 37°C for 30 min.

[0030] 3. Prepare 1 FMO tube with only CD19 surface antibody added as a control.

[0031] 4. Add 3 - 4 ml of PBS to terminate the reaction, shake and mix well.

[0032] 5. Centrifuge (300 G, 5 min, 20°C).

[0033] 6. Add 1 μl of CD19 surface antibody (BD, 566396) to each tube of cells, vortex and mix well.

[0034] 7. Incubate at 4°C for 30 min.

[0035] 8. Add 1 ml of Staining buffer to terminate the reaction, shake and mix well, and centrifuge (same conditions as above).

[0036] 9. Remove the supernatant, add 200 - 500 μl of Staining buffer, vortex and mix well, and load onto the machine.

[0037] 10. After collecting the data, set the gates for FCS / SSC, using the FMO tube as the boundary to define the position of high 2-NBDG uptake, and analyze the proportion of cells with high 2-NBDG uptake (high metabolism) among CD19+ B cells.

[0038] The results are as Figure 1 shown. Figure 1 A is a schematic diagram of the flow cytometry gating of B cells with high metabolism. 2-NBDG (2-(N-7-nitro-2,1,3-benzoxadiazol-4-ylamino)-2-deoxy-D-glucose) is an analog of glucose. After being taken up by cells, 2-NBDG will not be metabolized. 2-NBDG is fluorescently labeled, and after incubating with cells for a period of time, the fluorescence intensity can reflect the glucose uptake ability of cells.

[0039] That is, the B cell subset with high 2-NBDG uptake is the subset with high glucose uptake, and this subset has the characteristic of high metabolism. We call it B cells with high metabolism. As can be seen from Figure 1 B, the proportion of B cells with high metabolism in the peripheral blood of patients with autoimmune diabetes (including T1D and LADA patients) is much higher than that of healthy subjects (HD) and patients with type 2 diabetes (T2D), indicating that B cells in the peripheral blood of patients with autoimmune diabetes have high metabolism, which can be used as an index for diagnosing autoimmune diabetes and differentiating it from type 2 diabetes.

[0040] Perform a correlation analysis on the proportion of B cells with high metabolism and the characteristic clinical data of patients with type 1 diabetes. It is found that the proportion of B cells with high metabolism is negatively correlated with the fasting C-peptide level of patients ( Figure 2 A), the proportion of B cells with high metabolism is negatively correlated with the postprandial C-peptide level of patients ( Figure 2 B), suggesting that the worse the islet function of patients with a higher proportion of B cells with high metabolism; the proportion of B cells with high metabolism is positively correlated with glycated albumin ( Figure 2 C), glycated albumin can reflect the blood glucose control situation of patients in one month, and the proportion of B cells with high metabolism is positively correlated with glycated hemoglobin ( Figure 2 D), glycated hemoglobin can reflect the blood glucose control situation of patients in three months, which suggests that the worse the blood glucose control of patients with a higher proportion of B cells with high metabolism.

[0041] Diagnostic significance: The proportion of B cells with high metabolism can distinguish healthy people from patients with autoimmune diabetes and is a good molecular marker for diagnosing autoimmune diabetes.

[0042] Relationship with prognosis: In addition, autoimmune diabetes patients were divided into a subgroup with higher B-cell glucose uptake and a subgroup with lower B-cell glucose uptake according to the median value based on the descending order of the proportion of highly metabolically active B cells. If the 2-hour C-peptide level of a patient was less than 200 pmol / L, it indicated pancreatic islet function failure. It was found that the autoimmune diabetes patients in the subgroup with higher B-cell glucose uptake (the proportion of 2-NBDG-positive B cells > 40%) had a faster rate of pancreatic islet function failure ( Figure 3 ).

[0043] Such as Figure 4 , the area under the ROC curve (AUC) was 0.8536, indicating that the proportion of highly metabolically active B cells in the autoimmune diabetes group could accurately predict autoimmune diabetes. The optimal value on the ROC curve was the threshold. Considering sensitivity and specificity comprehensively, it meant maximizing sensitivity on the basis of maximum specificity. Based on this method, the optimal value of the ROC curve was 0.3892, that is, the threshold was 0.3892. At this node, the proportion of highly metabolically active B cells showed a sensitivity of 55% and a specificity of 100%, and a positive predictive value of 95% (confidence interval 0.7788 - 0.9284). It can be seen that the proportion of highly metabolically active B cells can be used to assist in the diagnosis of autoimmune diabetes well.

Claims

1. Use of a reagent for detecting the uptake amount of 2-NBDG in B cells in the preparation of a preparation for diagnosing and / or predicting the prognosis of autoimmune diabetes; the autoimmune diabetes includes LADA and T1DM.

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