CN116949168A - Application of CD73 in diagnosis of parkinsonism - Google Patents

Application of CD73 in diagnosis of parkinsonism Download PDF

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Publication number
CN116949168A
CN116949168A CN202310912160.2A CN202310912160A CN116949168A CN 116949168 A CN116949168 A CN 116949168A CN 202310912160 A CN202310912160 A CN 202310912160A CN 116949168 A CN116949168 A CN 116949168A
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gene
disease
protein
chip
parkinson
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杨国锋
赵媛
张译丹
张健
高雅
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Second Hospital of Hebei Medical University
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Second Hospital of Hebei Medical University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Abstract

The invention discloses application of CD73 in diagnosis of parkinsonism, and the invention discovers that the content of CD73 in parkinsonism patient serum is obviously reduced compared with healthy control population by researching the level of CD73 in parkinsonism patient serum and healthy control serum, and the CD73 level of PD patient serum is inversely related to H & Y stage and UPDRS-III score, thereby providing a new direction for effective diagnosis of parkinsonism patients and having wide application prospect in clinic.

Description

Application of CD73 in diagnosis of parkinsonism
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of CD73 in diagnosis of parkinsonism.
Background
Parkinson's Disease (PD) is the most common neurodegenerative disease causing dyskinesia in the elderly, with typical clinical central motor four signs of tremor, rigidity, hypokinesia and bradykinesia, postural gait instability. As the disease progresses, the motor symptoms of PD progress, which becomes a main cause of the decrease in quality of life and the increase in life dependence of the patient suffering from PD. Therefore, the screening of the markers related to the Parkinson's disease has important significance for diagnosis and subsequent treatment of the Parkinson's disease.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides application of CD73 in diagnosis of parkinsonism.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides the use of a reagent for detecting the level of CD73 expression in a sample for the preparation of a diagnostic, staged product for parkinson's disease.
Further, the reagent is selected from a probe specifically recognizing the CD73 gene, a primer specifically amplifying the CD73 gene, or a binding agent specifically binding to a protein encoded by the CD73 gene.
Further, the primer or probe is labeled with a labeling substance.
Further, the labeling substance includes a fluorescent substance, a radioisotope, or an enzyme.
Further, the product comprises a chip, a test paper, a kit or a nucleic acid membrane strip.
Further, the chip comprises a gene chip and a protein chip.
Further, the gene chip includes a probe for CD73 gene for detecting the transcription level of CD73 gene, and the protein chip includes a specific binding agent for CD73 protein.
Further, the kit comprises a gene detection kit and a protein detection kit.
Further, the gene detection kit comprises a reagent or a chip for detecting the transcription level of the CD73 gene, and the protein detection kit comprises a reagent or a chip for detecting the expression level of the CD73 protein.
Further, the kit also includes instructions.
Further, the kit further comprises a buffer solution.
Further, the kit further comprises a reagent for detecting the expression level of the CD73 gene or protein by an RT-PCR method, a qRT-PCR method, a biochip detection method, a southern blotting method, an in situ hybridization method and an immunoblotting method.
Further, the sample includes serum, tissue, exosomes.
Further, the sample is selected from serum.
Further, parkinson's disease is diagnosed when the level of CD73 in serum appears to be significantly down-regulated.
In a second aspect the invention provides a diagnostic, staged product for parkinson's disease comprising an agent for detecting the level of CD73 expression in a sample.
Further, the product comprises a chip, a test paper, a kit or a nucleic acid membrane strip.
Further, the sample includes serum, tissue, exosomes.
Further, the sample is selected from serum.
In a third aspect, the present invention provides a method of screening for a drug candidate for the treatment of parkinson's disease, said method comprising: treating a culture system expressing or containing the CD73 gene or a protein encoded by the CD73 gene with a substance to be screened; and detecting the expression or activity of the CD73 gene or a protein encoded thereby in the system; wherein the substance to be screened is a candidate drug for treating Parkinson's disease when the substance to be screened promotes the expression level or activity of the CD73 gene or a protein encoded by the same.
In a fourth aspect the invention provides the use of CD73 as a target in the screening of candidate drugs for the treatment of parkinson's disease.
Further, the method for screening candidate drugs for treating parkinson's disease comprises: treating a culture system expressing or containing the CD73 gene or a protein encoded by the CD73 gene with a substance to be screened; and detecting the expression or activity of the CD73 gene or a protein encoded thereby in the system; wherein the substance to be screened is a candidate drug for treating Parkinson's disease when the substance to be screened promotes the expression level or activity of the CD73 gene or a protein encoded by the same.
In a fifth aspect, the invention provides the use of CD73 in the construction of a computational model for diagnosis and staging of parkinson's disease.
A sixth aspect of the invention provides the use of CD73 in the construction of a system/device/computer readable storage medium for diagnosis and staging of parkinson's disease.
The invention has the advantages and beneficial effects that: according to the invention, through researching the levels of CD73 in the serum of the parkinsonism patient and the healthy control serum, the content of CD73 in the serum of the parkinsonism patient is obviously reduced compared with the healthy control population, and the serum CD73 level of the PD patient is inversely related to the H & Y stage and the UPDRS-III score (P is less than 0.01), so that a new direction is provided for the effective diagnosis of the parkinsonism patient, and the method has a wide application prospect in clinic.
Drawings
FIG. 1 is a graph of serum CD73 differential levels for a training set, wherein 1A is a graph of serum CD73 differential levels and 1B is a ROC graph;
fig. 2 is a graph of validation set serum CD73 ROC.
Detailed Description
The following provides definitions of some of the terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention provides application of a reagent for detecting CD73 expression level in a sample in preparation of a product for diagnosing and staging Parkinson's disease.
In the present invention, CD73 includes wild-type, mutant or fragments thereof. The term encompasses full length, unprocessed CD73, as well as any form of CD73 derived from processing in a cell, as well as naturally occurring variants (e.g., splice variants or allelic variants) of CD 73. The term encompasses, for example, human CD73 as well as CD73 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), gene IDs: 4907.
in the present invention, diagnosing, making a diagnosis, and variations of these terms, refers to the discovery, judgment, or cognition of an individual's health state or condition based on one or more signs, symptoms, data, or other information associated with the individual. The health status of an individual may be diagnosed as healthy/normal (i.e., no disease or condition present) or may be diagnosed as unhealthy/abnormal (i.e., there is an assessment of disease or condition or characteristic). The terms diagnostic, making a diagnosis, etc. include early detection of a disease in relation to a particular disease or condition; characteristics or classification of disease; discovery of progression, cure, or recurrence of disease; following treatment or therapy of an individual, a response to the disease is found.
In the present invention, the expression level or level refers to the absolute or relative amount of CD73 in the present invention, and the expression level of CD73 in the present invention may be determined by various techniques, and in particular, the absolute or relative amount of CD73 in the present invention may be detected by using methods well known to those skilled in the art.
The reagent is selected from a probe specifically recognizing the CD73 gene, a primer specifically amplifying the CD73 gene or a binding agent specifically binding to a protein encoded by the CD73 gene.
In the present invention, the probe specifically recognizing the CD73 gene may be DNA, RNA, DNA-RNA chimera, PNA or other derivative. The length of the probe is not limited, and any length may be used as long as it specifically hybridizes to the target nucleotide sequence and binds thereto. The probe may be as short as 10, 25, 20, 15, 13 or 10 bases in length. Also, the probes can be as long as 60, 80, 100, 150, 300 base pairs or more in length, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the probe length is usually at least 14 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15-25 base pairs, with the longest length generally not exceeding 30 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs to avoid affecting hybridization efficiency.
In the present invention, a primer refers to a single stranded polynucleotide capable of hybridizing to a nucleic acid and allowing polymerization of the complementary nucleic acid (typically by providing a free 3' -OH group). The primers are capable of achieving specific amplification of the target sequence. Specific amplification refers to the amplification of a target sequence,
primers or probes of the invention may be chemically synthesized using a phosphoimide solid support method or other well known methods. Many means known in the art may also be used for modification. Non-limiting examples of such modifications include methylation, capping, substitution with one or more analogs of the natural nucleotide, and modification between nucleotides, e.g., modification of uncharged linkers (e.g., methyl phosphate, phosphotriester, phosphoimide, carbamate, etc.), or modification of charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).
The primer or probe is labeled with a labeling substance.
The labeling substance includes, but is not limited to, a fluorescent substance, a radioisotope, or an enzyme.
Among them, the fluorescent substances include, but are not limited to, TAMRATTM, alexa555, alexa647, cy3, cy5 of cyanine dye series, fluorescein.
Radioisotopes include, but are not limited to, 32P, 33P, 35S.
Enzymes include, but are not limited to, alkaline phosphatase, horseradish peroxidase.
In the primer or the probe of the present invention labeled with a labeling substance, the labeling substance may be directly bound to the primer or the probe, or may be bound via a linker. The linker may be any linker commonly used in the art, and specifically, for example, a nucleic acid of 1 to 3 bases, more preferably a DNA of 1 to 3 bases, still more preferably a DNA of 2 bases, and particularly preferably 2 bases of adenine (A) -adenine (A).
As a method for labeling the primer or the probe according to the present invention with a fluorescent substance, a method generally carried out in this field may be used as long as it is according to a method known per se, and specifically, for example, a method of incorporating a nucleotide labeled with a fluorescein into a primer or a probe according to a method known in the art may be used.
The method of labeling the primer or probe according to the present invention with a radioisotope may be carried out according to a method generally known per se in the art, and specifically, for example, a method of labeling by incorporating a nucleotide labeled with a radioisotope may be mentioned. Specifically, a random primer method, a gap shift method, a5 '-end labeling method based on T4 polynucleotide kinase, a 3' -end labeling method based on terminal deoxynucleotidyl transferase, and the like can be mentioned.
The method of labeling the primer or probe of the present invention with an enzyme may be carried out according to a method known per se which is generally carried out in this field, and specifically, for example, a direct labeling method in which an enzyme molecule such as alkaline phosphatase or horseradish peroxidase is directly covalently bound to the primer or probe to be labeled may be mentioned.
In the present invention, binding agent refers to a naturally occurring or non-naturally occurring molecule that specifically binds to a target sequence. Examples of specific binding agents include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids.
In the present invention, a binding agent that specifically binds to a protein encoded by the CD73 gene, such as the protein CD73 receptor, lectin that binds to protein CD73, an antibody directed against protein CD73, a peptide antibody (peptide body) directed against protein CD73, a bispecific dual binding agent, or a bispecific antibody format. Specific examples of specific binding agents include peptides, peptidomimetics, aptamer, spiegelmer, darpin, ankyrin repeat proteins, kunitz-type domains, antibodies.
The product comprises a chip, test paper, a kit or a nucleic acid membrane strip.
In the present invention, a chip, also referred to as an array, refers to a solid support comprising attached nucleic acid or peptide probes. The array typically comprises a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using mechanical synthesis methods or light-guided synthesis methods that combine a combination of photolithographic methods and solid-phase synthesis methods. The array may comprise a planar surface or may be a bead, gel, polymer surface, fiber such as optical fiber, glass or any other suitable nucleic acid or peptide on a substrate. The array may be packaged in a manner that allows for diagnosis or other manipulation of the fully functional device.
The term "microarray" is an ordered arrangement of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The substrate may be a solid substrate, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid substrate, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any arrangement thereof.
In the invention, the chip comprises a gene chip and a protein chip; the gene chip comprises a solid phase carrier and a probe fixed on the solid phase carrier, wherein the probe comprises an oligonucleotide probe aiming at a CD73 gene and used for detecting the transcription level of the CD73 gene; the protein chip comprises a solid phase carrier and a specific antibody of CD73 protein fixed on the solid phase carrier; the gene chip may be used to detect the expression levels of a plurality of genes including the human CD73 gene (e.g., a plurality of genes associated with parkinson's disease). The protein chip may be used to detect the expression levels of a plurality of proteins including human CD73 protein (e.g., a plurality of proteins associated with parkinson's disease). By simultaneously detecting a plurality of markers related to the Parkinson's disease, the accuracy of diagnosing the Parkinson's disease can be greatly improved.
In the present invention, the kit may further include a fluorescent dye, and various known fluorescent dyes may be used. For example, a method using an intercalator (intercalator) having a labeling function, a method using a probe in which a fluorescent substance is bound to a nucleotide that hybridizes specifically to an amplified DNA sequence, and the like can be mentioned. Examples of intercalators include ethidium bromide, SYBR GreenI, which are unsaturated fluorescent dyes, resolight (manufactured by Roche Co., ltd.) and EvaGreen (manufactured by Biotim Co., ltd.) which are saturated fluorescent dyes. The intercalator is preferably SYBR Green I as an unsaturated fluorescent dye or EvaGreen, resolight as a saturated fluorescent dye, more preferably EvaGreen, resolight as a saturated fluorescent dye. The amount of the fluorescent dye to be used was recommended by the manufacturer and seller of the fluorescent dye to be used.
The kit also includes instructions, which may include instructions for obtaining a sample, treating a sample.
The kit may contain genomic DNA of bacteria used as a positive control for PCR and sterile water used as a negative control.
In the present invention, the components of the kit may be packaged in an aqueous medium or in a lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, flask, baud bottle, syringe, or other container in which one component may be placed, and preferably, an appropriate aliquot may be performed. Where more than one component is present in the kit, the kit will also typically contain a second, third or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in one vial. The kits of the invention will also typically include a container for holding the reagents, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.
The solid support of the kit may be, for example, a plastic, a silicon wafer, a metal, a resin, a glass, a membrane, particles, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film, a plate, or a slide. The biological sample may be, for example, a cell culture, a cell line, a tissue, an oral tissue, a gastrointestinal tissue, an organ, a cellular organelle, a biological fluid, a serum sample, a urine sample, or skin.
In the present invention, a nucleic acid membrane strip includes a substrate and a probe immobilized on the substrate; the substrate may be any substrate suitable for immobilization of probes including, but not limited to, nylon membranes, nitrocellulose membranes, polypropylene membranes, glass sheets, silica gel wafers, micro magnetic beads.
The chip, test paper, kit or nucleic acid membrane strip described in the present invention can be used to detect the expression levels of a plurality of genes or proteins including CD73 gene or protein and their expression products.
In the present invention, a sample or specimen has the same meaning and is used interchangeably. A specimen or sample refers to any substance, biological fluid, tissue, or cell obtained or otherwise taken from an individual. It includes blood (including whole blood, white blood cells, peripheral blood mononuclear cells, buffy coat, plasma, and serum), tissue, exosomes, sputum, tears, mucus, nasal washes, nasal aspirates, breath-like, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymphatic fluid, nipple aspirates, bronchial aspirates, synovial fluid, joint aspirates, ascites, cells, cell extracts, and cerebrospinal fluid. It also includes all of the experimentally isolated fractions described above. For example, a blood sample may be fractionated into serum or into fractions containing specific types of blood cells, such as red blood cells or white blood cells (white blood cells). If desired, the sample may be a combination of samples from an individual, such as a combination of tissue and fluid samples. The sample also includes a substance containing a homogeneous solid substance, such as a substance from a fecal sample, a tissue sample, or a biopsy. Samples also include materials derived from tissue culture or cell culture. Any suitable method for obtaining a biological sample may be utilized; exemplary methods include, for example, phlebotomy, wiping (e.g., oral wiping), and fine needle biopsy procedures. Samples may also be collected by, for example, microdissection (e.g., laser Capture Microdissection (LCM) or Laser Microdissection (LMD)), smear (e.g., PAP smear), or catheter lavage. Samples obtained from or derived from an individual include any such samples that are processed in any suitable manner after being obtained from an individual.
In an embodiment of the invention, the sample comprises serum, tissue, exosomes.
In a specific embodiment of the invention, the sample is selected from serum.
The invention provides application of CD73 in constructing a calculation model for diagnosing and staging Parkinson's disease.
In the present invention, the computational model comprises the expression level of CD 73. As the skilled person knows, the step of associating CD73 levels with a certain possibility or risk may be implemented and realized in different ways. Preferably, the measured concentrations of CD73 and one or more other markers are mathematically combined and the combined values are correlated with the underlying diagnostic problem. The determination of the marker values may be combined by any suitable prior art mathematical method.
The present invention provides the use of CD73 in the construction of a system/device/computer readable storage medium for diagnosis and staging of parkinson's disease.
In the present invention, implementation of the system may include manually, automatically, or a combination thereof to perform or complete selected tasks. Moreover, the actual instrumentation and equipment of the embodiments of the system according to the present invention could implement several selected tasks by hardware, by software, or by firmware or by a combination thereof using an operating system.
For example, the hardware used to perform the selected task may be a chip or a circuit. As software, the selected tasks may be implemented as a plurality of software instructions being executed by a computer using any suitable operating system. In the present invention, one or more tasks according to exemplary embodiments of the method and/or system as described herein may be performed by a processing unit, such as a computing platform for executing a plurality of instructions. Optionally, the processing unit comprises a volatile memory for storing instructions and/or data and/or a non-volatile memory for storing instructions and/or data, e.g. a magnetic hard disk and/or a removable medium. Optionally, a network connection is also provided. A display and/or a user input device such as a keyboard or mouse may also optionally be provided.
In the present invention, a computer-readable storage medium, such as computer-executable code, may take many forms, including but not limited to, tangible storage media, carrier wave media, or physical transmission media. Nonvolatile storage media includes, for example, optical or magnetic disks, such as any storage devices in any computer, volatile storage media including dynamic memory, main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier wave transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency and infrared data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, a cable or link transporting such a carrier wave, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
The invention is further illustrated below in connection with specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The principal features of the invention may be used in various embodiments without departing from the scope of the invention.
Examples
1 data and method
1.1 study object
Samples of this study were taken in PD patients hospitalized in the geriatrics of the second hospital of the university of henry in the river north medical science from 11 months 2019 to 4 months 2023, wherein 33 cases of PD patients were taken in the training set as a case group, of which 16 women (48.5%) and 17 men (51.5%); 0 smokers; 35 healthy physical viewers who received the contemporaneous physical examination center were also included as a healthy control group, of which 20 females (57.1%), 15 males (42.9%); 0 smokers.
97 cases of the verification set included PD patients were a case group in which 51 women (52.6%), 46 men (47.4%), 10 smokers (10.3%); 71 healthy physical viewers who received the contemporaneous physical examination center were also included as a healthy control group, of which 31 females (43.7%), 40 males (56.3%), and 14 smokers (19.7%).
1.2 inclusion criteria
(1) Meets the diagnosis and diagnosis standard of 2016 type Chinese parkinsonism; (2) the course of the disease reaches more than 1 year; (3) the patient has one or more of the following characteristics and is obvious, including postural instability, resting tremor, muscle stiffness, non-primary vision disorders, and the like; (4) the clinical data is complete; (5) the patient was known to the family, signed informed consent, and passed through the Council of ethics of the home (ethics review resolution number: 2022-R039).
1.3 exclusion criteria
(1) Patients with secondary parkinsonism; (2) patients with parkinsonism; (3) patients with severe center of gravity, liver, kidney, and gastrointestinal dysfunction; (4) providing a patient with unclear and detailed medical history; (5) infected persons in approximately 1 month; (6) patients suffering from immune system diseases; (7) patients with chronic infectious diseases; (8) an anti-inflammatory agent and an immunomodulator are used in about 1 month; (9) patients with malignant tumor history; recently, there are trauma or cranium operators.
1.4 study content and method
Collecting inclusion object data includes: (1) basic data: gender, smoking history, and course of the patient in the case group. (2) Case group patient condition assessment: the motor function of the patient is evaluated by adopting a unified parkinsonism rating scale part 3 (motor examination) (the unified Parkinson's disease rating scale part III, UPDRS-III) (all of which are evaluated in a state of ' off ' when the patient stops temporarily), the scale is 14, each scale is 0-4 points according to the severity of the corresponding symptoms, and the lower score indicates the better quality of life; patient dyskinesia severity was assessed using H & Y staging, and patients were classified according to their motor symptoms into mild PD groups (stage i H & Y, stage ii) and moderate PD groups (stage iii-v H & Y). All of the above assessments were jointly attended by 2 neurologists. (3) Serum CD73 results: all the inclusion persons were used to empty vein blood and sent to the full-automatic biochemical analyzer of clinical laboratory at the second hospital of university of Hebei medical science to examine the obtained 5' -NT value.
1.5 statistical methods
Statistical analysis was performed using SPSS software version 26.0, with t-test for normal distribution of data meteringIndicating that otherwise, the median (quartile spacing) is employed [ M (Q1, Q3)]Representing, performing rank sum test; the counting data is checked by x 2In examples (percent) [ n (%)]Performance; binary Logistic regression is adopted to analyze the relevance of each risk factor and PD; the predictive value adopts the analysis of a working characteristic curve of a subject to obtain the area under the curve (area under the curve, AUC), confidence interval, sensitivity, specificity and cut-off value; pearson's method (Pearson) for case group serum CD73 and H&And (5) carrying out correlation analysis on the Y-stage and UPDRS-III scores. One-way ANOVA for analysis of different H&The change of CD73 level in serum of PD patients with stage Y has statistical significance by taking P < 0.05 as a difference.
2 experimental results
2.1 general data analysis of case group and healthy control group
The study validation set co-incorporates 168 study subjects. Of these, 97 cases and 71 healthy controls were used. The sex distribution of the two groups of subjects was balanced (P > 0.05) and there was no significant difference in smoking history (P > 0.05). In addition, the disease course of the case group was 2.0 (1.0,4.0) years, the H & Y stage was 2.0 (2.0, 3.0), and the UPDRS-III score was 18.66.+ -. 0.70 score. CD73 expression was significantly reduced (P < 0.01) in peripheral blood of PD patients (table 1).
TABLE 1 general data analysis of the validation set case group and healthy control group
Note that: a is t test; b is chi-square test; c is rank sum check
2.2 Logistic regression analysis of serum CD73 and other risk factors in relation to Parkinson's disease
After a Logistic regression analysis, the results showed that the lower serum CD73 levels, the greater the risk of developing PD (or=0.208, p < 0.01) after gender and smoking history were adjusted (table 2).
TABLE 2 Logistic regression analysis of the correlation of serum CD73 and other risk factors with PD
Note that: * As a healthy control group
2.3 differential expression of serum CD73 in PD
In the training set, ROC curves were drawn with healthy control as positive samples and case as negative samples, and the result showed that serum CD73 had AUC of 0.844 (fig. 1) for PD prediction.
The pooled serum CD73 was verified to have an AUC of 0.845, an optimal sensitivity of 90.1% and a specificity of 67.0% for PD prediction (table 3, fig. 2).
TABLE 3 predictive ROC curve of validation set serum CD73 versus PD
Note that: a represents U/L
General data analysis of 2.4 mild and moderate PD groups
Grouping the study subjects in case groups according to H & Y stage, stage i, stage ii being mild PD group (n=50); stage iii-v is the moderately severe PD group (n=47). There were no statistical differences in gender and smoking history (P > 0.05) between the two groups of patients. The moderately severe PD group had a longer course than the slightly PD group, with a higher UPDRS-iii score and a significantly reduced serum CD73 level (P < 0.05) (table 4).
Table 4 validation set general data analysis for mild and moderate PD groups
Note that: a is t test; b is chi-square test; c is rank sum check
Correlation analysis of serum CD73 from 2.5PD patients with H & Y stage, UPDRS-III score
The serum CD73 levels of PD patients were inversely correlated (P < 0.01) with H & Y stage and UPDRS-III scores as determined by Pearson correlation analysis (Table 5).
TABLE 5 correlation analysis
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. The use of a reagent for detecting the expression level of CD73 in a sample for the preparation of a product for diagnosis and staging of parkinson's disease.
2. The use according to claim 1, wherein the reagent is selected from the group consisting of a probe specifically recognizing the CD73 gene, a primer specifically amplifying the CD73 gene, or a binding agent specifically binding to a protein encoded by the CD73 gene;
preferably, the primer or probe is labeled with a labeling substance;
preferably, the labeling substance includes a fluorescent substance, a radioisotope, or an enzyme.
3. The use according to claim 1, wherein the product comprises a chip, a test strip, a kit or a nucleic acid membrane strip.
4. The use according to claim 3, wherein the chip comprises a gene chip, a protein chip;
preferably, the gene chip comprises a probe for CD73 gene for detecting the transcription level of CD73 gene, and the protein chip comprises a specific binding agent for CD73 protein;
preferably, the kit comprises a gene detection kit and a protein detection kit;
preferably, the gene detection kit comprises a reagent or chip for detecting the transcription level of the CD73 gene, and the protein detection kit comprises a reagent or chip for detecting the expression level of the CD73 protein;
preferably, the kit further comprises instructions;
preferably, the kit further comprises a buffer.
5. The use according to claim 4, wherein the kit further comprises reagents for detecting the expression level of CD73 gene or protein by RT-PCR, qRT-PCR, biochip assay, southern blotting, in situ hybridization, immunoblotting;
preferably, the sample comprises serum, tissue, exosomes;
preferably, the sample is selected from serum.
6. A product for diagnosis and staging of parkinson's disease, said product comprising an agent for detecting the level of CD73 expression in a sample;
preferably, the product comprises a chip, a test paper, a kit or a nucleic acid membrane strip;
preferably, the sample comprises serum, tissue, exosomes;
preferably, the sample is selected from serum.
7. A method of screening for a candidate agent for treating parkinson's disease, said method comprising: treating a culture system expressing or containing the CD73 gene or a protein encoded by the CD73 gene with a substance to be screened; and detecting the expression or activity of the CD73 gene or a protein encoded thereby in the system; wherein the substance to be screened is a candidate drug for treating Parkinson's disease when the substance to be screened promotes the expression level or activity of the CD73 gene or a protein encoded by the same.
Use of cd73 as a target in screening candidate drugs for the treatment of parkinson's disease;
preferably, the method for screening candidate drugs for treating parkinson's disease comprises: treating a culture system expressing or containing the CD73 gene or a protein encoded by the CD73 gene with a substance to be screened; and detecting the expression or activity of the CD73 gene or a protein encoded thereby in the system; wherein the substance to be screened is a candidate drug for treating Parkinson's disease when the substance to be screened promotes the expression level or activity of the CD73 gene or a protein encoded by the same.
Application of CD73 in constructing a calculation model for diagnosis and stage of Parkinson's disease.
Use of cd73 in constructing a system/device/computer readable storage medium for diagnosis and staging of parkinson's disease.
CN202310912160.2A 2023-07-25 2023-07-25 Application of CD73 in diagnosis of parkinsonism Pending CN116949168A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130217033A1 (en) * 2007-10-24 2013-08-22 Faron Pharmaceuticals Oy Biomarker for monitoring development of diseases and assessing the efficacy of therapies
CN103278634A (en) * 2013-05-16 2013-09-04 中国科学院近代物理研究所 Application of CD73 as stem cell surface marker of renal clear cell carcinoma
CN106434939A (en) * 2016-10-18 2017-02-22 乐卫东 Diagnosis kit for Parkinson's disease, and application of diagnosis kit

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US20130217033A1 (en) * 2007-10-24 2013-08-22 Faron Pharmaceuticals Oy Biomarker for monitoring development of diseases and assessing the efficacy of therapies
CN103278634A (en) * 2013-05-16 2013-09-04 中国科学院近代物理研究所 Application of CD73 as stem cell surface marker of renal clear cell carcinoma
CN106434939A (en) * 2016-10-18 2017-02-22 乐卫东 Diagnosis kit for Parkinson's disease, and application of diagnosis kit

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