Application of biomarker in preparation of product for predicting epilepsy

Abstract

The invention relates to the technical field of biological medicine, and discloses an application of biomarkers in preparation of a product for predicting epilepsy, the biomarkers are CXCL17 and GPR35, and a detection result is related to the combined expression level of CXCL17 and GPR35 in a biological sample. The invention further discloses a kit for assisting in predicting epilepsy occurrence and monitoring the drug treatment effect. The invention provides a product capable of applying the combined expression level of CXCL17 and GPR35 for the first time, the product can carry out rapid, objective, high-sensitivity and high-specificity prediction in the early stage of epilepsy occurrence, and after definite diagnosis of epilepsy, by monitoring the compensatory increase of CXCL17 and GPR35 in the chronic stage, the epilepsy can be quickly and objectively predicted in a high-sensitivity and high-specificity manner. The invention discloses an important role in an epilepsy nerve protection mechanism as a protective molecule.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a new use of biomarkers, and more particularly to the application of chemotactic factor CXCL17 and / or its receptor GPR35 in the preparation of a product for predicting the occurrence of epilepsy. BACKGROUND

[0002] Epilepsy is a common central nervous system disease. There are more than 20 kinds of antiepileptic drugs on the market, but existing evidence shows that about 30% of epilepsy patients are not sensitive to common drugs on the market, leading to the final development of drug-resistant epilepsy. Given the chronic progression and repeated seizures of epilepsy, early prediction of the occurrence of epilepsy has become a major challenge in clinical treatment.

[0003] Currently, the techniques for predicting the occurrence of epilepsy mainly rely on electroencephalogram, neuroimaging examination, biomarker analysis and machine learning methods. However, the existing technology has limitations in the timeliness and sensitivity of predicting the occurrence of epilepsy, especially the evaluation by electroencephalogram and imaging examination can only be carried out after the onset of epilepsy, and often requires a long observation period to make a judgment. Therefore, the existing diagnostic methods are mostly descriptive and auxiliary, and cannot make accurate and timely prediction before the onset of epilepsy, which may miss the opportunity for early intervention and treatment, and may delay the patient's condition.

[0004] Most of the existing technologies focus on post-seizure detection or prediction based on certain indirect markers, and most of the methods rely on complex equipment, long-term monitoring or require a large amount of clinical data support. There is still a lack of effective means to achieve early, accurate and real-time prediction, therefore, developing a technology that can achieve early, accurate and real-time prediction of seizures is still an important topic in current medical research.

[0005] Therefore, it is urgent to develop an efficient and sensitive technology that can achieve early and accurate prediction before the onset of epilepsy. Such technology will provide timely intervention opportunities for clinical practice, help individualized treatment and improve the quality of life of epilepsy patients. At present, there is still a lack of effective products that can quickly and objectively predict the occurrence of epilepsy in clinical practice, which provides great research and application potential for the present application. SUMMARY

[0006] The purpose of the present application is to provide the application of a biomarker in the preparation of a product for predicting the occurrence of epilepsy, to solve the technical problem of the lack of efficient and sensitive prediction or determination of the therapeutic effect of epilepsy in the prior art, for the auxiliary diagnosis of epilepsy, the assessment of disease severity or the monitoring of therapeutic effect.

[0007] To solve the above technical problems, the present application specifically provides the following technical solutions: The application provides application of a biomarker in preparation of a product for predicting epilepsy, and the biomarker is CXCL17 and GPR35. The detection result is related to combined expression levels of the CXCL17 and the GPR35 in the biological sample.

[0008] As a preferred scheme of the application, the biological sample is mouse brain tissue, peripheral blood, serum, plasma or cerebrospinal fluid.

[0009] As a preferred scheme of the application, the product is a detection kit.

[0010] The application further provides a detection kit, which is used for assisting in predicting epilepsy. The kit realizes the diagnosis function by detecting expression levels of the CXCL17 and the GPR35 and performing combined interpretation.

[0011] As a preferred scheme of the application, the kit comprises a solid-phase carrier coated with a capture antibody, a detection antibody, a standard sample, a sample diluent, a washing solution, a substrate developing solution and a termination solution. The capture antibody is a specific antibody for a target protein CXCL17 or GPR35. The detection antibody is a labeled antibody for the same target protein and different from the capture antibody in terms of a combined epitope. The standard sample is a recombinant CXCL17 protein and / or a recombinant GPR35 protein with a known concentration, and is used for drawing a standard curve.

[0012] As a preferred scheme of the application, the label of the labeled antibody is any one of horseradish peroxidase, alkaline phosphatase, biotin or fluorescein. The washing solution is a phosphate buffer solution containing Tween 20. The termination solution is a sulfuric acid or hydrochloric acid solution.

[0013] The application further provides application of the kit in predicting epilepsy or monitoring a drug treatment effect.

[0014] As a preferred scheme of the application, the method comprises the following steps. The sample is jointly detected by the CXCL17 detection antibody and the GPR35 detection antibody in the kit, so as to obtain concentration data of the CXCL17 and the GPR35 in the sample. The concentration data of the CXCL17 and the concentration data of the GPR35 are jointly compared with preset values, so as to obtain a judgment result.

[0015] As a preferred scheme of the present application, the preset value is a joint interpretation criterion or individual previous joint detection data.

[0016] As a preferred scheme of the present application, the joint interpretation criterion is: CXCL17 concentration is greater than or equal to 1300 pg / mL and GPR35 concentration is greater than or equal to 4.0 pg / mL.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application first proposes that CXCL17 and receptor GPR35 are constantly highly expressed in the brain tissue and the periphery of patients with epilepsy, and the expression amount can be used as a biological index for predicting the occurrence of epilepsy, so that a product for rapidly judging the occurrence of epilepsy in the early stage can be prepared to overcome the defects of hysteresis and passivity of the existing clinical detection product. The present application specifically provides a detection kit for predicting the occurrence of epilepsy, and discloses a use method and a joint interpretation criterion. The sensitivity of the joint interpretation criterion is 83.3%, the specificity is as high as 71.4%, the cut-off value is 0.4455, the specificity is significantly higher than that of single index detection, the false positive rate can be effectively reduced, the index is positively correlated with the development degree of the disease, the drug responsiveness of patients at the molecular level in the early stage of treatment or even before treatment can be accurately typed, and the patients who have been diagnosed can be dynamically monitored, which has important clinical value and market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only exemplary, and other drawings can be obtained by the provided drawings without creative labor for those skilled in the art.

[0019] Figure 1 A statistical chart of GPR35 expression level in serum samples of patients with epilepsy compared with non-epilepsy control group under ELISA detection in Example 1 of the present application is provided. Figure 2 A statistical chart of CXCL17 expression level in serum samples of patients with epilepsy compared with non-epilepsy control group under ELISA detection in Example 1 of the present application is provided. Figure 3 A statistical chart of ROC curve analysis of GPR35 and CXCL17 in distinguishing patients with epilepsy (n = 42) and non-epilepsy control in Example 1 of the present application is provided. Figure 4ROC curve analysis statistical graph for differentiating epilepsy patients (n = 42) and non-epilepsy controls by using two indicators of the present application into a binary Logistic regression model to establish a joint predictor; Figure 5 Representative Western blotting graph of GPR35 protein levels in the hippocampus of mice at different time points in Example 5 of the present application in kainic acid-induced seizure or control group; Figure 6 Statistical graph of Western blotting quantification of GPR35 protein levels in the hippocampus of mice at different time points in Example 5 of the present application in kainic acid (KA) induced seizure or control group; Figure 7 Statistical graph of GPR35 gene knockout (GPR35 KO ) on KA-induced seizure in Example 5 of the present application; Figure 8 Statistical graph of the average number of seizures per day and the average duration of chronic epilepsy (after 4 weeks) in Example 5 of the present application in KA-treated wild-type (WT) and GPR35 KO mice; Figure 9 Statistical graph of the mRNA expression of inflammatory factors in Example 5 of the present application in KA-treated WT and GPR35 KO mice; Figure 10 Statistical graph of the protein expression level of cytokine TNF-α in glutamate-induced BV2 cells under LV-GPR35 overexpression or control treatment in Example 5 of the present application; Figure 11 Statistical graph of the protein expression level of cytokine IL-1β in glutamate-induced BV2 cells under LV-GPR35 overexpression or control treatment in Example 5 of the present application; Figure 12 Statistical graph of the protein expression level of cytokine IL-6 in glutamate-induced BV2 cells under LV-GPR35 overexpression or control treatment in Example 5 of the present application; Figure 13 Statistical graph of the protein expression level of cytokine IL-10 in glutamate-induced BV2 cells under LV-GPR35 overexpression or control treatment in Example 5 of the present application; Figure 14 Statistical graph of the distribution clustering analysis of GPR35 in the hippocampus of intractable epilepsy patients characterized using human single-cell sequencing data sets in Example 6 of the present application; Figure 15Figure 6. Cluster analysis plot of GPR35 distribution in hippocampus of refractory epilepsy patients characterized using human single-cell sequencing datasets in Example 6. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0021] The present application provides an application of a biomarker in preparing a product for predicting the occurrence of epilepsy, wherein the biomarker is CXCL17 (chemokine 17) and receptor GPR35 (ligand G protein-coupled receptor 35).

[0022] The chemokine 17 and the ligand G protein-coupled receptor 35 are constantly expressed in a biological sample.

[0023] The biological sample is brain tissue, peripheral blood, serum, plasma or cerebrospinal fluid.

[0024] The biological sample is taken from an epilepsy patient.

[0025] The product is an enzyme-linked immunosorbent assay kit.

[0026] The present application also includes a detection kit for assisting in predicting the occurrence of epilepsy, which realizes the diagnosis function by detecting the expression levels of CXCL17 and GPR35 and performing joint interpretation.

[0027] The kit can directly detect the chemokine 17 and the ligand G protein-coupled receptor 35 in brain tissue, peripheral blood, serum, plasma or cerebrospinal fluid.

[0028] The kit comprises: 1. A solid phase carrier coated with a capture antibody: the capture antibody is a specific antibody against CXCL17 or GPR35.

[0029] 2. A detection antibody: a labeled antibody against the same target protein (CXCL17 or GPR35) and different from the capture antibody in binding epitope, and the label is horseradish peroxidase, alkaline phosphatase, biotin or fluorescein.

[0030] 3. A standard: a series of known concentrations of recombinant CXCL17 protein and / or recombinant GPR35 protein for drawing a standard curve.

[0031] 4. Sample diluent: for diluting the biological sample.

[0032] 5. Wash solution: phosphate buffer solution containing Tween 20.

[0033] 6. Substrate solution: color developing substrate corresponding to the marker, such as TMB substrate solution.

[0034] 7. Stop solution: sulfuric acid or hydrochloric acid solution.

[0035] The application also provides the use of the above-mentioned kit in evaluating the severity of epilepsy or monitoring the effect of drug treatment.

[0036] The application also provides a method for using the kit, comprising the following steps: Taking a peripheral blood sample of a patient; Detecting the peripheral blood sample by using the detection kit, drawing a standard curve, marking CXCL17 and / or GPR35, drawing a concentration curve of the marker, and comparing the concentration curve with the standard curve.

[0037] The peripheral blood is the venous blood of the patient, wherein the content of the chemokine 17 and the ligand G protein-coupled receptor 35 in the serum sample is detected by extracting the serum in the peripheral blood and using a product capable of detecting the chemokine 17 and the ligand G protein-coupled receptor 35 in the serum sample.

[0038] If the content exceeds the set value, it can be judged that the patient has a good treatment response.

[0039] The product has high relevance and high accuracy, and can directly detect the peripheral blood. Compared with electroencephalogram and imaging examination and long-term drug observation, the use mode of the product is relatively simple, easy to use, and convenient for rapid judgment of the patient.

[0040] DETAILED DESCRIPTION: The application will be further described below in conjunction with examples, but the scope of protection of the application is not limited thereto.

[0041] Example 1: CXCL17 and GPR35 as diagnostic markers for predicting the occurrence of epilepsy 1. Sample collection: Collecting fasting venous blood of clinically diagnosed epilepsy patients and healthy volunteers, separating serum or whole blood, and storing at -80℃.

[0042] 2. Detection method: Using commercial ELISA kit (detecting human CXCL17 and GPR35), strictly following the instructions, detecting the concentration of CXCL17 and GPR35 in all serum samples.

[0043] 3. Statistical analysis: The results are shown in Figure 1 and Figure 2 . Figure 1This is a statistical graph showing the expression level of GPR35 in serum samples from epilepsy patients compared to the non-epileptic control group, as detected by ELISA. Figure 2 This is a statistical graph showing the expression levels of CXCL17 in serum samples from epilepsy patients compared to the non-epileptic control group, as detected by ELISA. Analysis was performed using a two-sided unpaired t-test. Data represent mean ± SEM.

[0044] from Figure 1 It was found that, according to ELISA, the expression level of GPR35 in serum samples of epilepsy patients (n = 42) was increased compared with that of the non-epileptic control group (n = 35).

[0045] from Figure 2 It was found that, according to ELISA, the expression level of CXCL17 in serum samples of epilepsy patients (n = 42) was increased compared with that of the non-epileptic control group (n = 35).

[0046] The results showed that the serum CXCL17 and GPR35 concentrations in the epilepsy group were significantly higher than those in the healthy control group (P < 0.05). Epilepsy leads to a simultaneous increase in the expression levels of serum GPR35 and CXCL17, and the simultaneous increase in the expression levels of serum GPR35 and CXCL17 is significantly associated with the occurrence of epilepsy.

[0047] Figure 3 ROC curve analysis of GPR35 and CXCL17 in distinguishing between epileptic patients (n = 42) and non-epileptic controls.

[0048] Figure 4 The ROC curve analysis of the two indicators in the binary logistic regression model to establish a joint predictive factor in distinguishing between epileptic patients (n=42) and non-epileptic controls was statistically plotted.

[0049] ROC curve analysis showed that the AUC value of CXCL17 for diagnosing epilepsy was 0.8429, and the AUC value of GPR35 was 0.6646. Incorporating both indicators into a binary logistic regression model to establish a joint predictive factor increased the AUC value to 0.854 (95% CI: 0.772–0.935), demonstrating good diagnostic potential in distinguishing between epileptic patients (n = 42) and non-epileptic controls (n = 35), with both sensitivity and specificity superior to single indicators.

[0050] 4. Conclusion: CXCL17 and GPR35 are excellent biomarkers for predicting the occurrence of epilepsy, and their combined use has even greater value. ROC curve analysis assessed that GPR35 and CXCL17 have good diagnostic potential in distinguishing between epileptic patients (n = 42) and non-epileptic controls (n = 35).

[0051] Example 2 The present embodiment provides a double-index combined detection ELISA kit for detecting the concentrations of CXCL17 and GPR35 in human serum or plasma. The kit comprises the following components: 1. Solid phase carrier coated with capture antibody: the capture antibody is a specific antibody against CXCL17 or GPR35.

[0052] 2. Detection antibody: a labeled antibody against the same target protein (CXCL17 or GPR35) and different from the capture antibody in binding epitope, the label being horseradish peroxidase, alkaline phosphatase, biotin or fluorescein.

[0053] 3. Standard: a series of known concentrations of recombinant CXCL17 protein and / or recombinant GPR35 protein for drawing a standard curve.

[0054] 4. Sample diluent: for diluting biological samples.

[0055] 5. Washing solution: such as phosphate buffer containing Tween 20.

[0056] 6. Substrate developing solution: a developing substrate corresponding to the label, such as TMB substrate solution.

[0057] 7. Stop solution: such as sulfuric acid or hydrochloric acid solution.

[0058] Example 3 The steps for detecting the levels of CXCL17 and GPR35 in human serum samples using the kit of the present application are as follows: 1. Sample preparation: collect peripheral venous blood from the subject to be tested, centrifuge to separate serum or whole blood, and store at -80°C for testing. Restore to room temperature before testing.

[0059] 2. Sample addition: set up blank wells, standard wells and sample wells respectively. Add 50 μL of different concentrations of standard to the standard wells, and 50 μL of serum to be tested to the sample wells.

[0060] 3. Incubation: incubate at 37°C for 60 minutes. Wash the plate 5 times.

[0061] 4. Add detection antibody: add 50 μL of corresponding detection antibody to each well (add CXCL17 detection antibody to CXCL17 detection plate, and add GPR35 detection antibody to GPR35 detection plate). Incubate at 37°C for 60 minutes. Wash the plate 5 times.

[0062] 5. Add enzyme complex: add 50 μL of streptavidin-HRP to each well. Incubate at 37°C for 30 minutes in the dark. Wash the plate 5 times.

[0063] 6. Color development: add 90 μL of TMB substrate solution to each well, and develop color at 37°C in the dark for 15-20 minutes.

[0064] 7. Termination with assay: Add 50 μL of termination solution to each well and mix gently. Immediately measure the absorbance value of each well at 450 nm wavelength using a microplate reader.

[0065] Result analysis 1. Standard curve drawing: Draw the standard curve with the concentration of the standard as the abscissa and the corresponding absorbance value as the ordinate, and obtain the regression equation (usually four-parameter logistic curve fitting).

[0066] 2. Concentration calculation: Substitute the OD value of the sample well into the standard curve equation to calculate the concentration of CXCL17 and GPR35 in the sample.

[0067] 3. Result interpretation: According to the standard curve, calculate the concentration of CXCL17 and GPR35 in the sample.

[0068] The present application determines the optimal cutoff value through ROC curve analysis. The specific method is as follows: taking patients diagnosed with epilepsy (n = 42) as the experimental group and healthy volunteers (n = 35) as the control group, draw the ROC curve of CXCL17 and GPR35 for diagnosing epilepsy. The optimal cutoff value is determined by the Youden index (sensitivity + specificity - 1) maximum principle.

[0069] The results show that the optimal cutoff value of CXCL17 is 1225 pg / mL (at this time the sensitivity is 100% and the specificity is 48.57%), and the optimal cutoff value of GPR35 is 3.678 pg / mL (at this time the sensitivity is 92.86% and the specificity is 31.43%).

[0070] To further improve the specificity of diagnosis, the joint interpretation standard proposed by the present application is: when the serum CXCL17 concentration is ≥ 1300 pg / mL and the GPR35 concentration is ≥ 4.0 pg / mL, that is, it exceeds the preset value, it is determined as a positive result.

[0071] For patients who have not been diagnosed, the preset value is calculated, the sensitivity of this joint interpretation standard is 83.3%, the specificity is as high as 71.4%, the cut-off value is 0.4455, and its specificity is significantly higher than that of single index detection (see Table 1), which can effectively reduce the false positive rate, and is especially suitable for predicting the occurrence of epilepsy as an auxiliary means. Figure 4 , Table 1), which can effectively reduce the false positive rate, and is especially suitable for predicting the occurrence of epilepsy as an auxiliary means.

[0072] The greater the concentration data of CXCL17 and GPR35, the higher the risk of epilepsy occurrence can be determined, and the indicators are positively correlated with the degree of disease development, which can accurately classify the drug responsiveness of patients at the molecular level in the early stage of treatment or even before treatment.

[0073] Example 4: Dynamic monitoring For patients who have been diagnosed, the preset value is the joint detection data of the patient's previous individual.

[0074] The combined expression level of CXCL17 and GPR35 gradually increases in the chronic phase of epilepsy and tends to be stable after reaching the peak, which may be related to the neuroprotective mechanism triggered by epilepsy, and has important diagnostic and therapeutic significance. Regularly detect the level of CXCL17 and / or GPR35 before and after drug treatment. If the level significantly increases after treatment, it suggests that the treatment may be effective; if the level remains unchanged or decreases, it suggests poor efficacy or poor prognosis.

[0075] In Figure 4 The area under the curve analysis is shown in Table 1: Table 1 The prediction probability of the combined prediction molecule is shown in Table 2: Table 2 Greater than or equal to this value is positive a ]] Sensitivity 1 - Specificity Greater than or equal to this value is positive a ]] Sensitivity 1 - Specificity 0.0000000 1.000 1.000 0.6096853 0.667 0.286 0.0211872 1.000 0.971 0.6177996 0.667 0.257 0.0218829 1.000 0.943 0.6264675 0.667 0.229 0.0250251 1.000 0.914 0.6348102 0.667 0.200 0.0312483 1.000 0.886 0.6442663 0.667 0.171 0.0362565 1.000 0.857 0.6475299 0.643 0.171 0.0420819 1.000 0.829 0.6492921 0.619 0.171 0.0608394 1.000 0.800 0.6731081 0.619 0.143 0.0771986 1.000 0.771 0.6986565 0.595 0.143 0.0839567 1.000 0.743 0.7156539 0.595 0.114 0.0991245 1.000 0.714 0.7347095 0.571 0.114 0.1127160 1.000 0.686 0.7432970 0.548 0.114 0.1241716 1.000 0.657 0.7514848 0.524 0.114 0.1382434 1.000 0.629 0.7582286 0.524 0.086 0.1641442 1.000 0.600 0.7864207 0.500 0.086 0.1939228 1.000 0.571 0.8169466 0.476 0.086 0.2222897 1.000 0.543 0.8229646 0.452 0.086 0.2425501 0.976 0.543 0.8280328 0.452 0.057 0.2547510 0.976 0.514 0.8315882 0.452 0.029 0.2745369 0.952 0.514 0.8493880 0.452 0.000 0.2939899 0.952 0.486 0.8709917 0.429 0.000 0.3173358 0.929 0.486 0.8806236 0.405 0.000 0.3347302 0.929 0.457 0.8941154 0.381 0.000 0.3460671 0.905 0.457 0.9035226 0.357 0.000 0.3556846 0.905 0.429 0.9096742 0.333 0.000 0.3628734 0.905 0.400 0.9163804 0.310 0.000 0.3831513 0.881 0.400 0.9203147 0.286 0.000 0.3991018 0.881 0.371 0.9263870 0.262 0.000 0.4075584 0.881 0.343 0.9343295 0.238 0.000 0.4219563 0.857 0.343 0.9383601 0.214 0.000 0.4350032 0.857 0.314 0.9388652 0.190 0.000 0.4424889 0.833 0.314 0.9395995 0.167 0.000 0.4455091 0.833 0.286 0.9419280 0.143 0.000 0.4597226 0.810 0.286 0.9501121 0.119 0.000 0.4910719 0.786 0.286 0.9579429 0.095 0.000 0.5205967 0.762 0.286 0.9612236 0.071 0.000 0.5391934 0.738 0.286 0.9714641 0.048 0.000 0.5490722 0.714 0.286 0.9863055 0.024 0.000 0.5795811 0.690 0.286 1.0000000 0.000 0.000 a. The minimum boundary value is the minimum measured test value minus 1, and the maximum boundary value is the maximum measured test value plus 1. All other boundary values are the average of two consecutive ordered measured test values.

[0076] Example 5: Exploring changes in GPR35 expression in animal models and functional studies By using KA to induce seizures in mice, the control group received intraperitoneal injection of the same amount of normal saline. The acute seizure stage was evaluated by the Racine scale, and only animals showing IV to VI seizures were included in the subsequent analysis. We set five time points for evaluating GPR35 in each group: 3 days, 1 week, 2 weeks, 4 weeks, and 8 weeks after KA induction or control group. The results are shown in Figure 5 As Figure 6 shown, Figure 5 is a representative Western blotting statistical chart of GPR35 protein levels in the hippocampus of mice at different time points after kainic acid-induced seizures or control group, Figure 6 is a representative Western blotting quantitative statistical chart of GPR35 protein levels in the hippocampus of mice at different time points after kainic acid-induced seizures or control group. The results show that GPR35 starts to increase after modeling, and tends to be stable after reaching the peak expression at 1 month.

[0077] To determine whether GPR35 has a function in the occurrence of epilepsy, we used CRISPR / Cas9 technology to generate GPR35 whole gene knockout mice (GPR35 KO), to verify the role of GPR35 in epileptogenesis. We will induce chronic epilepsy in GPR35 KO and WT mice with KA (Kainic acid) to prepare a chronic epilepsy model for experiments ( Figure 7 ). In the chronic phase (4 weeks), we will evaluate seizure activity on electroencephalogram (EEG) by in vivo EEG recording and detect the expression of inflammatory factors by PCR. The results are shown in Figure 8-9 , which show that EEG shows increased seizure severity in GPR35 KO epileptic mice ( Figure 8 ), and higher mRNA expression of pro-inflammatory factors ( Figure 9 ). These results find that the absence of GPR35 increases the severity of chronic seizures and is involved in the neuroinflammatory pathophysiology of epilepsy, indicating its involvement in the pathogenesis of epilepsy. In summary, the compensatory increase in the expression of GPR35 in the chronic phase plays an important role as a protective molecule in the neuroprotective mechanism of epilepsy.

[0078] Example 6: Exploring the effect of overexpression of GPR35 on inflammatory factors in a cell model First, we constructed an LV-GPR35 overexpression vector by cloning the GPR35 gene into an appropriate plasmid as the experimental group, and used a vector without the GPR35 gene as the control group. We packaged the virus by HEK293T cells, produced LV-GPR35 overexpression virus, infected primary microglial cells with LV-GPR35 overexpression virus, and screened stable strains. Then, we induced the stable strain cells with glutamate (10 mM) for 12 hours to prepare a cell model. Next, we used enzyme-linked immunosorbent assay (ELISA) to detect the levels of cytokines (TNF-a, IL-1b, IL-6, IL-10) in the culture supernatant, with 4 replicates per group (n = 4). Finally, we analyzed the differences in cytokine levels between the LV-GPR35 overexpression group and the control group by statistical methods. The results are shown in Figure 10-13 , which show that overexpression of GPR35 significantly reduces the secretion of pro-inflammatory factors (TNF-a, IL-1b, and IL-6) and increases the secretion of anti-inflammatory factor IL-10 under glutamatergic stimulation.

[0079] The present inventors first used human single-cell sequencing (snRNA-seq) datasets (GSE140393, GSE190452) to characterize the distribution of GPR35 in the hippocampus of patients with refractory epilepsy, cluster analysis identified 9 major cell types with representative marker genes, and cell population analysis revealed significant differences between refractory epilepsy patients and controls, with decreased neurons and increased microglia in refractory epilepsy patients ( Figure 14-15 ).

[0080] Among them, GPR35 is significantly up-regulated in microglia cells of refractory epilepsy patients and control group, and is not significantly up-regulated in other cell types. Further research found that the protein expression level of chemokine CXCL17 and its receptor GPR35 in serum samples of epilepsy patients was significantly higher than that of healthy controls.

[0081] Further receiver operating characteristic curve analysis (ROC curve) shows that the combination of CXCL17 and GPR35 has high diagnostic efficiency for distinguishing epilepsy patients from healthy people (wherein the area under the curve AUC of the index chemokine CXCL17 is approximately 0.85), showing the potential as an ideal diagnostic biomarker.

[0082] In addition, the embodiment of the present application further verifies the expression change and function of GPR35 in animal models; the knockout of GPR35 gene in mice significantly increases the susceptibility to epilepsy; and it is verified in a cell model that overexpression effectively reduces the expression of pro-inflammatory factors and increases the expression of anti-inflammatory factors.

[0083] The combined expression level of CXCL17 and GPR35 gradually increases in the chronic stage of epilepsy, which may be closely related to the neuroprotective mechanism triggered by epilepsy, and has important clinical diagnostic and therapeutic significance. Overcome the lag and passive defects of existing clinical detection products, realize high sensitivity and high specificity diagnosis, and help to accurately classify the drug responsiveness of epilepsy patients, and provide strong support for the development of individualized treatment plan.

[0084] The above examples are only exemplary embodiments of the present application and are not used to limit the present application, and the protection scope of the present application is defined by the claims. Those skilled in the art can make various modifications or equivalent replacements to the present application within the spirit and protection scope of the present application, and such modifications or equivalent replacements shall also be considered to fall within the protection scope of the present application.

Claims

1. The application of a biomarker in the preparation of products for predicting the occurrence of epilepsy, characterized in that, The biomarkers are CXCL17 and GPR35; The detection results were correlated with the co-expression levels of CXCL17 and GPR35 in the biological samples.

2. The application of a biomarker according to claim 1 in the preparation of products for predicting the occurrence of epilepsy, characterized in that, The biological samples are mouse brain tissue, peripheral blood, serum, plasma, or cerebrospinal fluid.

3. The application of a biomarker according to claim 2 in the preparation of products for predicting the occurrence of epilepsy, characterized in that, The product in question is a test kit.

4. A test kit according to any one of claims 1-3, characterized in that, The kit is used to help predict the occurrence of epilepsy; The kit achieves diagnostic functionality by detecting and jointly interpreting the expression levels of CXCL17 and GPR35.

5. The detection kit according to claim 4, characterized in that, The kit includes a solid-phase carrier coated with capture antibodies, detection antibodies, standards, sample diluent, washing buffer, substrate chromogenic solution, and stop solution; The capture antibody is a specific antibody against the target protein CXCL17 or GPR35. The detection antibody is a labeled antibody that targets the same target protein but has a different binding epitope than the capture antibody. The standards are recombinant CXCL17 protein and / or recombinant GPR35 protein at known concentrations, and are used to plot a standard curve.

6. The detection kit according to claim 5, characterized in that, The marker for the labeled antibody is any one of horseradish peroxidase, alkaline phosphatase, biotin, or fluorescein. The washing solution is a phosphate buffer containing Tween 20; The terminating solution is a sulfuric acid or hydrochloric acid solution.

7. The use of a kit according to any one of claims 4-6 in determining the occurrence of epilepsy or monitoring the effect of drug treatment.

8. The application according to claim 7, characterized in that, Includes the following steps: The sample was subjected to joint detection using the CXCL17 detection antibody and GPR35 detection antibody in the kit to obtain the concentration data of CXCL17 and GPR35 in the sample. The concentration data of CXCL17 and the concentration data of GPR35 are compared with preset values ​​to obtain a determination result.

9. The application according to claim 8, characterized in that, The preset value is either the joint interpretation standard or the individual's previous joint detection data.

10. The application according to claim 9, characterized in that, The joint interpretation criteria are as follows: The concentration of CXCL17 is ≥ 1300 pg / mL and the concentration of GPR35 is ≥ 4.0 pg / mL.

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