CN113125758A - Application of C/EBP beta in preparation of product for diagnosing or assisting in diagnosing acute ischemic stroke - Google Patents
Application of C/EBP beta in preparation of product for diagnosing or assisting in diagnosing acute ischemic stroke Download PDFInfo
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- 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
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- G—PHYSICS
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- 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
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
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- G01N2800/00—Detection or diagnosis of diseases
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- G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
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Abstract
The invention provides a biomarker C/EBP beta for diagnosing or assisting to diagnose acute ischemic stroke of mammals. The research results of the inventor show that the expression level of the C/EBP beta protein in the peripheral plasma of the cerebral ischemia mouse and the AIS patient in the acute phase is obviously reduced compared with that in the normal physiological state, and the expression level of the C/EBP beta in the peripheral plasma of the AIS can singly or jointly predict the adverse outcome risk of the patient. Compared with the traditional clinical model, the diagnostic efficacy can be obviously improved after the peripheral plasma C/EBP beta protein expression level factor is added. Furthermore, the C/EBP β protein is present in the plasma in the form of exosomes. In conclusion, C/EBP beta in plasma is a biomarker for judging the prognosis effect of AIS patients. The blood examination method is easy to accept by the examinee, and can be an effective means for early diagnosis, disease progress judgment and prognosis evaluation of AIS patients.
Description
Technical Field
The invention relates to an application of C/EBP beta in preparing a product for diagnosing or assisting in diagnosing acute ischemic stroke in the biomedical field.
Background
The transcription factor CCAAT/enhancer binding protein beta (C/EBP beta) is an important transcription factor belonging to the C/EBP family members associated with inflammatory responses to neurodegenerative or brain injury, which regulate activated microglia genes, promoter and enhancer regions binding various cytokines or pro-inflammatory genes. The C/EBP beta participates in the functions of regulating cell growth, differentiation and the like in a normal physiological state, is mainly expressed in microglia in the pathological process of brain injury and neurodegenerative diseases, can mediate microglial disorder and trigger inflammatory reaction, and plays an important role in accelerating the progress of the disease course of nervous system diseases.
Neurovascular disease is becoming more prevalent due to the increasing burden of vascular risk factors and the aging problem of prominent human mouth. The acute ischemic stroke accounts for 87 percent of all strokes, and is a serious disease which is recognized by the world to seriously threaten human health due to high morbidity, high recurrence rate, high mortality rate and high disability rate. Ultra-early or early (within 6 hours of onset), individualized treatment is critical for prognosis in patients with acute stroke. There are several problems that exist: (1) acute ischemic stroke is diagnosed by clinical evaluation and neuroimaging, and needs a long time; (2) many patients with transient ischemic attacks are asymptomatic at the time of arrival at the hospital and do not allow accurate neurological assessment; (3) even if the patient's basic characteristics at the time of stroke are similar, there may be a large difference in prognosis. These factors can delay diagnosis and targeted treatment of the patient, resulting in missed optimal treatment sessions. Although blood biomarkers have been used for many years in the diagnosis of other vascular diseases, no biomarker related to stroke has been found to be applied clinically, and the accuracy of biomarkers reported so far is far lower than the requirements of clinical practice.
Therefore, the development of rapid and convenient blood biomarker diagnosis indexes of acute ischemic stroke patients has an important prompting function for early diagnosis and subsequent treatment of stroke.
Disclosure of Invention
The invention aims to solve the technical problem of how to diagnose or assist in diagnosing acute ischemic stroke.
In order to solve the technical problems, the invention provides a biomarker for diagnosing or assisting to diagnose acute ischemic stroke of mammals, wherein the biomarker is transcription factor CCAAT/enhancer binding protein beta (C/EBP beta).
The invention also provides any one of the following applications of the substance for detecting the content of the biomarker in the sample:
u1, and application in preparing products for diagnosing or assisting in diagnosing acute ischemic stroke of mammals;
u2, and application in preparing products for predicting prognosis of acute ischemic stroke of mammals;
u3 and application thereof in preparing products for screening or assisting in screening acute ischemic stroke patients of mammals.
In the above application, the sample is peripheral plasma.
In the above application, the substance for detecting the content of C/EBP β in the sample may comprise a reagent and/or an apparatus for detecting the content of C/EBP β, such as a reagent and an apparatus for detecting the content of C/EBP β by an ELISA method.
In order to solve the technical problems, the invention also provides a product, which is any one of the following products:
v1, a product for diagnosing or aiding in the diagnosis of acute ischemic stroke in mammals;
v2, a product for predicting the prognosis of acute ischemic stroke in mammals;
v3, products for screening or auxiliary screening of mammals for acute ischemic stroke patients;
the product comprises reagents and/or instruments for detecting the content of the biomarkers.
In the above product, the sample is peripheral plasma.
Any of the above mammals can be a human or a mouse.
The inventor firstly discovers that the C/EBP beta protein is expressed in peripheral plasma of healthy people and Acute Ischemic Stroke (AIS) patients, and the expression level of the C/EBP beta protein in the plasma can improve the 3-month prognosis and the prediction energy efficiency of the treatment effect of a traditional model on the stroke patients.
Drawings
FIG. 1 is a graph showing the expression level of C/EBP β protein in peripheral plasma of mice measured by ELISA in example 1 of the present invention. The test objects are peripheral plasma of a normal mouse and peripheral plasma of MCAO model mice 3 days after cerebral ischemia. Data shown are expressed as mean ± standard deviation, representing a significance analysis of P <0.001 in mice 3 days after cerebral ischemia compared to normal mice.
FIG. 2 is a graph showing the expression level of C/EBP β protein in plasma of healthy persons and AIS patients measured by ELISA in example 2 of the present invention. The AIS patient is an acute ischemic stroke patient. Data shown are expressed as mean ± sd representing P <0.05 for AIS patients versus healthy persons for significance analysis.
FIG. 3 is a graph showing the comparison of the expression level of C/EBP β protein in peripheral plasma of two groups of patients with mRS ≦ 2 dichotomy for AIS patient in example 2 of the present invention. Data shown are expressed as mean ± sd, representing mRS >2 compared to AIS patients with mRS ≦ 2 for significance analysis with P < 0.01.
FIG. 4 is a graph showing the detection of C/EBP β protein expression in plasma exosomes of healthy humans and AIS patients in example 3 of the present invention. Panel a of figure 4 is a nanoparticle tracer analysis of exosomes. Fig. 4B shows the exosome morphology identification by transmission electron microscopy, with scale 200 nm. FIG. 4 is a C diagram of a Westernblot assay for exosome protein markers; positive control and exosomes extracted from plasma are indicated, respectively. FIG. 4 is a D-diagram of Westernblot for measuring the expression level of C/EBP β protein in exosomes of healthy humans and AIS patients.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.
The C57BL/6J male mice in the following examples are of a standard strain, provided by experimental animal technology ltd, viton, beijing, with an animal license number: SCXK (Jing) 2019-.
The results of the quantitative tests in the following examples were averaged.
Example 1 change in C/EBP beta protein expression in peripheral plasma of cerebral ischemic mice
Subject: c57BL/6J male mice (10-12 weeks old, 22-24 g in weight, Specific Pathogen Free (SPF) class of cleaning grade) were randomly divided into MCAO model mouse group and normal control mouse group, each group containing 10 mice, and the treatment of each group was as follows:
MCAO model mouse groups: a Middle cerebral artery occlusion Model (MCAO) was prepared by a wire-embolization method in which mice were fasted overnight before surgery but water was not prohibited. The specific operation is as follows: after being weighed, the mouse is induced and anesthetized by 4 percent of enflurane, a small animal respirator anesthesia machine is used, and 1 to 2 percent of enflurane is mixed with 70 percent of N2O and 30% of O2Anesthesia is maintained. Monitoring anus during operationWarm, blood sugar and blood qi, and ensure that all indexes are maintained in a normal range. During modeling, blood flow was monitored in the middle cerebral artery distribution area of mice using laser Doppler (PERIMED, PF 5001). The mouse is supine on the operating table, and the right common carotid artery, external carotid artery and internal carotid artery are separated by taking the incision in the middle of the neck. And (5) electrocoagulating and cutting off the thyroid artery and the occipital artery on the right side respectively. Two 9/0 nylon threads are ligated to the external carotid artery, wherein the proximal end is not tightly ligated and the distal end is tightly ligated to cut off the blood vessel at the distal end by electrocoagulation. The suture is tied at the proximal end of the common carotid artery without tight ligation. The distal end of the internal carotid artery was occluded by a mouse artery clamp to block blood flow. A small opening is cut between two ligation sites of the external carotid artery by using a microshear, a thread plug (701956PK5 Re; Doccol Corporation) with the diameter of 0.19mm at the head end is inserted, the ligation thread at the proximal end is loosened, and the thread plug enters the middle cerebral artery through the internal carotid artery until meeting slight resistance and the plug entering depth is about 10-12 mm. The ligature at the proximal end of the external carotid artery is tightened to fix the suture plug, and the suture on the common carotid artery is untied to restore blood flow. After suturing the skin, the mice are laid on the side and placed in a new breeding cage, after 45min, the mice are narcotized again, the thread plugs are pulled out to realize blood flow reperfusion, then the skin is sutured, and the mice are placed in the breeding cage for normal breeding for 3 days to separate the peripheral plasma of the mice.
Normal control mouse group: the same MCAO model mouse group was treated without surgery.
Separation of mouse peripheral plasma: peripheral blood was collected from the MCAO model mouse group and the normal control mouse group by abdominal aortic puncture, respectively. The specific mode is as follows: after anesthetizing the mice with chloral hydrate, they were placed on a dissecting plate in the supine position, and the limbs were fully stretched and fixed. The skin and peritoneum were cut open along the midline of the abdomen, leaving the abdominal cavity fully exposed. The abdominal aorta and the abdominal main vein were visualized by gently pushing the tube through filter paper. Selecting a centripetal end 1-3 mm from a bifurcation part of an abdominal aorta into left and right frontal arteries as a puncture point, puncturing the abdominal aorta with a 12-gauge injection needle with the inclined plane downwards to a depth of about 5mm, extracting 500 mu L of peripheral blood, slightly pumping the peripheral blood into a centrifugal tube containing EDTA-K2 anticoagulant, slightly reversing the peripheral blood for 8-10 times, and uniformly mixing. Separating plasma within 1h after blood collection, centrifuging the blood sample at room temperature of 800g for 10min, collecting the supernatant to a new EP tube, centrifuging the blood sample at the temperature of 4 ℃ for 10min at 16,000g, collecting the supernatant to a new EP tube, and collecting the obtained MCAO model mouse peripheral plasma and normal control mouse peripheral plasma for subsequent experiments.
Detection of the expression level of the peripheral plasma C/EBP β protein (GeneBank Gene ID:12608, updated at 2021, 5/9): detection was performed by ELISA according to the instructions. The ELISA detection kit adopts a Mouse (Mouse) CCAAT enhancer binding protein beta (C/EBP beta) ELISA detection kit (SY-M09127) produced by Shanghai win-win biotechnology, Inc.
The results of ELISA tests on peripheral plasma of MCAO model mice and peripheral plasma of normal control mice are shown in figure 1, and compared with the normal control mice, the expression level of C/EBP beta protein in the plasma of MCAO model mice 3 days after cerebral ischemia is obviously reduced.
Example 2 changes in C/EBP β expression in AIS patients' peripheral plasma and its impact on the predictive outcome of stroke patients in a traditional model
Subject: the department of medicine university Xuanwu hospital department of capital department neurology is the research base, and neurology receives and cures Acute Ischemic Stroke (AIS) patient throughout the year to establish the specimen bank, can provide abundant case data for this research. The study patients' blood was derived from AIS patients who received treatment at the martial hospital, university of capital medical, between 2015 and 2019. The normal control blood samples are all from health examination population of the Xuanwu hospital of the university of medical science of capital. All specimens were approved by the ethical committee of the department of capital medical university, the martial arts hospital, and all studies involving human specimens were approved by the patient or their principal.
Collecting clinical data: the main clinical data collection part comprises AIS patients and normal people, and both parts need to contain general data: including the patient's name, gender, age, occupation, past medical history, family history, female menstrual history, and marriage and childbirth history. AIS patients are mainly organized by hospitalization data. The clinical characteristics of the patient include clinical characteristics such as blood routine examination, MRI detection, mRS score, NIHSS score, sICH score, routine auxiliary examination and the like besides the general data. Meanwhile, follow-up visit agreement of all people is obtained, and the inspection result data is perfectly stored.
In this example, the inclusion criteria for AIS patients were: age 18-80 years; ② the clinical consideration is ischemic stroke: paroxysmal, focal, clinical symptoms manifested as neurological deficit (major symptoms include speech impairment, akinesia, cognition, gaze impairment, visual field impairment, and visual neglect); ③ the disease is in 4.5h, and the apoplexy symptom lasts at least 30min, and the disease is not improved obviously before the treatment; fourthly, the clinical manifestation is the symptom of the anterior circulation infarction, and the NIHSS score is more than or equal to 4 points; removing other cerebrovascular diseases such as cerebral hemorrhage, brain tumor and the like by CT image result, and having no early signs of large-area cerebral infarction; sixth, the patient (either the guardian or the immediate relative) signs an informed consent.
Exclusion criteria: the method comprises the following steps of (1) having a history of intracranial hemorrhage, having head trauma in the last 3 months, having gastrointestinal or urinary hemorrhage in the last 3 weeks, having major surgical operations in the last 2 weeks, and having arterial puncture at a part which is not easy to compress for hemostasis in the last 1 week; ② the myocardial infarction of the cerebral infarction patients in about 3 months, not including old cava infarction but not remaining with nerve defect; ③ severe heart, liver, kidney or diabetes; oral anticoagulant, INR>1.5; platelet count<100×109L, blood glucose<50 mg/dL; contraction pressure>185mmHg, or diastolic pressure>110 mmHg; seventhly, the patient who can not be examined by nuclear magnetic resonance has claustrophobia, heart valve replacement or the placement of false teeth made of metal material; eighty percent pregnancy; ninthly, unwilling to sign informed consent, unwilling to receive follow-up visits or the researcher deems inappropriate for the clinical researcher.
The inclusion criteria for healthy persons were: the age is 18-80 years old, and people with normal indexes are male and female.
AIS patient peripheral plasma collection: blood was collected, once intravenously (5 mL/tube) the first time after admission to each AIS patient for subsequent experimental studies. The blood collecting tube for plasma samples is an ethylene diamine tetraacetic acid dipotassium (EDTA-K2) anticoagulation tube, the separation and freezing storage of the plasma are completed within 2h, and the plasma samples are placed into a refrigerator at minus 80 ℃ for long-term storage after being subpackaged (1 mL/tube).
Detection of the expression level of peripheral plasma C/EBP β protein (GeneBank Gene ID:1051, updated at 2021, month 5 and 18): detection was performed by ELISA according to the instructions. The ELISA detection kit adopts a Human (Human) CCAAT enhancer binding protein beta (C/EBP beta) ELISA detection kit (SY-ELA07329) produced by Shanghai win-win biotechnology and technology Limited.
(1) Altered C/EBP beta protein expression in peripheral plasma after AIS patients
The AIS patients (257) peripheral plasma were isolated and subjected to ELISA test, and the results are shown in fig. 2, and the expression level of plasma C/EBP β protein of AIS patients was significantly reduced compared to that of healthy persons (88) peripheral plasma.
(2) Effect of C/EBP beta expression in AIS patient peripheral plasma on patient prognosis prediction by traditional model
The protein expression level of the peripheral plasma C/EBP beta of 300 AIS patients classified according to the mRS score after 3 months (179 AIS patients with mRS less than or equal to 2 and 121 AIS patients with mRS >2) is respectively subjected to statistical analysis with the mRS neurological function score and the risk efficacy predicted by a traditional clinical model. The results are shown in table 1 and fig. 3, and the AIS patients were classified as baseline characteristics according to mRS ≤ 2 after 3 months, and the demographic characteristics, past medical history, stroke characteristics, treatment, clinical biological characteristics, result evaluation, biological evaluation, and C/EBP β protein expression levels of the two groups of patients were compared. The level of C/EBP β expression in the peripheral plasma of AIS patients with mRS >2 is significantly reduced compared to AIS patients with mRS ≦ 2. Table 2 suggests that the expression level of C/EBP β in AIS patient peripheral plasma can be used alone or in combination to predict the risk of adverse outcome in patients, wherein adverse outcome means mRS >2 after 3 months in patients. I.e. when the expression level of C/EBP β protein in peripheral plasma is higher at the time of admission of the AIS patient, the risk of mRS >2 is lower 3 months after the patient, indicating a better neurological function. Table 3 shows that compared with the traditional clinical model, the AIS patient diagnosis efficacy can be remarkably improved after the addition of the peripheral plasma C/EBP beta protein level expression factor (AUC:78.3 vs.82.8; P < 0.05).
TABLE 1AIS patients baseline characteristics by 3-month mRS two-class
TABLE 2 risk of adverse outcome (3 month mRS >2) in patients with AIS C/EBP β in peripheral plasma alone or in combination
Note: model 1 refers to the estimation of the poor prognosis OR value of a patient from the C/EBP beta protein level in the plasma of the patient alone; model 2 refers to the age of inclusion, admission NIHSS score, hypertension, lymphocyte count, triglyceride and C/EBP β white blood levels in plasma to estimate the poor prognostic OR value for the patient. The variables included in the model were screened by lasso regression (&, continuous variables; # categorical variables).
A model construction step: considering the collinearity existing among clinical variables, the independent variable coefficient in the baseline data is punished by adopting lasso regression, and finally, the age of the included variable, the NIHSS score of admission, hypertension, lymphocyte count and triglyceride level are obtained to obtain a higher AIC value under the condition of smaller collinearity, so that the variables are included in the clinical model.
Two of the methods used to predict the risk of a 3-month poor outcome in a patient (C/EBP β levels in the patient's peripheral plasma as a negative correlation factor with the patient's 3-month poor prognosis):
(ii) when the levels of C/EBP β in peripheral plasma were raised by one unit per liter at the time of admission to the AIS patient, the risk of a poor prognosis (mRS >2) was increased by 0.988-fold (i.e. decreased by 1.012-fold) for the patient at 3 months;
② when AIS patients are admitted C/EBP beta <51.26pg/mL in peripheral plasma, the patient's risk of poor prognosis (mRS >2) for 3 months is increased 1.679-fold compared to patients with C/EBP beta >51.26 pg/mL.
Both models suggest: when the expression level of C/EBP β protein is higher in peripheral plasma at the time of admission of the AIS patient, the risk of mRS >2 is lower, i.e. the neurological function is better, after 3 months of the patient.
TABLE 3 increase of C/EBP β in AIS patient peripheral plasma the efficacy of the traditional clinical model to predict poor prognosis risk in patients
Wherein Clinical model refers to the patient age, hypertension, lymphocyte count, triglyceride, and admission NIHSS score inclusion analysis, and Clinical model + C/EBP β refers to the patient age, hypertension, lymphocyte count, triglyceride, admission NIHSS score, and C/EBP β protein expression level inclusion analysis. mRS >2 after 3 months in AIS patients was taken as poor prognosis, AUC indicates prognostic risk efficacy.
Example 3 expression of C/EBP β protein in exosomes in AIS patients plasma
Subject: healthy and AIS patients, inclusion criteria were as described in example 2.
And (3) separating plasma exosomes, namely extracting exosomes in plasma by exclusion and ultrafiltration according to the instruction by using an Exosucur kit (Enzekantai).
Identifying and analyzing the biological characteristics of exosomes (1) identifying the form of exosomes: and (3) dropping 20 mu L of the exosome heavy suspension on a special copper net for an electron microscope, and standing for 3 min. After a sample is fixed, negatively dyeing the sample with uranyl acetate, standing the sample at room temperature for 5min, sucking residual redundant liquid with absorbent filter paper, standing the sample under a 60W incandescent lamp for 2-3 min, and after the sample is dried, placing the sample under a transmission electron microscope (HITACHI, H-7650) to observe the shape of the sample; (2) particle diameter identification of exosomes: nanoparticle Tracking Analysis (NTA) measures exosome particle size and concentration. After fluorescent silica nanoparticles with known diameters are mixed with an exosome sample, a laser with the wavelength of 532nm is selected on a ZetaView nanoparticle tracking analyzer (PMX-110), and a sample mixing system is detected by using a scattering channel and a PE fluorescent channel. Drawing a standard working curve according to the diameters of the series of silicon dioxide nano particles and the scattered light intensity, correcting the refractive index difference of the exosome, converting the scattered light intensity of the exosome into a diameter parameter, and calculating the particle diameter of the exosome; (3) identification and analysis of exosome proteins: exosome suspensions were quantitated for total protein using BCA protein quantitation kit (thermoscientificic, #23227), followed by SDS-PAGE electrophoresis, wet rotation for 1.5h, 5% BSA blocked for 2h at room temperature, TBS-T wash followed by incubation of exosome protein positive markers: CD9(Proteintech, #60232-1-Ig), Tsg101(Absin, # Abs115706), CD63(Santa Cruz, # sc-5275), exosome-protein negative markers: calnexin (Proteintech, #10427-2-AP), and the target protein C/EBP β (Cell signaling technology, #3087) were incubated overnight at 4 ℃. After washing with TBST buffer, the corresponding secondary antibody (Cell Signaling Technology, #7074, #7076) was added, incubated at room temperature for 1h, followed by development and photography using an electrochemiluminescence apparatus (Olympus, Fluro Chen 2.0).
Plasma exosomes of healthy people and AIS patients were isolated and identified, and the expression of C/EBP β protein in exosomes was detected. The result is shown in fig. 4, exosomes are successfully extracted from plasma, the average particle size is 94.3nm, clear vesicle morphological structures can be seen through transmission electron microscope observation, and exosome surface protein markers are also successfully detected. In addition, both healthy and AIS patients were detected to express C/EBP β protein by the Westernblot method, and the expression level of C/EBP β protein in AIS patients plasma was significantly lower than that of healthy humans in case of consistent total protein loading (15 μ g).
The research results show that the expression level of the C/EBP beta protein in the peripheral plasma of the cerebral ischemia mouse and the AIS patient in the acute phase is obviously reduced compared with that in the normal physiological state, and the expression level of the C/EBP beta protein in the peripheral plasma of the AIS can singly or jointly predict the adverse outcome risk of the patient. Compared with the traditional clinical model, the diagnostic efficacy can be obviously improved after the peripheral plasma C/EBP beta protein expression level factor is added. Furthermore, the C/EBP β protein is present in the plasma in the form of exosomes. In conclusion, C/EBP beta in plasma is a biomarker for judging the prognosis effect of AIS patients. The blood examination method is easy to accept by the examinee, and can be an effective means for early diagnosis, disease progress judgment and prognosis evaluation of AIS patients.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (8)
1. A biomarker for diagnosing or assisting to diagnose acute ischemic stroke of mammals, wherein the biomarker is C/EBP beta.
2. The biomarker of claim 1, wherein the mammal is a human or a mouse.
3. Use of a substance for detecting the level of a biomarker according to claim 1 or 2 in a sample, wherein the substance is selected from the group consisting of:
u1, and application in preparing products for diagnosing or assisting in diagnosing acute ischemic stroke of mammals;
u2, in preparing mammal acute ischemic stroke prognosis or auxiliary prognosis product;
u3 and application thereof in preparing products for screening or assisting in screening acute ischemic stroke patients of mammals.
4. The use according to claim 3, wherein the sample is peripheral plasma.
5. The biomarker of claim 2 or 3, wherein the mammal is a human or a mouse.
6. A product, characterized in that it is any one of the following:
v1, a product for diagnosing or aiding in the diagnosis of acute ischemic stroke in mammals;
v2, a mammalian acute ischemic stroke prognosis or prognosis-assisting product;
v3, products for screening or auxiliary screening of mammals for acute ischemic stroke patients;
the product comprises reagents and/or instruments for detecting the level of the biomarkers of claim 1 or 2.
7. The product of claim 6, wherein the sample is peripheral plasma.
8. The biomarker of claim 6 or 7, wherein the mammal is a human or a mouse.
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