A cytokine group and its application

Abstract

The present invention discloses a cytokine group and application thereof. The cytokine group comprises IFN-γ, IL-17 and IFN-α cytokines. The application of the cytokine group is to use the cytokine group for detection, for example, to use the concentration data of the cytokines for detection of non-disease diagnosis, and obtain the concentration ratio of each cytokine before and after the intervention of the sample to be tested; if the concentration ratio of each cytokine is greater than the respective threshold value, the intervention is invalid, and if the concentration ratio of at least one cytokine is less than the respective threshold value, the intervention is effective; the cytokine group changes in the human body and can be used to judge the state of the sample.

Description

Technical Field

[0001] The invention relates to a cytokine group and application thereof, and belongs to the field of biotechnology-assisted detection. Background Art

[0002] Tuberculosis (TB) is a chronic, wasting infectious disease that has become a global problem threatening human health. The 2021 Global Tuberculosis Report released by WHO shows that in 2020, the number of people with latent tuberculosis infection in the world was close to 2 billion, with 9.87 million new tuberculosis patients and an incidence rate of 127 / 100,000. The estimated number of cases and incidence rates continued to show a downward trend, but the rate of decline was slower than in previous years. Excluding the impact of the COVID-19 epidemic, tuberculosis was once the number one cause of death from a single infectious source and the 13th leading cause of death in the world. Today, the evaluation of the effectiveness of tuberculosis treatment mainly depends on symptom improvement, sputum bacteria turning negative, and lesion absorption. For patients with mild symptoms or negative sputum bacteria, the evaluation of treatment effectiveness mainly depends on lesion absorption. However, the process of lesion absorption is long and difficult to observe. If the patient's condition cannot be accurately judged, it is difficult to guide clinicians to adjust the treatment plan in a timely manner. Summary of the invention

[0003] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a cytokine panel that changes in the human body and can be used to determine the state of a sample.

[0004] The second object of the present invention is to provide a quantity of the above-mentioned cytokine panel, and to judge the status of the sample through the cytokine panel.

[0005] The third object of the present invention is to provide an application of the above cytokine panel, wherein the cytokine panel is used in detection.

[0006] The first object of the present invention can be achieved by adopting the following technical solution: a cytokine group includes IFN-γ, IL-17 and IFN-α cytokines.

[0007] The second object of the present invention can be achieved by adopting the following technical scheme: the amount of a cytokine group, the amount of the cytokine group is the concentration of IFN-γ, IL-17 and IFN-α cytokines in serum respectively.

[0008] The third object of the present invention can be achieved by adopting the following technical scheme: an application of a cytokine group, which is to use the cytokine group for detection; the cytokine group includes IFN-γ, IL-17 and IFN-α cytokines.

[0009] Furthermore, the concentration of each cytokine in serum is obtained, and the concentration data is used in non-disease diagnosis tests.

[0010] Furthermore, the concentration ratio of each cytokine in the test sample after intervention and before intervention is obtained; if the concentration ratio of each cytokine is greater than the respective cut-off value, the intervention is ineffective, and if the concentration ratio of at least one cytokine is less than or equal to the respective cut-off value, the intervention is effective.

[0011] Further, the intervention is a non-therapeutic intervention.

[0012] Furthermore, the concentration of each cytokine in serum is obtained, and the concentration data is used to detect the therapeutic effect of tuberculosis.

[0013] Furthermore, the concentration ratio of each cytokine in the test sample after treatment and before treatment is obtained; if the concentration ratio of each cytokine is greater than the respective cut-off value, the treatment is ineffective, and if the concentration ratio of at least one cytokine is ≤ the respective cut-off value, the treatment is effective.

[0014] Furthermore, the cut-off value of IFN-γ is 0.64, the cut-off value of IL-17 is 0.71, and the cut-off value of IFN-α is 0.65.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The cytokine group of the present invention changes in the human body and can be used to determine the status of the sample;

[0017] 2. The cytokine panel of the present invention is used for detection with high accuracy;

[0018] 3. The application of the cytokine panel of the present invention reveals the changes of IFN-γ, IL-17 and IFN-α cytokines, providing a new guiding direction for detection. DETAILED DESCRIPTION

[0019] Below, in conjunction with specific implementation modes, the present invention is further described:

[0020] An application of a cytokine panel is to use the cytokine panel in detection; the cytokine panel includes IFN-γ, IL-17 and IFN-α cytokines, specifically as follows:

[0021] The concentration of each cytokine in serum was obtained, and the concentration data were used to detect the therapeutic effect of pulmonary tuberculosis, and the concentration ratio of each cytokine in the test sample before and after treatment was obtained; if the concentration ratio of each cytokine was greater than the respective cut-off value, the treatment was ineffective, and if the concentration ratio of at least one cytokine was less than or equal to the respective cut-off value, the treatment was effective; the cut-off value of IFN-γ was 0.64, the cut-off value of IL-17 was 0.71, and the cut-off value of IFN-α was 0.65.

[0022] How this technology works:

[0023] Evaluating whether the tuberculosis condition is controlled and improved from the microscopic level of the body's anti-tuberculosis immune response can shorten the time for evaluating the effect of anti-tuberculosis treatment and improve the sensitivity and accuracy of anti-tuberculosis treatment effect evaluation. The immune response during the onset of tuberculosis is mainly mediated by immune cells such as macrophages and T cells and the cytokines they express and secrete. Cytokines in serum can be produced by a variety of cells, and cytokines have a certain regulatory effect on the immune activity of immune cells. During the treatment of pulmonary tuberculosis, along with the control of tuberculosis and the improvement of the condition, the expression levels of IFN-γ, IL-17 and IFN-α cytokines in the patient's serum will also change accordingly. This change can reflect the effect of anti-tuberculosis treatment to a certain extent.

[0024] Example:

[0025] 1. Sample selection:

[0026] Patient inclusion criteria: patients clinically diagnosed with pulmonary tuberculosis. Exclusion criteria: (1) history of tuberculosis or drug-resistant tuberculosis; (2) other lung diseases; (3) other infectious diseases; (4) autoimmune diseases (such as HIV, rheumatoid arthritis, ankylosing spondylitis, asthma, Sjögren's syndrome, systemic lupus erythematosus, scleroderma, polymyositis, and dermatomyositis; (5) taking immunosuppressants or hormone drugs in the past three months; (6) organic lesions of organs such as liver and kidney, blood diseases, malignant tumors, etc.; (7) pregnant or lactating women.

[0027] 2. Sample collection and preservation:

[0028] All subjects had chest X-rays taken before treatment and 2 months after intensive treatment, and 3 mL of venous blood was drawn. The venous blood was placed in an anticoagulant tube, left to stand for 1 hour, centrifuged at 2000 rpm for 20 minutes, and the serum was divided into cryopreservation tubes and stored at -80°C. A total of 56 cases were enrolled.

[0029] 3. Determination of cytokine concentration in serum:

[0030] After the serum was thawed, it was centrifuged and the supernatant was taken. 25 μL of the capture microsphere mixture in the cytokine combined detection kit (immunofluorescence method, Segi Bio) was added to the flow cytometer tube, 25 μL of the gradient diluted standard was added to the standard tube, and 25 μL of serum was added to the sample tube; 25 μL of fluorescence detection reagent was added to all tubes, vortexed and mixed, and incubated at room temperature in the dark for 2.5 h; 1 mL of PBS was added after the reaction was completed, and after centrifugation at 200 g for 5 min, the supernatant was discarded and 200 μL of PBS was added. The samples were detected by flow cytometer (BD, FACSAriaT MⅡ) in the order of standard tube and sample tube, and the software provided by the kit was used to make the standard curve, and the concentrations of interleukins IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IFN-α in the serum were analyzed.

[0031] 4. Data processing:

[0032] The chest X-ray lesions at 2 months after treatment were compared with those before treatment. Patients with unchanged or enlarged lesions were classified as the ineffective group (N=25), and patients with absorbed lesions were classified as the effective group (N=31). Ratio value: the ratio of the serum cytokine concentration at 2 months after treatment to the concentration before treatment.

[0033] SPSS 20.0 statistical software was used for statistical analysis, and the cutoff value and test efficiency of Ratio were explored based on the receiver operating characteristic curve (ROC). The results are shown in Table 1:

[0034] Table 1 Analysis of the efficacy of 12 cytokine ratio values ​​in evaluating the effectiveness of tuberculosis treatment

[0035]

[0036]

[0037] Combination: For a single cytokine, the cutoff value of the cytokine is taken as the cutoff value. If the Rati o value is greater than the cutoff value, the treatment is considered ineffective, that is, it is positive for the cytokine. The 12 cytokines are combined in series, and the 8 combinations with good test efficiency are as follows:

[0038] A: IFN-γ, IL-17, IFN-α;

[0039] B: IL-6, TNF, IFN-γ, IL-17, IL-1β;

[0040] C: IL-6, IFN-α;

[0041] D: IL-6, IL-8;

[0042] E: IL-6, IFN-γ, IL-17;

[0043] F: IFN-γ, IL-17, IL-8;

[0044] G: IFN-α, IL-8;

[0045] H: IL-12P70, IL-8;

[0046] If the Ratio values ​​of all cytokines in the combination are greater than the cut-off value, it is positive, which means the treatment is ineffective; otherwise, it is negative, which means the treatment is effective. The test efficiency analysis of the combination is shown in Table 2:

[0047] Table 2 Power analysis of cytokine tandem combinations for evaluating the effectiveness of tuberculosis treatment

[0048]

[0049]

[0050] Among them, group A can judge the treatment effect of pulmonary tuberculosis at 2 months of treatment without relying on sputum negative conversion (patients must be sputum positive patients, accounting for only about 30%-50%), symptom improvement (patients must have obvious symptoms, which is somewhat subjective) and imaging results (slow improvement and low sensitivity). This method has the advantages of wide applicability, objectivity and accuracy. At the same time, it reveals the changes of IFN-γ, IL-17 and IFN-α cytokines during the treatment of pulmonary tuberculosis, providing a new guiding direction for the evaluation of the treatment effect of refractory tuberculosis such as extrapulmonary tuberculosis and multidrug-resistant tuberculosis, and for faster evaluation of the treatment effect.

[0051] For those skilled in the art, various other corresponding changes and modifications can be made according to the technical solutions and concepts described above, and all of these changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. Use of a reagent for detecting a group of cytokines in the preparation of a reagent for detecting the therapeutic effect of pulmonary tuberculosis, characterized in that: The cytokine group consists of IFN-γ, IL-17 and IFN-α cytokines; the concentration ratio of each cytokine in the tested serum after treatment and before treatment is obtained; if the concentration ratio of each cytokine is greater than the respective cut-off value, the treatment is ineffective; if the concentration ratio of each cytokine is less than or equal to the respective cut-off value, the treatment is effective; the cut-off value of IFN-γ is 0.64, the cut-off value of IL-17 is 0.71; and the cut-off value of IFN-α is 0.65.

Images