Application of selenoprotein P in diagnosis or prediction of moyamoya disease

Abstract

The present invention discloses the application of selenoprotein P in diagnosing or predicting moyamoya disease, and specifically provides the application of a reagent for detecting selenoprotein P in preparing a product for diagnosing or predicting moyamoya disease. The present invention provides a product, system, and device for diagnosing or predicting moyamoya disease. The present invention first discovered that the expression level of selenoprotein P in the plasma of patients with moyamoya disease is reduced. By detecting selenoprotein P, it is possible to diagnose whether a subject suffers from moyamoya disease or has a risk of suffering from moyamoya disease, and has broad market application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of selenoprotein P in diagnosing or predicting moyamoya disease. Background Art

[0002] Moyamoya disease (MMD) is a group of occlusive diseases characterized by progressive stenosis or occlusion of the bilateral internal carotid artery terminals and their large branches, accompanied by abnormal neovascularization at the skull base. The cause is unknown, and the above manifestations caused by other known symptoms are called Moyamoya syndrome. The name "Moyamoya" comes from the fact that during cerebral angiography, the base of the brain shows a blurred reticular shadow due to abnormal capillary proliferation, which is like a puff of smoke from smoking, hence the name. Its clinical manifestations are mainly divided into two categories: hemorrhage and ischemia. The onset age has a bimodal distribution of about 5 and 40 years old. Ischemia is the main clinical manifestation in children, and ischemia and hemorrhage in adults are basically the same probability. The essence of this disease is the occlusion of the main trunk of the brain base artery with compensatory vascular proliferation.

[0003] The current method of diagnosing moyamoya disease is imaging examination, which can be used to evaluate the patient's cerebral blood flow reserve before surgery and formulate a treatment plan; determine the patency of blood vessels during surgery and judge the effect of vascular reconstruction; evaluate the efficacy of postoperative patients and improve the surgical procedure. The pathological changes of moyamoya disease are progressive thickening of the vascular endothelium and the formation of moyamoya-like small blood vessels. The widely recognized pathological changes are smooth muscle fiber proliferation and displacement, which are related to changes in the internal environment caused by many cytokines and receptors, such as basic fibroblast growth factor, angiopoietin-2, platelet growth factor, prostaglandins in cerebrospinal fluid, and transforming growth factor related to connective tissue production. Studies have confirmed that the cerebral blood flow reserve of patients with moyamoya disease is lower than that of normal people. Changes in cerebral hemodynamics lead to changes in the internal environment, which causes corresponding changes in cytokines and receptors, and then leads to changes at the cellular level. The formation of moyamoya-like small blood vessels is of great significance for the emergence of collateral circulation, which in turn affects the severity of patients' symptoms. Studies have suggested that patients with moyamoya disease have inflammatory changes in both the internal carotid artery and the external carotid artery, and extensive inflammatory lesions of cerebral blood vessels occur, which are not limited. It is considered that moyamoya disease may not be a single cerebral vascular stenosis. The treatment of moyamoya disease is still unclear, and there is no ideal method at home and abroad. Internal medicine uses vasodilators and antibiotics for symptomatic treatment, but the effect is not ideal. Surgery is the main treatment. Although more than ten surgical methods have been developed since the Japanese discovered moyamoya disease in 1961, the core of all of them is to lead the external carotid artery to the skull. At present, the direct anastomosis of the superficial temporal artery to the middle cerebral artery and the indirect anastomosis of EDAS are mainly used to treat moyamoya disease internationally.

[0004] At present, the diagnosis method of moyamoya disease mainly relies on imaging examinations in large tertiary hospitals. It is necessary to make an appointment for relevant examinations in the outpatient department or inpatient department. The process is cumbersome and the cost is high. Some examinations (such as DSA and CTA) are not only expensive, but also patients need to endure certain pain. Although the use of contrast agents makes the diagnosis of lesions more accurate, it will have a certain impact on the patient's kidney function. In addition, some patients are allergic to contrast agents. Therefore, these diagnostic methods have many inconveniences. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0006] The present invention provides the use of a reagent for detecting selenoprotein P in a sample in preparing a product for diagnosing or predicting whether a subject suffers from moyamoya disease.

[0007] Further, the sample includes bone marrow, blood, blood cells, serum, plasma, peripheral blood, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free-floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, stool, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, washing fluid, lavage fluid, aspirate, scrape, bone marrow sample, tissue biopsy sample, surgical sample, secretion and / or excretion.

[0008] Furthermore, the sample includes blood, serum, plasma, and peripheral blood.

[0009] Furthermore, the sample is plasma.

[0010] Furthermore, the subject includes a human or a non-human mammal.

[0011] Furthermore, the subject is a human.

[0012] Furthermore, the selenoprotein P is present in the subject's sample as a marker.

[0013] The terms "marker", "biomarker" as used herein refer to a molecular species that is an indicator of a disease or effect mentioned in this specification. The molecular species may be the metabolite itself found in a sample of a subject. Moreover, the biomarker may also be a molecular species derived from the metabolite. In such a case, the actual metabolite will be chemically modified in the sample or during the assay, and as a result of the modification, a chemically different molecular species (i.e., analyte) will be the molecular species to be assayed. In this case, it should be understood that the analyte represents the actual metabolite and has the same ability as an indicator of each disease. Moreover, the biomarker according to the present invention does not necessarily correspond to a molecular species. Instead, the biomarker may contain stereoisomers or enantiomers of a compound. In addition, a biomarker may also represent the sum of isomers of biological species of isomeric molecules. The isomers will in some cases show the same analytical characteristics and therefore cannot be distinguished by a variety of analytical methods (including those used in the attached embodiments described below). However, these isomers will share at least the same overall formula parameters. As used herein, a marker refers to an entity or moiety whose presence or level is characteristic of a particular state or event. In some embodiments, the presence or level of a particular marker may be characteristic of the presence or stage of a disease, disorder, or condition.

[0014] Selenoprotein P (SELENOP) is a selenoprotein in plasma, mainly produced in the liver. The "P" in SELENOP comes from its presence in plasma. SELENOP contains selenocysteine, a selenocysteine ​​analogue that contains Se instead of sulfur. Selenoprotein P is a bifunctional protein and one of the main members of the selenoprotein family. It is mainly synthesized by the liver and secreted into the peripheral blood, and then transports selenium to non-liver tissues for their use to synthesize other selenoproteins. At the same time, selenoprotein P also has antioxidant enzyme activity and plays an important role in protecting the body from oxidative damage.

[0015] As used herein, the term "sample" typically refers to a sample obtained or derived from a biological source of interest as described herein (eg, a tissue or an organism or a cell culture).

[0016] In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the sample is or includes a biological tissue or fluid. In some embodiments, the sample may be or include bone marrow; blood; plasma; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free-floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washing fluids or lavage fluids, such as catheter lavage fluids or bronchoalveolar lavage fluids; aspirates; scrapings; bone marrow samples; tissue biopsy samples; surgical samples; feces, other body fluids, secretions and / or excretions; and / or cells from them, etc. In some embodiments, the sample is or includes cells obtained from an individual. In some embodiments, the cells obtained are or include cells from the individual from which the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any appropriate means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of: biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluids (e.g., blood, lymph, feces, etc.), etc. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample).

[0017] Furthermore, the reagent for selenoprotein P includes primers or probes for specifically detecting the expression level of a gene encoding selenoprotein P, or a reagent that specifically binds to selenoprotein P.

[0018] The term "sample" is used herein to refer to any single-stranded oligonucleotide sequence that can be used as a primer in, for example, PCR techniques. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. Factors involved in determining the appropriate length of a primer are readily known to those skilled in the art.

[0019] Furthermore, the reagents include reagents for detecting the presence, absence and / or amount of genes, fragments and selenoprotein P encoding selenoprotein P in a sample by PCR reaction, RT-PCR derivatization reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, double hybridization probe, composite probe, ISH, microarray, Southern blot, Northern blot, multi-analyte spectrum test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay or slot blot assay.

[0020] As used herein, the term "detecting the amount of..." refers to determining at least one characteristic feature of a biomarker to be determined by the method of the present invention in a sample. Characteristic features according to the present invention refer to features that characterize the physical and / or chemical properties (including biochemical properties) of a biomarker. Such properties include, for example, molecular weight, viscosity, density, charge, spin, optical activity, color, fluorescence, chemiluminescence, elemental composition, chemical structure, ability to react with other compounds, ability to elicit a response (e.g., induction of a reporter gene) in a biological readout system, etc. The value of the property can be used as a characteristic feature and can be determined according to techniques known in the art. Moreover, the characteristic feature can be any feature derived from the value of the physical and / or chemical property of the biomarker by standard operations (e.g., mathematical calculations such as multiplication, division, or logarithmic calculus). Most preferably, at least one characteristic feature allows the determination and / or chemical identification of the at least one biomarker and its amount. Therefore, the characteristic value preferably also contains information related to the abundance of the biomarker from which the characteristic value is derived. For example, the characteristic value of a biomarker can be a peak in a mass spectrum. Such peaks contain characteristic information of the biomarker, ie, m / z information, as well as intensity values ​​related to the abundance (ie, its amount) of the biomarker in the sample.

[0021] The present invention provides a product for predicting or diagnosing whether a subject suffers from moyamoya disease, the product comprising a reagent for detecting the expression level of a gene encoding selenoprotein P in a sample, or a reagent for detecting the expression level of selenoprotein P in a sample.

[0022] Furthermore, the product includes the reagent described above, a kit, a chip, a nucleic acid membrane strip or a test paper containing the reagent.

[0023] Furthermore, the kit includes a gene detection kit and a protein detection kit. The gene detection kit includes a reagent or a chip for detecting the transcription level of a gene encoding selenoprotein P, and the protein detection kit includes a reagent or a chip for detecting the expression level of selenoprotein P.

[0024] Furthermore, the chip includes a gene chip and a protein chip. The gene chip includes an oligonucleotide probe targeting a selenoprotein P gene for detecting the transcription level of the gene encoding selenoprotein P, and the protein chip includes a selenoprotein P-specific antibody or ligand.

[0025] Furthermore, the test strips include gene detection test strips and protein detection test strips.

[0026] Furthermore, the test paper includes chromatography test paper.

[0027] The term "probe" refers to a substance that can specifically bind to a target substance to be detected in a sample. The types of probes are those commonly used in the art and are not limited. They include but are not limited to peptide nucleic acid (PNA), locked nucleic acid (LNA), peptide, polypeptide, protein, ribonucleic acid or deoxyribonucleic acid.

[0028] The term "expression level" refers to the amount of a measurable gene product produced by a gene in a subject's sample, wherein the gene product may be a transcription product or a translation product, and the term "expression level" also refers to the amount of selenoprotein P in a subject's sample. According to the understanding of those skilled in the art, the gene expression level can be expressed by measuring the messenger RNA level of the gene or the protein level of the gene.

[0029] In some embodiments, the expression level of the gene encoding selenoprotein P can be measured by measuring the mRNA level encoded by the gene or measuring the protein level encoded by the gene, that is, the level of selenoprotein P or its variants. Selenoprotein P variants include all physiologically relevant post-translational chemical modifications of the protein, such as glycosylation, phosphorylation, acetylation, etc., but must have the function of the protein.

[0030] In an embodiment of the present invention, the expression level of selenoprotein P in the sample includes the selenoprotein P level of any mammal and the expression level of the gene encoding selenoprotein P, and the mammal includes but is not limited to domestic or farm animals (cows, horses, pigs, sheep, goats, dogs, cats or rodents), primates and humans. In a specific embodiment, the selenoprotein P is a human protein.

[0031] Methods for determining or comparing selenoprotein P levels include protein chip analysis, immunoassay, ligand binding analysis, MALDI-TOF (matrix desorption / ionization time-of-flight mass spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, bidirectional immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), immunoblotting and ELISA (enzyme-linked immunosorbent assay), but are not limited to these.

[0032] As used herein, the term "primer" refers to an oligonucleotide that can serve as a starting point for DNA synthesis under suitable conditions. These conditions include inducing the synthesis of primer extension products complementary to the nucleic acid chain in the presence of four different nucleoside triphosphates and an extender (e.g., DNA polymerase or reverse transcriptase) in a suitable buffer at a suitable temperature. Primer extension can also be carried out in the absence of one or more nucleoside triphosphates, in which case an extension product of limited length will be produced.

[0033] The terms "chip" and "substrate" are used interchangeably and refer to any surface to which capture probes can be attached (e.g., solid arrays, beads, coverslips, etc.). More details about suitable substrates are disclosed in U.S. Provisional Patent Application No. 62 / 980,073, entitled "Pipeline for Analysis of Analytes," filed on February 21, 2020, with attorney docket number 104371-5033-PR01, which is hereby incorporated by reference.

[0034] The present invention provides application of selenoprotein P in constructing a system or device for diagnosing or predicting moyamoya disease.

[0035] The term "subject" as used herein includes, but is not limited to, humans, non-human primates, and non-human vertebrates, such as wild, domestic, and farm animals, including any mammals, such as cats, dogs, cows, sheep, pigs, horses, rabbits, rodents such as mice and rats. The terms "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject, particularly humans, who require diagnosis, treatment, or therapy.

[0036] The terms "assess," "detect," "measure," "evaluate," and "determine" are used interchangeably to refer to any form of measurement, including determining whether an element is present or not and including both quantitative and qualitative measurements. The assessment may be relative or absolute.

[0037] The present invention provides a system or device for diagnosing or predicting moyamoya disease using selenoprotein P. The system or device includes a processing module for detecting the coding gene of the selenoprotein P or the expression level of the selenoprotein P according to the detection results, analyzing the obtained values, and obtaining the diagnostic or predictive values ​​of the above-mentioned selenoprotein P.

[0038] The term "system" as used herein relates to different tools that are operatively connected to each other. The tools may be applied in a single device or may be physically separate devices that are operatively connected to each other. The tool for comparing the characteristic values ​​of biomarkers is preferably based on the aforementioned comparison algorithm. The data storage medium, i.e., the memory, preferably comprises a data set or a database, wherein each stored data set indicates a medical condition or effect referred to in the present invention. Therefore, the system of the present invention allows to identify whether the data set stored in the data storage medium comprises a test data set. Therefore, the system of the present invention can implement the method of the present invention. In a preferred embodiment of the system, a tool for determining the characteristic value of selenoprotein P of a sample is included.

[0039] Furthermore, the system or device includes a judgment module, which is used to compare the result of the processing module with a set value.

[0040] Furthermore, the system or device includes a detection module for detecting the expression level of a selenoprotein P-related coding gene or protein in a sample to obtain a detection result.

[0041] Furthermore, the system or device also includes a memory or a processor.

[0042] Furthermore, the memory is used to store a computer program for execution.

[0043] As used herein, the terms "memory" and "data storage medium" are synonymous and encompass data storage media based on a single physical entity, such as a CD, CD-ROM, hard disk, optical storage medium or floppy disk. Moreover, the term further includes data storage media consisting of physically separate entities that are effectively connected to each other in a manner that provides the aforementioned data set, preferably in a manner suitable for query retrieval.

[0044] Furthermore, the computer program is used to control the processor to execute operations of the processing module, the judgment module, and the detection module.

[0045] The term "data set" is synonymous with "computer program" and refers to a collection of data that can be physically and / or logically grouped together. Therefore, the data set can be executed in a single data storage medium, or in physically separated data storage media that are effectively connected to each other. Preferably, the data set is executed by a database. Therefore, the database used herein contains a data set in an appropriate storage medium. Moreover, the database preferably also includes a database management system. The database management system is preferably a network-based, hierarchical or object-oriented database management system. Moreover, the database can be a joint database or a centralized database. More preferably, the database is executed as a distributed (joint) system (e.g., as a client-server-system). More preferably, the database is constructed to allow the retrieval algorithm to compare the test data set and the data set contained in the data set. Specifically, by using such an algorithm, it is possible to retrieve similar or identical data sets (e.g., query retrieval) indicating the medical conditions or effects shown above in the database. Therefore, if the same or similar data set can be identified in the data set, the test data set is related to the medical condition or effect. Therefore, the information obtained from the data set can be used as, for example, a reference value for the above-mentioned method of the present invention. More preferably, the data set contains the characteristic values ​​of all biomarkers contained in any of the above groups.

[0046] The terms "reference value" and "set value" are synonymous and refer to characteristic characteristic values ​​of individual biomarkers that can be associated with a medical condition (i.e., the presence or absence of a disease, disease state, or effect mentioned herein). Preferably, the reference value is a threshold value (e.g., an amount or a ratio of amounts) of a biomarker, wherein a value above the threshold value or substantially the same as the threshold value found in the sample to be studied indicates the presence of a medical condition, and a value below the threshold value found in the sample to be studied indicates the absence of a medical condition. It should be understood that, also preferably, the reference value may be a threshold value of a biomarker, wherein a value below the threshold value or substantially the same as the threshold value found in the sample to be studied indicates the presence of a medical condition, and a value above the threshold value found in the sample to be studied indicates the absence of a medical condition.

[0047] The term "module" refers to computer program logic used to provide specified functionality. Therefore, a module can be implemented in hardware, firmware, and / or software. In one embodiment, a program module is stored on a storage device, loaded into memory, and executed by a processor.

[0048] The present invention provides the use of any of the above-mentioned reagents for detecting selenoprotein P in a sample in the preparation of products for diagnosing or predicting carotid artery stenosis or occlusion, abnormal neovascularization of the skull base, intimal thickening, smooth muscle fiber hyperplasia and displacement.

[0049] Furthermore, the carotid artery includes the internal carotid artery.

[0050] Furthermore, the internal carotid artery includes the terminal end of the internal carotid artery and large branch vessels of the internal carotid artery.

[0051] Furthermore, the carotid artery stenosis or occlusion includes stenosis or occlusion of the terminal of the internal carotid artery and stenosis or occlusion of large branches of the internal carotid artery.

[0052] The term "processor" as used herein is intended to include any processing device, for example, a processing device including a CPU (central processing unit). The term "processor" as used herein is intended to include a memory associated with a processor or CPU, for example, RAM, ROM, etc. The term "processor" may refer to more than one processing device. Other processing devices on a computer cluster or in a multi-processor computer server may share elements associated with the processing device. Therefore, as described herein, a software component including instructions or codes for performing the method of the present invention may be stored in one or more associated memories or storage devices (for example, ROM, fixed or removable memory), and when ready to use, partially or completely loaded into a memory (for example, RAM) and executed by a CPU. Storage devices may further be used to store program code, a genome sequence database, etc. Storage devices may be computer storage devices of any suitable form, including traditional hard disk drives, solid-state drives, or ultra-fast disk arrays. In some embodiments, storage devices include network attached storage devices, which may be operably connected to a plurality of similar computer servers including a computing cluster.

[0053] The present invention provides a method for predicting whether a subject has carotid artery stenosis or occlusion, abnormal neovascularization of the skull base, vascular intima thickening, smooth muscle fiber hyperplasia and displacement for non-diagnostic purposes. The method comprises the step of detecting the expression level of a gene or protein encoding selenoprotein P in a sample of the subject.

[0054] Furthermore, the carotid artery includes the internal carotid artery.

[0055] Furthermore, the internal carotid artery includes the terminal end of the internal carotid artery and large branch vessels of the internal carotid artery.

[0056] Furthermore, the carotid artery stenosis or occlusion includes stenosis or occlusion of the terminal of the internal carotid artery and stenosis or occlusion of large branches of the internal carotid artery.

[0057] The term "diagnosis" as used herein refers to an assessment of whether a subject suffers from moyamoya disease. As will be appreciated by those skilled in the art, such an assessment, although preferably correct for 100% of the subjects studied, may not usually be correct for 100% of the subjects studied. However, the term requires that a statistically significant proportion of subjects be correctly assessed and thus diagnosed. Whether the proportion is statistically significant can be immediately determined by those skilled in the art using various well-known statistical evaluation tools (e.g., confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc.). Specific details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The p value is preferably 0.2, 0.1, 0.05, 0.01 or 0.001.

[0058] The present invention provides a method for screening drugs for treating or preventing moyamoya disease for non-therapeutic and non-diagnostic purposes, the method comprising the step of detecting the expression level of a coding gene or protein of selenoprotein P in a sample of a subject.

[0059] The term "comparison" refers to determining whether the measured value of a biomarker is substantially the same as or different from a reference value. Preferably, the value of the biomarker is considered to be different from the reference value if the observed difference is statistically significant, and the statistical significance can be determined by statistical techniques mentioned elsewhere in this specification. If the difference is not statistically significant, the value of the biomarker is substantially the same as the reference value. Based on the above comparison, it can be assessed whether the subject has a disease.

[0060] The computer described herein may further include a computing system that further includes a back-end component (e.g., a data server), a middleware component (e.g., an application server), a front-end component (e.g., a client computer with an operator / physician graphical interface, or a web browser through which the physician / operator can interact with the embodiments of the patient subject matter described herein), or any combination thereof. In some embodiments, the components of the computer system may be interconnected by a network by any form or medium of digital data communication (e.g., a communication network). In some embodiments, the communication network may include or exclude: cellular networks (3G, 4G, 5G), personal area networks (wireless personal area networks, such as infrared, ZigBee, Bluetooth, and ultra-wideband or UWB, and wired connections, such as USB or FireWire), local area networks (LANs, such as Ethernet (IEEE802.3) and Wi-Fi / WLAN (IEEE 802.11)), and wide area networks (WANs), such as the Internet.

[0061] Advantages and beneficial effects of the present invention:

[0062] The present invention discloses the application of selenoprotein P in diagnosing or predicting moyamoya disease, and specifically provides the application of a reagent for detecting selenoprotein P in preparing a product for diagnosing or predicting moyamoya disease. The present invention provides a product, system, and device for diagnosing or predicting moyamoya disease. The present invention first discovered that the expression level of selenoprotein P in the blood of moyamoya disease patients is reduced. By detecting selenoprotein P, it is possible to diagnose whether a subject suffers from moyamoya disease or has a risk of suffering from moyamoya disease, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is a bar graph of the differential expression of selenoprotein P in the test set between healthy people and moyamoya disease patient samples;

[0064] Figure 2 This is a bar graph verifying the differential expression of selenoprotein P in healthy people and moyamoya disease patient samples;

[0065] Figure 3 This is the ROC curve of the test set selenoprotein P for the diagnosis of moyamoya disease;

[0066] Figure 4 This is the ROC curve diagram for validating selenoprotein P in the diagnosis of moyamoya disease. DETAILED DESCRIPTION

[0067] Before describing the present method and composition, it should be understood that the present invention is not limited to the specific method or composition described, as they can certainly vary. In addition, the terminology used herein is only used for the purpose of describing specific embodiments and should not limit the scope of the present invention, because the scope of the present invention is limited to the scope limited by the attached claims.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood in the art to which the invention belongs. Although any methods and materials similar or equivalent to the methods and materials described can be used to implement or test the present invention, some possible and preferred methods and materials are now described. All publications mentioned are cited and incorporated herein to disclose and describe the methods and / or materials related to the cited publications. It is understood that the content disclosed herein covers any content that conflicts with the disclosure of the incorporated publications.

[0069] It should be noted that, herein and in the appended claims, the term "one or more" refers to a number greater than one. For example, the term "one or more" encompasses any of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, fifty or more, 100 or more, or even more numbers, unless the context clearly indicates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, a reference to an "RNA sequence" includes one or more peptides and equivalents, such as polypeptides known to the skilled person, and the like.

[0070] Example 1

[0071] 1. Information on experimental research subjects

[0072] 1) Test set: 33 healthy controls and 95 patients with moyamoya disease;

[0073] 2) Validation set: 33 healthy controls and 95 patients with moyamoya disease;

[0074] 3) Clinical inclusion criteria: patients with obvious symptoms (transient ischemic attack, cerebral infarction or cerebral hemorrhage); confirmed by imaging examination (CTA or DSA); aged 18-60 years.

[0075] 4) Clinical exclusion criteria: patients aged <18 years or aged >60 years, with missing clinical data.

[0076] 5) Clinical characteristics of the control population and patients with moyamoya disease

[0077] There were no differences in clinical characteristics between the control population and patients with MMD.

[0078] 2. Experimental methods

[0079] The extraction and detection steps of selenoprotein P in peripheral blood;

[0080] Selenoprotein P was detected by ELISA using the CUSABIO (SEPP1) enzyme-linked immunosorbent assay kit purchased from Wuhan Huamei Bioengineering Co., Ltd., China.

[0081] Before the experiment begins, all reagents should be equilibrated to room temperature; when diluting reagents or samples, they must be mixed evenly, and foaming should be avoided as much as possible during mixing. Plasma samples were diluted 1:1000 with sample diluent before testing.

[0082] 1) Move all reagents to room temperature (18-25°C) to equilibrate for at least 30 minutes, and prepare the reagents for use.

[0083] 2) Sample addition: Set up standard wells and test sample wells respectively. Add 100 μl of standard or test sample to each well, shake gently to mix, cover with plate sticker, and incubate at 37°C for 2 hours.

[0084] 3) Discard the liquid, spin dry, and do not wash.

[0085] 4) Add 100 μl of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0086] 5) Discard the liquid in the wells, spin dry, and wash the plate 3 times. Soak for 2 minutes each time, 200 μl / well, spin dry.

[0087] 6) Add 100 μl of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour.

[0088] 7) Discard the liquid in the wells, spin dry, and wash the plate 5 times. Soak for 2 minutes each time, 200 μl / well, spin dry.

[0089] 8) Add 90 μl of substrate solution to each well and incubate at 37°C in the dark for 15-30 minutes.

[0090] 9) Add 50 μl of stop solution to each well to terminate the reaction.

[0091] 10) Within 5 minutes after the reaction is terminated, the optical density (OD value) of each well is measured in sequence at a wavelength of 450 nm using a microplate reader.

[0092] 11) Analyze through professional curve making software and calculate the sample concentration.

[0093] Diagnostic performance analysis:

[0094] The R package "pROC" was used to draw the receiver operating characteristic (ROC) curve, and the selenoprotein P levels that showed significant differences between healthy people and moyamoya disease subjects were analyzed. The AUC values, sensitivity, and specificity in the test set and validation set were obtained to determine its diagnostic efficacy for moyamoya disease.

[0095] 3. Experimental results

[0096] 1) Differential expression analysis

[0097] Test set: The analysis results of differential expression in the test set are as follows Figure 1 As shown in the figure, compared with healthy people, the expression level of selenoprotein P in patients with moyamoya disease was significantly decreased.

[0098] Validation set: The analysis results of differential expression in the validation set are as follows Figure 2 As shown in the figure, compared with healthy people, the expression level of selenoprotein P in patients with moyamoya disease was significantly decreased.

[0099] 2) ROC curve analysis of selenoprotein P in the diagnosis of moyamoya disease

[0100] Taking selenoprotein P as the variable, the expression level data of the healthy population and the moyamoya disease patient samples in the test set were used to draw the ROC curve of the healthy population and the moyamoya disease patient. The results are as follows: Figure 3 As shown, the results show that the ROC curve drawn for the test set samples shows an AUC value of 0.706, a Cut off value of 16.69, a sensitivity of 0.515, and a specificity of 0.821. The specific data are shown in Table 1, indicating that selenoprotein P can be used as a biomarker for the diagnosis of moyamoya disease.

[0101] Table 1

[0102]

[0103] Taking selenoprotein P as the variable, the expression level data of the validation set of healthy people and moyamoya disease patients were used to draw the ROC curve of healthy people and moyamoya disease patients. The results are as follows Figure 4 As shown, the results showed that the ROC curve drawn for the validation set samples showed an AUC value of 0.737, a Cut off value of 20.17, a sensitivity of 0.576, and a specificity of 0.768. The specific data are shown in Table 2, which verified that selenoprotein P can be used as a biomarker for the diagnosis of moyamoya disease.

[0104] Table 2

[0105]

[0106] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting selenoprotein P in a sample in the preparation of a product for diagnosing or predicting whether a subject suffers from moyamoya disease, wherein the reagent for detecting selenoprotein P in a sample is a reagent for detecting the protein expression level of selenoprotein P in a sample, and the sample is blood, serum, plasma, or peripheral blood.

2. The use according to claim 1, characterized in that: The sample is plasma.

3. The use according to claim 1, characterized in that: The subject includes a human or a non-human mammal.

4. The use according to claim 1, characterized in that: The subject is a human.

5. The use according to claim 1, characterized in that: The selenoprotein P reagent is a reagent that specifically binds to selenoprotein P.

6. The use according to claim 1, characterized in that: The reagents include reagents for detecting the presence, absence and / or amount of selenoprotein P in a sample by microarray, multi-analyte profiling test, enzyme-linked immunosorbent assay, radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay or slot blot assay.

7. The use according to claim 1, characterized in that: The reagents include reagents for detecting the presence, absence and / or amount of selenoprotein P in a sample by TMA.

8. Use of a reagent for detecting the protein expression level of selenoprotein P in a sample in constructing a system or device for diagnosing or predicting moyamoya disease, wherein the sample is blood, serum, plasma, or peripheral blood.

9. A system or device for diagnosing or predicting moyamoya disease using selenoprotein P, characterized in that: The system or device includes a detection module for detecting the expression level of selenoprotein P protein in a sample to obtain a detection result, wherein the sample is blood, serum, plasma, or peripheral blood; The system or device includes a processing module for detecting the expression level of the selenoprotein P according to the detection result, analyzing the obtained value, and obtaining the diagnostic or predictive value of the above selenoprotein P.

10. The system or device according to claim 9, comprising a judgment module for comparing the result of the processing module with a set value.

11. The system or device of claim 9, further comprising a memory or a processor.

12. The system or device of claim 11, wherein the memory is used to store a computer program for execution.

13. The system or device according to claim 12, wherein the computer program is used to control the processor to execute operations of the processing module, the judgment module, and the detection module.

14. A method for screening drugs for treating or preventing moyamoya disease for non-therapeutic and non-diagnostic purposes, characterized in that: The method comprises the step of detecting the protein expression level of selenoprotein P in a sample of a subject, wherein the sample is blood, serum, plasma or peripheral blood.

Images