Use of the marker in the manufacture of a diagnostic or aid-to-diagnosis product for ischemic stroke

Abstract

The application discloses application of a marker in preparation of a product for diagnosing or assisting in diagnosing ischemic cerebral stroke, the marker being CLDN7 and / or PPM1B, and it is found through verification of clinical samples that the marker has high diagnostic efficiency on ischemic cerebral stroke, can be used as a biomarker for diagnosing and differentiating ischemic cerebral stroke, and has the advantages of high accuracy, good specificity, high sensitivity and the like, provides a brand-new thought and strategy for early diagnosis of ischemic cerebral stroke in the field, and has good clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to the application of biomarkers in the preparation of products for the diagnosis or auxiliary diagnosis of ischemic stroke. More specifically, the biomarkers are CLDN7 and / or PPM1B. Background Technology

[0002] Ischemic stroke (IS) refers to the necrosis of brain tissue caused by insufficient blood supply to the brain due to stenosis or occlusion of the arteries supplying the brain (carotid and vertebral arteries). It is a sudden neurological deficit caused by localized cerebral ischemia and permanent cerebral infarction (e.g., positive DWI). Clinical studies show that many factors can induce ischemic stroke. Common causes (listed in order of incidence) include atherosclerotic occlusion of large arteries, cerebral embolism (embolic infarction), non-embolic infarction of small deep perforating arteries (lacunar infarction), and watershed infarction caused by distal arterial stenosis and decreased cerebral blood flow (hemodynamic stroke). Diagnosis of ischemic stroke relies primarily on clinical diagnosis. CT or MRI can help rule out hemorrhage, confirm the diagnosis, and determine the extent of the lesion. For some patients, acute-phase thrombolytic therapy may be helpful. Depending on the cause, carotid endarterectomy or stent implantation, antiplatelet therapy, or warfarin can help reduce the incidence of recurrent stroke.

[0003] The increasing burden of vascular risk factors and the prominent problem of population aging have led to the increasing prevalence of neurovascular diseases. Among them, ischemic stroke accounts for 87% of all strokes. Due to its high incidence, high recurrence rate, high mortality rate, and high disability rate, it is a major disease that is recognized worldwide as a serious threat to human health. Ultra-early or early (within 6 hours of onset) individualized treatment is crucial for the prognosis of patients with acute stroke. Currently, there are several problems: (1) Ischemic stroke is diagnosed through clinical assessment and neuroimaging, which takes a long time; (2) Many patients with transient ischemic attacks are asymptomatic when they arrive at the hospital, making accurate neurological function assessment impossible; (3) Even if patients have similar basic characteristics at the time of stroke, their prognosis may vary greatly. These factors can delay the diagnosis and targeted treatment of patients, leading to missed opportunities for optimal treatment. Although blood biomarkers have been used for many years in the diagnosis of other vascular diseases, no blood biomarkers for stroke have been applied clinically, and the accuracy of currently reported biomarkers is far below the requirements of clinical practice.

[0004] Therefore, developing rapid and convenient blood biomarker diagnostic indicators for ischemic stroke patients is of great clinical significance for the early diagnosis and subsequent treatment of ischemic stroke. Summary of the Invention

[0005] In view of this, in order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide biomarkers that can be used to diagnose or assist in the diagnosis of ischemic stroke, wherein the biomarkers are CLDN7 and / or PPM1B.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides the use of reagents for detecting the expression levels of CLDN7 and / or PPM1B in samples in the preparation of products for diagnosing or assisting in the diagnosis of ischemic stroke.

[0008] Furthermore, the reagents include those used to detect the expression levels of CLDN7 and / or PPM1B in samples using sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, chromatography technology, and mass spectrometry technology.

[0009] Furthermore, the reagents include reagents for detecting the expression levels of CLDN7 and / or PPM1B mRNA in the sample, and reagents for detecting the expression levels of proteins and / or peptides encoded by CLDN7 and / or PPM1B in the sample.

[0010] Furthermore, the reagents for detecting the expression levels of CLDN7 and / or PPM1B mRNA in the sample include probes that specifically recognize CLDN7 and / or PPM1B, and primers that specifically amplify CLDN7 and / or PPM1B.

[0011] Furthermore, the reagents used to detect the expression levels of proteins and / or peptides encoded by CLDN7 and / or PPM1B in the sample include antibodies that specifically bind to CLDN7 and / or PPM1B, antibody fragments, and affinity proteins.

[0012] Furthermore, the sequences of the primers for specifically amplifying CLDN7 are shown in SEQ ID NO:5-SEQ ID NO:6;

[0013] The sequences of the primers for specifically amplifying PPM1B are shown in SEQ ID NO:7-SEQ ID NO:8.

[0014] Furthermore, the sample is a blood sample.

[0015] Furthermore, the sequencing technologies include (but are not limited to): first-generation sequencing, second-generation sequencing, and third-generation sequencing. First-generation sequencing, also known as Sanger sequencing, is a sequencing technology that utilizes DNA polymerase synthesis reactions. First-generation sequencing is a sequencing technology based on the Sanger method. Second-generation sequencing is based on massive parallel analysis (MPS), which can simultaneously complete the synthesis of complementary strands of sequencing templates and the acquisition of sequence data. Third-generation sequencing is based on single-molecule sequencing and massive parallel sequencing technologies.

[0016] Furthermore, the nucleic acid hybridization technology refers to the process by which complementary nucleotide sequences (DNA and DNA, DNA and RNA, RNA and RNA, etc.) form non-covalent bonds through Watson-Crick base pairing, thereby forming stable homologous or heterologous double-stranded molecules, also known as nucleic acid hybridization.

[0017] Furthermore, the aforementioned nucleic acid amplification technology refers to a general term for a large class of technical methods. Currently, nucleic acid amplification technologies include conventional PCR, real-time fluorescence PCR, and isothermal nucleic acid amplification technology, which can specifically amplify trace amounts of target DNA by millions of times, thereby greatly improving the ability to analyze and detect DNA molecules. It can detect single DNA molecules or samples containing only one target DNA molecule per 100,000 cells.

[0018] Furthermore, the protein immunoassay technique refers to a class of methods that include radioimmunoassay, direct, indirect or comparative enzyme-linked immunosorbent assay, enzyme immunoassay, fluorescence immunoassay, Western blotting, immunoprecipitation, and immunoassay based on any particle (e.g., using gold particles, silver particles or latex particles, magnetic particles or quantum dots) to detect target analytes, and can be performed in the form of microtiter plates or strips.

[0019] Furthermore, the chromatographic technique refers to a method for separating and analyzing various components in a complex mixture. It utilizes the fact that different substances have different partition coefficients in a system composed of a stationary phase and a mobile phase. When the two phases move relative to each other, these substances move together with the mobile phase and undergo repeated partitioning between the two phases, thereby achieving separation of the substances.

[0020] Furthermore, the mass spectrometry technique refers to a method that uses electric and magnetic fields to separate and detect moving ions (charged atoms, molecules or molecular fragments, isotopic ions, fragment ions, rearranged ions, multiply charged ions, metastable ions, negative ions, and ions generated by ion-molecule interactions) according to their mass-to-charge ratio. Measuring the accurate mass of the ions allows for the determination of their compound composition.

[0021] A second aspect of the present invention provides a product for diagnosing or assisting in the diagnosis of ischemic stroke.

[0022] Furthermore, the product contains reagents for detecting the expression levels of CLDN7 and / or PPM1B in the sample.

[0023] In some implementations, the product includes chips, reagent kits, test strips, and high-throughput sequencing platforms.

[0024] In some embodiments, the reagents for detecting the expression levels of CLDN7 and / or PPM1B in the sample include reagents for detecting the expression levels of CLDN7 and / or PPM1B mRNA in the sample, and reagents for detecting the expression levels of proteins and / or peptides encoded by CLDN7 and / or PPM1B in the sample.

[0025] In some embodiments, the reagent for detecting the expression level of CLDN7 and / or PPM1B mRNA in the sample includes a probe that specifically recognizes CLDN7 and / or PPM1B, and primers that specifically amplify CLDN7 and / or PPM1B. In some preferred embodiments, the sequences of the primers that specifically amplify CLDN7 are shown in SEQ ID NO:5-SEQ ID NO:6, and the sequences of the primers that specifically amplify PPM1B are shown in SEQ ID NO:7-SEQ ID NO:8.

[0026] In some embodiments, the reagents used to detect the expression levels of proteins and / or peptides encoded by CLDN7 and / or PPM1B in the sample include antibodies that specifically bind to CLDN7 and / or PPM1B, antibody fragments, and affinity proteins.

[0027] Furthermore, the chip can be prepared using conventional biochip preparation methods known to those skilled in the art, including (but not limited to): using a solid-phase support of a modified glass slide or silicon wafer, the 5' end of the probe containing an amino-modified polydT string, preparing the oligonucleotide probe into a solution, and then using a spotting instrument to spot it onto the modified glass slide or silicon wafer, arranging it into a predetermined sequence or array, and then fixing it by leaving it overnight, thus obtaining the chip described in this invention.

[0028] Furthermore, the kit also includes instructions for use or a label, a positive control, a negative control, a buffer, an adjuvant, or a solvent, and one or more containers for containing the compositions contained in the kit. The compositions may be in liquid form or lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials, including glass or plastic. The instructions for use or label details how to use the kit to test samples and its use in diagnosing or assisting in the diagnosis of ischemic stroke.

[0029] Furthermore, the kit may also include a variety of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed in this invention. Any reagent that is based on the detection of CLDN7 and / or PPM1B to diagnose or assist in the diagnosis of ischemic stroke is included within the scope of protection of this invention.

[0030] Furthermore, the kit includes a qPCR kit, an immunoblotting kit, an immunochromatographic assay kit, a flow cytometry kit, an immunohistochemistry kit, an ELISA kit, and an electrochemiluminescence assay kit.

[0031] The primers included in the products described in this invention can be prepared by chemical synthesis. They can be appropriately designed using methods well-known to those skilled in the art, with reference to known information, and prepared by chemical synthesis, and are not limited to the primers shown in SEQ ID NO:5-SEQ ID NO:6 and SEQ ID NO:7-SEQ ID NO:8 as described in this invention. The antibodies in the products described in this invention can be antibodies or fragments thereof of any structure, size, immunoglobulin class, origin, etc., as long as they bind to the target protein. The antibodies or fragments thereof included in the products of this invention can be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody that retains the binding activity of the antibody against an antigen, or a peptide containing a portion of an antibody. Antibody fragments may include F(ab′)2, Fab′, Fab, single-chain Fv (scFv), disulfide-bonded Fv (dsFv) or polymers thereof, dimerized V regions (biantibodies), or peptides containing CDRs. Antibodies can be obtained by methods well-known to those skilled in the art. For example, mammalian cell expression vectors that retain whole or part of the target protein or integrate the polynucleotides encoding them can be prepared as antigens. After immunizing animals with an antigen, immune cells are obtained from the immunized animals and fused with cancer cells to obtain hybridomas. Antibodies are then collected from the hybridoma cultures. Finally, monoclonal antibodies against the marker protein can be obtained by antigen-specific purification of the obtained antibodies using the marker protein or a portion thereof, which was used as the antigen.

[0032] A third aspect of the present invention provides a method for screening candidate drugs for the treatment of ischemic stroke.

[0033] Furthermore, the method includes the following steps:

[0034] (1) Contact the analyte with a system containing or expressing CLDN7 and / or PPM1B;

[0035] (2) Detect the expression levels of CLDN7 and / or PPM1B in the system;

[0036] (3) Select substances that can reduce CLDN7 expression level and / or increase PPM1B expression level as candidate drugs for the treatment of ischemic stroke.

[0037] In some implementations, the system includes (but is not limited to): a cellular system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

[0038] A fourth aspect of the present invention provides a diagnostic system for diagnosing or assisting in the diagnosis of ischemic stroke.

[0039] Furthermore, the diagnostic system includes:

[0040] (1) Ischemic stroke assessment device: including a control unit and a storage unit, used to assess whether the subject has an ischemic stroke;

[0041] (2) Information communication terminal devices that are interconnected: used to provide data on the expression levels of CLDN7 and / or PPM1B in samples from subjects;

[0042] The control unit of the ischemic stroke assessment device includes the following four units:

[0043] 1) Data receiving unit: used to receive data transmitted from the information communication terminal device regarding the expression levels of CLDN7 and / or PPM1B in the sample;

[0044] 2) Discriminant value calculation unit: It calculates the discriminant value based on the discrimination of the expression levels of CLDN7 and / or PPM1B in the sample received by the data receiving unit and the expression levels of CLDN7 and / or PPM1B stored in the storage unit as explanatory variables;

[0045] 3) Discriminant value benchmark evaluation unit: It evaluates the risk of ischemic stroke in the subject based on the discriminant value calculated by the discriminant value calculation unit;

[0046] 4) Evaluation result sending unit: It sends the evaluation results of the subject obtained by the discriminant benchmark evaluation unit to the information communication terminal device.

[0047] Furthermore, the sample is a blood sample from the subject.

[0048] The biomarker CLDN7 (claudin 7) described in this invention has a Gene ID of 1366 in NCBI (https: / / www.ncbi.nlm.nih.gov / ), specifically located at 17p13.1.

[0049] The biomarker PPM1B (protein phosphatase, Mg2+ / Mn2+dependent1B) described in this invention has a Gene ID of 5495 in NCBI (https: / / www.ncbi.nlm.nih.gov / ), specifically located at 2p21.

[0050] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0051] This invention is the first to discover that CLDN7 and / or PPM1B are closely related to ischemic stroke and can be used as biomarkers for the diagnosis and differentiation of ischemic stroke. It has the advantages of high accuracy, good specificity and high sensitivity, providing a new idea and strategy for the early diagnosis of ischemic stroke in this field and has good clinical application value. Attached Figure Description

[0052] Figure 1 This is a graph showing the results of differentially expressed genes identified in the screening set;

[0053] Figure 2 A graph showing the differential expression of CLDN7 between patients with ischemic stroke and normal controls in the screening set;

[0054] Figure 3 A graph showing the differential expression of PPM1B between patients with ischemic stroke and normal controls in the screening set;

[0055] Figure 4 The ROC curve results for CLDN7 in the screening set for the diagnosis of ischemic stroke are shown in the figure, where the vertical axis represents sensitivity and the horizontal axis represents specificity.

[0056] Figure 5 The ROC curve results for PPM1B in the screening set for the diagnosis of ischemic stroke are shown in the figure, where the vertical axis represents sensitivity and the horizontal axis represents specificity.

[0057] Figure 6 The ROC curve results for CLDN7+PPM1B in the screening set for the diagnosis of ischemic stroke are shown in the figure, where the vertical axis represents sensitivity and the horizontal axis represents specificity.

[0058] Figure 7 Figure A shows the real-time amplification curve of the internal control GAPDH gene and the product dissolution curve. Figure B shows the product dissolution curve.

[0059] Figure 8Figure A shows the real-time amplification curve of the internal control ACTB gene and the product dissolution curve. Figure B shows the product dissolution curve.

[0060] Figure 9 Figure A shows the real-time amplification curve of the CLDN7 gene and the product dissolution curve. Figure B shows the product dissolution curve.

[0061] Figure 10 Figure A shows the real-time amplification curve of the PPM1B gene and the product melting curve. Figure B shows the product melting curve.

[0062] Figure 11 A graph showing the differential expression of CLDN7 between patients with ischemic stroke and normal controls in the validation set;

[0063] Figure 12 A graph showing the differential expression of PPM1B between patients with ischemic stroke and normal controls in the validation set;

[0064] Figure 13 The ROC curve results for CLDN7 in the validation set for diagnosing ischemic stroke are shown in the figure, where the vertical axis represents sensitivity and the horizontal axis represents specificity.

[0065] Figure 14 The ROC curve results of PPM1B for diagnosing ischemic stroke in the validation set are shown in the figure, where the vertical axis represents sensitivity and the horizontal axis represents specificity.

[0066] Figure 15 The ROC curve results for CLDN7+PPM1B in the validation set for the diagnosis of ischemic stroke are shown in the figure. The vertical axis represents sensitivity and the horizontal axis represents specificity. Detailed Implementation

[0067] Unless otherwise defined, all technical and scientific terms used in the context of this invention have the same meaning as understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention; furthermore, some terms are explained below.

[0068] As used herein, the term "expression level" refers to the degree to which a specific biomarker sequence is transcribed from its genomic locus, i.e., the concentration of the biomarker in one or more blood samples analyzed. In a specific embodiment of the invention, the specific biomarker is CLDN7 and / or PPM1B.

[0069] As used herein, the term "biomarker" refers to a molecular indicator possessing specific biological, biochemical, or other characteristics that can be used to determine the presence or absence of a particular disease or condition and / or the severity of a particular disease or condition. In a specific embodiment of the invention, the particular disease or condition refers to ischemic stroke. In this invention, the term "biomarker" specifically refers to a compound, preferably a gene, that is differentially present (i.e., increased or decreased) in a biological sample from a subject or group of subjects with a first phenotype (e.g., having the disease), compared to a biological sample from a subject or group of subjects with a second phenotype (e.g., no disease). This term generally refers to the presence / concentration / content of one gene or the presence / concentration / content of two or more genes; in a specific embodiment of the invention, the biomarker is CLDN7 and / or PPM1B.

[0070] Biomarkers can be present differentially at any level, but are generally present at levels that are increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, and at least 140%. %, at least 150%, or more; or generally present at levels reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (i.e., absent), preferably, the biomarker is statistically significant (P < 0.05).

[0071] As used herein, the term "sample" refers to a composition obtained from or derived from a subject (e.g., an individual of interest) containing cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. For example, a sample refers to any sample derived from a subject of interest that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, tissue culture fluid, tissue extracts, homogenized tissue, tumor tissue, cell extracts, and combinations thereof. As a preferred embodiment, the sample is selected from blood, serum, and plasma; as another preferred embodiment, the sample is selected from tissue. In a specific embodiment of the invention, the sample is preferably a blood sample derived from a subject.

[0072] As used herein, the term "subject" refers to any animal, including both human and non-human animals. Non-human animals include all vertebrates, such as mammals like non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; as well as non-mammals such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0073] As used herein, the term "treatment" refers to any treatment involving humans or animals (e.g., applied by a veterinarian) that achieves certain desired therapeutic effects, such as inhibiting the development of a disease (including slowing its progression, stopping its development), improving the condition, and curing the disease. It also includes treatment as a preventative measure (e.g., prevention). Use in patients who have not yet developed the disease but are at risk of developing it is also included in the term "treatment."

[0074] As used herein, the term "differential expression" refers to a change in the expression level of a biomarker in a target sample compared to a control sample, which may be a sample from healthy individuals. This change can be either upregulated (i.e., an increase in the concentration of the biomarker in the target sample) or downregulated (i.e., a decrease in the concentration of the biomarker or its disappearance in the target sample). In a specific embodiment of the invention, the biomarker is CLDN7 and / or PPM1B.

[0075] As used herein, the term "primer" refers to an oligonucleotide that can be used in amplification methods such as polymerase chain reaction (PCR) to amplify a nucleotide sequence based on a polynucleotide sequence corresponding to a target gene (e.g., the sequence of CLDN7 and / or PPM1B or a portion thereof). Typically, at least one PCR primer used to amplify a polynucleotide sequence is sequence-specific to that polynucleotide sequence. The exact length of a primer depends on a variety of factors, including temperature, primer source, and the method used. For example, for diagnostic applications, oligonucleotide primers typically contain at least 10, 15, 20, or 25 or more nucleotides, depending on the complexity of the target sequence, but they may contain fewer or more nucleotides. The factors involved in determining the appropriate primer length are well known to those skilled in the art.

[0076] As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific, intended target biomolecule. In some embodiments, the term "probe" here means any molecule or molecule associated with, which can bind indirectly or directly, covalently or nonvalently, to any substrate and / or reaction product and / or protease disclosed herein, and whose association or binding can be detected using the methods disclosed herein. In some embodiments, the probe is a fluorescent probe, an antibody, or an absorbance-based probe. If it is an absorbance-based probe, the chromophore pNA (p-nitroaniline) may be used as a probe for detecting and / or quantifying the target nucleic acid sequences disclosed herein. In some embodiments, the probe may be a nucleic acid sequence comprising a fluorescent molecule or substrate that becomes fluorescent upon exposure to an enzyme, and the nucleic acid sequence is complementary to a fragment of a nucleic acid sequence.

[0077] As used herein, the term "antibody" refers to a specific immunoglobulin targeting an antigenic site. In this invention, an antibody is an antibody that specifically binds to the biomarker peptides and / or proteins described herein. Antibodies can be manufactured according to conventional methods in the art. The forms of antibodies include polyclonal or monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (e.g., bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-binding site of the antibody, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody exhibits the desired biobinding activity.

[0078] As used in this article, the term "peptide" refers to a compound composed of amino acids linked by peptide bonds, including the full length of the peptide or amino acid fragments. The expression level of the gene-encoded peptide can be normalized based on the amount of total protein in the sample or the amount of peptide encoded by the housekeeping gene.

[0079] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0080] Example 1: Screening of biomarkers related to the diagnosis of ischemic stroke

[0081] 1. Screening Method

[0082] 1.1 Source of the filter set and methods of preprocessing

[0083] In this embodiment, the screening set for biomarkers related to the diagnosis of ischemic stroke was obtained from the GeneExpression Omnibus (GEO) database (https: / / www.ncbi.nlm.nih.gov / geo / ). Microarray data and clinical information from the GSE146882 dataset were downloaded from the GEO database as the screening set for this study. The sample size in the screening set was ischemic stroke:control group = 10:10. All samples were derived from blood samples of the subjects.

[0084] Adapter processing was performed on raw data using the automatic paired-end sequence detection mode of the FASTP software. Data trimming and quality control were implemented using a minimum N base number threshold of 5, a minimum read length threshold of 15, a base quality threshold of Q15, a low-quality base percentage threshold of 40%, a sliding window filtering method with a window average quality threshold of Q20, and the software's default parameters were used to output high-quality sequencing data for subsequent analysis. The obtained clean data was aligned to the human reference genome using ICGC software (version GRCh38.d1.vd1). Data from the GEO database were standardized using the RMA method, and genes were annotated using the platform's annotation file GPL23178. The data were then merged, deduplicated, and averaged.

[0085] 1.2 Differentially expressed genes associated with ischemic stroke

[0086] Differentially expressed genes were analyzed using the "limma" package (version 3.36.5) in R software. Differentially expressed genes were compared between blood samples from ischemic stroke patients and control groups to obtain differentially expressed genes (DEGs) between the ischemic stroke group and the control group. The selection criteria for DEGs were adj.P.Val < 0.05 and |log2FC| > 1. The results were visualized using a volcano plot.

[0087] 2. Screening Results

[0088] The analysis results of differentially expressed genes in the screening set are shown below. Figure 1 The results showed that, compared with the control group, a total of 9,985 differentially expressed genes were identified in the ischemic stroke group, of which 4,377 were upregulated and 5,581 were downregulated. The top 25 genes with the highest differential expression among the upregulated and downregulated genes were displayed in the form of a heatmap.

[0089] In the screening set, the blood biomarkers CLDN7 and PPM1B involved in this invention showed significantly different expression in ischemic stroke (see...). Figure 2 and Figure 3 Among them, CLDN7 was significantly upregulated in ischemic stroke, while PPM1B was significantly downregulated in ischemic stroke.

[0090] Example 2: Preliminary Verification of the Diagnostic Efficacy of the Screened Diagnostic Biomarkers for Ischemic Stroke 1. Verification Analysis Methods

[0091] The receiver operating characteristic (ROC) curves were plotted using the R package "pROC". The AUC values, sensitivity, and specificity of the biomarkers CLDN7, PPM1B, and CLDN7+PPM1B, which showed significant differential expression between the ischemic stroke group and the control group and were selected in Example 1, were analyzed to determine their diagnostic efficacy for ischemic stroke.

[0092] In evaluating the diagnostic efficacy of individual biomarkers CLDN7 and PPM1B for ischemic stroke, gene expression levels (Log2 expression levels) were used. The point corresponding to the highest Youden index was selected as the cutoff value; that is, the optimal cutoff threshold was determined by the point with the highest Youden index. When evaluating the diagnostic efficacy of the combined biomarker CLDN7+PPM1B for ischemic stroke, a logistic regression analysis was first performed on CLDN7+PPM1B. In the logistic regression analysis, the independent variable was CLDN7+PPM1B, and the dependent variable was the prevalence of ischemic stroke. The probability of each individual having ischemic stroke could be calculated using the fitted regression curve. Different probability cutoff thresholds were determined to obtain the predictive results. The optimal probability cutoff threshold was determined by the point with the highest Youden index. Based on the determined probability cutoff threshold, the AUC, sensitivity, and specificity of CLDN7+PPM1B detection in the screening set could be calculated. The AUC values, sensitivity, and specificity of CLDN7, PPM1B, and CLDN7+PPM1B were analyzed to determine their diagnostic efficacy.

[0093] 2. Verification analysis results

[0094] The diagnostic efficacy results for CLDN7, PPM1B, and CLDN7+PPM1B in the screening set are shown below. Figure 4 , Figure 5 , Figure 6 As shown in Table 1 below, the results indicate that CLDN7, PPM1B, and CLDN7+PPM1B all demonstrated high diagnostic efficacy for ischemic stroke in the screening set.

[0095] Table 1. Diagnostic efficacy of CLDN7, PPM1B, and CLDN7+PPM1B.

[0096]

[0097] Example 3 further validated the expression of the screened diagnostic biomarkers using clinically collected samples.

[0098] 1. Research Subjects

[0099] This study collected blood samples from 27 patients with ischemic stroke and 14 healthy controls at the Second Affiliated Hospital of Shandong First Medical University. The collected blood samples were used for subsequent analysis. The inclusion and exclusion criteria for the ischemic stroke patients are as follows:

[0100] Inclusion criteria:

[0101] ① All patients were diagnosed with ischemic stroke according to the "Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke in China";

[0102] ② Patients and their families are aware of the research and cooperate by signing the informed consent form;

[0103] ③ The Medical Ethics Committee of the Second Affiliated Hospital of Shandong First Medical University supported and approved this study;

[0104] ④ The patient had not received treatment prior to the test;

[0105] ⑤ All researchers underwent color Doppler ultrasound, CT, MRI and other examinations;

[0106] ⑥ The time from the onset of illness to admission is ≤3 days.

[0107] Exclusion criteria:

[0108] ① History of cardiovascular and cerebrovascular diseases;

[0109] ② Patients with severe liver or kidney disease, malignant hematological diseases, or tumors;

[0110] ③Those who have language, cognitive, consciousness, emotional, or mental impairments, poor compliance, and are unable to cooperate in completing tasks.

[0111] The normal control group refers to the population that underwent routine physical examinations at the Second Affiliated Hospital of Shandong First Medical University. There were no statistically significant differences in age, gender, and BMI between them and the ischemic stroke patient group, and none of them had any cerebrovascular diseases.

[0112] The clinical information of the 41 subjects is shown in Table 2 below.

[0113] Table 2. Basic clinical information of the subjects participating in this study.

[0114]

[0115]

[0116] 2. Main reagents for the experiment

[0117] The main reagents used in this study are shown in Table 3 below.

[0118] Table 3 List of Reagents Used

[0119]

[0120] 3. Main experimental instruments

[0121] The main experimental instruments used in this study are shown in Table 4 below.

[0122] Table 4 List of Instruments Used

[0123] Instrument Name Instrument Model factory centrifuge Centrifuge 5424R Eppendorf NanoVue Plus 28956057 BIOCHROM LTD Real-time PCR instrument Gene-9660 BIOER

[0124] 4. Real-Time PCR Detection

[0125] 4.1 Primer Design

[0126] The primer sequences for Real Time PCR detection of the target genes CLDN7 and PPM1B are shown in Table 5 below. The primers were synthesized by BOMIDE.

[0127] Table 5 Primer sequences

[0128]

[0129] 4.2 Experimental Methods

[0130] 4.2.1 Extracting total RNA from samples

[0131] ① Add 0.75 mL of lysis buffer RLS to every 0.25 mL of liquid sample (blood sample), and pipette the liquid sample several times to help lyse the cells in the sample. Repeat every 5-10 × 10⁻⁶ mL. 6 Add at least 0.75 mL of lysis buffer RLS to each cell. The final volume ratio of lysis buffer RLS to liquid sample is always 3:1.

[0132] ② Add 0.75 mL of lysis buffer RLS to the EP tube, then add 0.25 mL of blood sample, shake vigorously for 30 seconds to mix, and incubate at 15-30℃ for 10 minutes to allow complete decomposition of ribosomes.

[0133] ③ Add 0.2 mL of chloroform to every 0.75 mL of lysis buffer RLS, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes.

[0134] ④ Centrifuge at 4℃ and 12000rpm for 10 minutes. The sample will separate into three layers: a lower organic phase, a middle layer, and an upper colorless aqueous phase. RNA is present in the upper aqueous phase. The volume of the aqueous phase layer is approximately 70% of the volume of the added RLS. Transfer the aqueous phase to a new tube for the next step.

[0135] ⑤ Add 1 volume of 70% ethanol and mix by inverting (precipitation may occur at this point). Transfer the resulting solution and any possible precipitate together into the adsorption column RA (the adsorption column is fitted inside the collection tube).

[0136] ⑥ Centrifuge at 12000 rpm for 45 seconds, discard the waste liquid, and put the adsorption column back into the collection tube.

[0137] ⑦ Add 0.5 mL of protein removal solution RE, centrifuge at 12000 rpm for 45 s, and discard the waste liquid.

[0138] ⑧ Add 0.5 mL of rinsing buffer RW, centrifuge at 12000 rpm for 45 s, and discard the waste liquid.

[0139] ⑨ Add 0.5 mL of rinsing buffer RW, centrifuge at 12000 rpm for 45 s, and discard the waste liquid.

[0140] ⑩ Place the adsorption column RA back into the collection tube and centrifuge at 13000 rpm for 2 min to remove as much of the washing solution as possible, so as to avoid residual ethanol in the washing solution inhibiting the downstream reaction.

[0141] Remove the adsorption column RA and place it in an RNase-free centrifuge tube. Add 30-50 μL of RNase-free water (preheating in a 65-70°C water bath beforehand will improve the effect) to the center of the adsorption membrane, based on the expected RNA yield. Incubate at room temperature for 2 minutes, then centrifuge at 12000 rpm for 1 minute. If more RNA is needed, the resulting solution can be added back to the adsorption column and centrifuged for 1 minute, or an additional 30 μL of RNase-free water can be added and centrifuged for 1 minute. Combine the two eluents.

[0142] 4.2.2 Reverse transcription to synthesize mRNA and cDNA

[0143] mRNA reverse transcription was performed using the FastKing cDNA First-Strand Synthesis Kit (catalog number: KR116). First, genomic DNA was removed. In a test tube, 2.0 μL of 5×g DNA Buffer, 1 μg of Total RNA, and RNase-Free ddH2O were added to bring the total volume to 10 μL. The tube was heated at 42°C for 3 min. Then, 2.0 μL of 10×King RT Buffer, 1.0 μL of FastKing RT Enzyme Mix, 2.0 μL of FQ-RT Primer Mix, and 5.0 μL of RNase-Free ddH2O were added to the same test tube, bringing the total volume to 20 μL. The mixture was then heated at 42°C for 15 min and then at 95°C for 3 min. For long-term storage, the synthesized cDNA should be stored at -20°C or lower.

[0144] 4.2.3 Real-Time PCR

[0145] 4.2.3.1 Quantitative Detection of mRNA Fluorescence

[0146] 4.2.3.1.1 Instruments and Analytical Methods

[0147] Using a Gene-9660 real-time PCR instrument, 2 -△△CT The method is used to perform relative quantitative analysis of the data.

[0148] 4.2.3.1.2 Operation Procedure

[0149] ①Reaction system

[0150] Amplification was performed using SuperReal PreMix Plus (SYBR Green) (catalog number: FP205), and the experimental procedures were followed according to the product instructions. The RealTime reaction system is shown in Table 6 below.

[0151] Table 6 Reaction System

[0152] reagents Usage 2×SuperReal PreMix Plus 10μL Upstream primer (10 μM) 0.6μL Downstream primer (10 μM) 0.6μL <![CDATA[50×ROX Reference Dye △ ]]> 2μL DNA template 2μL Sterile distilled water 4.8μL

[0153] ②Amplification procedure

[0154] The amplification program is as follows: 95℃ for 15 min, (95℃ for 10 sec, 55℃ for 30 sec, 72℃ for 32 sec) × 40 cycles, 95℃ for 15 sec, 60℃ for 60 sec, 95℃ for 15 sec.

[0155] ③ Primer screening

[0156] After mixing the cDNA from each sample, the mixture was used as a template for 10-fold serial dilution. 2 μL of each diluted sample was then used as a template for amplification using the target gene primer and the internal reference gene primer, respectively. Melting curve analysis was performed at 60-95℃, and primers were screened based on high amplification efficiency and a single peak in the melting curve.

[0157] ④ Sample Real-Time PCR Detection

[0158] Each sample's cDNA was diluted 3-10 times, and 2 μL was used as a template for amplification using the target gene primer and the internal control gene primer, respectively (see Table 7 below). Melting curve analysis was performed at 60-95℃.

[0159] Table 7. Sample Real-Time PCR Detection Design

[0160] template Sample cDNA Sample cDNA Number of repeated detection channels 3 3 Primers Target gene primers Internal reference gene primers

[0161] 5. Data Statistics

[0162] The raw ct values ​​exported from the program were sorted according to the sample loading order to obtain the raw ct values ​​of three replicates for each gene in each sample. The average ct values ​​of the target gene and the internal reference gene in the three replicates were calculated in Excel. The expression of the target gene relative to the internal reference gene in the ischemic stroke group and the control group were calculated respectively. Statistical analysis was performed using GraphPad software, and the difference between the two groups was analyzed using a t-test.

[0163] 6. Relative quantitative analysis of each sample

[0164] Based on the original RealTime PCR test results, according to 2 -△△ct The relative quantitative calculation formula is used to calculate the relative quantitative results of the target gene for each sample, that is, the difference in the mRNA transcription level of the target gene between each sample in the ischemic stroke group and the control group. The calculation formula is as follows:

[0165]

[0166] 7. Experimental Results

[0167] 7.1 Results of RNA concentration detection and 1.5% agarose RNA electrophoresis

[0168] The results of RNA concentration detection and 1.5% agarose RNA electrophoresis are shown in Table 8 below.

[0169] Table 8. Results of RNA concentration and purity

[0170]

[0171]

[0172] 7.2 Real-Time PCR Detection Results and Analysis of Each Sample

[0173] Real-time amplification curves and melting curves of amplification products for each sample are shown below. Figures 7-10 As shown.

[0174] This study further validated the expression levels of CLDN7 and PPM1B using collected actual clinical samples. The statistical results are as follows: Figure 11 , Figure 12 As shown, the results indicated that, compared with the control group, the expression level of CLDN7 was significantly upregulated and the expression level of PPM1B was significantly downregulated in the ischemic stroke group.

[0175] Example 4 uses clinically collected samples to further validate the diagnostic efficacy of the screened diagnostic biomarkers.

[0176] 1. Experimental Methods

[0177] Using the validation method described above, the AUC values, sensitivity, and specificity of biomarkers CLDN7, PPM1B, and CLDN7+PPM1B in the validation set (the validation set composed of actual clinical samples collected in Example 3) were analyzed to determine their diagnostic efficacy for ischemic stroke.

[0178] 2. Verification analysis results

[0179] The diagnostic efficacy results for CLDN7, PPM1B, and CLDN7+PPM1B in the validation set are shown below. Figure 13 , Figure 14 , Figure 15 As shown in Table 9 below, the results indicate that in the validation set, CLDN7, PPM1B, and CLDN7+PPM1B all demonstrated high diagnostic efficacy for ischemic stroke. This suggests that, based on actual clinical samples collected in clinical practice, CLDN7, PPM1B, and CLDN7+PPM1B also possess high diagnostic efficacy and can serve as diagnostic markers for ischemic stroke.

[0180] Table 9. Diagnostic efficacy of CLDN7, PPM1B, and CLDN7+PPM1B.

[0181]

[0182] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of reagents for detecting the expression levels of CLDN7 and / or PPM1B in samples in the preparation of products for the diagnosis or auxiliary diagnosis of ischemic stroke.

2. The application according to claim 1, characterized in that, The reagents include those used to detect the expression levels of CLDN7 and / or PPM1B in samples using sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, chromatography technology, and mass spectrometry technology.

3. The application according to claim 2, characterized in that, The reagents include reagents for detecting the expression levels of CLDN7 and / or PPM1B mRNA in the sample, and reagents for detecting the expression levels of proteins and / or peptides encoded by CLDN7 and / or PPM1B in the sample.

4. The application according to claim 3, characterized in that, The reagents used to detect the expression levels of CLDN7 and / or PPM1B mRNA in the sample include probes that specifically recognize CLDN7 and / or PPM1B, and primers that specifically amplify CLDN7 and / or PPM1B.

5. The application according to claim 3, characterized in that, The reagents used to detect the expression levels of proteins and / or peptides encoded by CLDN7 and / or PPM1B in the sample include antibody fragments or affinity proteins that specifically bind to CLDN7 and / or PPM1B.

6. The application according to claim 4, characterized in that, The sequences of the primers for specifically amplifying CLDN7 are shown in SEQ ID NO:5-SEQ ID NO:6; The sequences of the primers for specifically amplifying PPM1B are shown in SEQ ID NO:7-SEQ ID NO:

8.

7. The application according to claim 1, characterized in that, The sample is a blood sample.

8. A diagnostic system for diagnosing or assisting in the diagnosis of ischemic stroke, characterized in that, The diagnostic system includes: (1) Ischemic stroke assessment device: including a control unit and a storage unit, used to assess whether the subject has an ischemic stroke; (2) Information communication terminal devices that are interconnected: used to provide data on the expression levels of CLDN7 and / or PPM1B in samples from subjects; The control unit of the ischemic stroke assessment device includes the following four units: 1) Data receiving unit: used to receive data transmitted from the information communication terminal equipment regarding the expression levels of CLDN7 and / or PPM1B in the sample; 2) Discriminant value calculation unit: It calculates the discriminant value based on the discrimination of the expression level of CLDN7 and / or PPM1B in the sample received by the data receiving unit and the expression level of CLDN7 and / or PPM1B stored in the storage unit as explanatory variables; 3) Discriminant value benchmark evaluation unit: It evaluates the risk of ischemic stroke in the subject based on the discriminant value calculated by the discriminant value calculation unit; 4) Evaluation result sending unit: It sends the evaluation results of the subject obtained by the discriminant benchmark evaluation unit to the information communication terminal device.

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