Application of SPP1 gene in preparation of product for auxiliary diagnosis or cerebral infarction diagnosis

Abstract

The invention belongs to the technical field of disease diagnosis products, and discloses application of an spp1 gene in preparation of a product for auxiliary diagnosis or cerebral infarction diagnosis. Through construction of a mouse cerebral infarction model and single cell data analysis, it is found that in brain tissues of mice in a cerebral infarction group, the spp1 gene as a specific up-regulation differential gene shows a significant up-regulation trend in many cell types, and clinical samples are further collected for verification. The result shows that the spp1 gene has significant up-regulation in a peripheral blood sample of a cerebral infarction patient, has high diagnosis efficiency, provides a new direction for cerebral infarction diagnosis, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of disease diagnosis products, and relates to application of a spp1 gene in preparation of a product for auxiliary diagnosis or diagnosis of cerebral infarction. BACKGROUND

[0002] Cerebral infarction, also known as ischemic stroke, is a kind of irreversible damage to brain tissue caused by abnormal local blood circulation due to various factors, accompanied by cerebral ischemia, hypoxia and brain tissue necrosis. Due to its high incidence and high disability rate, cerebral infarction has been recognized as a global public health problem and has caused huge economic burden worldwide. As a high-risk disease in the elderly population, there are about 3 million cases of cerebral infarction worldwide each year, of which more than 50% of patients eventually die, and the survivors suffer from irreversible neurological damage sequelae. Although the elderly are the main patient group of cerebral infarction, recent studies have found that the age of onset of cerebral infarction is gradually becoming younger. According to reports, young cerebral infarction patients account for 10%-14% of all cerebral infarction patients. The current biomarkers for the diagnosis of cerebral infarction still have deficiencies in specificity and sensitivity. Therefore, it is particularly important to find biomarkers with high specificity and high sensitivity. The development of these biomarkers not only can improve the early diagnosis ability of cerebral infarction, but also can provide the basis for developing personalized treatment plans, thereby improving the prognosis of patients.

[0003] In clinical practice, the detection of cerebral infarction mainly relies on imaging technology and biomarker detection. Although these technologies play an important role in the diagnosis of cerebral infarction, they also have certain limitations. In terms of imaging technology, CT technology and MRI technology are mainly used. Although non-contrast computed tomography technology in CT technology can quickly diagnose cerebral infarction, its sensitivity to mild cerebral infarction is low, especially in the early stage of onset (sensitivity less than 20% within the first 3 hours). Although diffusion weighted imaging technology in MRI technology is the gold standard for detecting acute ischemic cerebral infarction and has high sensitivity, it takes a long time, is not suitable for patients with metal implants and claustrophobia. In addition, biomarkers S100β, neuron-specific enolase and glial fibrillary acidic protein can be used to assist in the diagnosis of cerebral infarction and evaluate the severity of injury, but they have insufficient specificity and sensitivity, are difficult to be released into the blood rapidly after the occurrence of cerebral infarction, and cannot accurately distinguish cerebral infarction from other similar diseases.

[0004] A biomarker is an endogenously produced biomolecule and can be used to detect an objective indicator of abnormal biological processes and can also help to detect whether a pharmacological intervention can be used to reduce tissue organ damage caused by pathological processes. Specifically, in patients with cerebral infarction, an effective biomarker can help to detect the main pathological processes of cerebral infarction in an early, accurate and easy way, i.e. from blood flow interruption to cell damage, inflammatory response, apoptosis, ischemia-reperfusion injury, and finally to tissue repair and remodeling. Therefore, changes in the expression level of the biomarker can be detected in damaged cells of the patient. The existing biomarkers for detecting cerebral infarction, S100β, neuron-specific enolase and glial fibrillary acidic protein, are insufficient in sensitivity and specificity. Therefore, it is very important to continue to find biomarkers with high sensitivity and high specificity for the prevention and damage classification of clinical cerebral infarction. SUMMARY

[0005] To solve the above technical problems, the present application provides an application of a spp1 gene in the preparation of a product for assisting in the diagnosis or diagnosis of cerebral infarction.

[0006] In a first aspect of the present application, a reagent for detecting the expression level of a spp1 gene in a sample is provided for use in the preparation of a product for assisting in the diagnosis or diagnosis of cerebral infarction, wherein the expression level of the spp1 gene is significantly up-regulated in the sample taken from a patient with cerebral infarction.

[0007] In some embodiments of the present application, the sample is selected from blood.

[0008] In some embodiments of the present application, the expression level is the mRNA expression level of the spp1 gene.

[0009] In some embodiments of the present application, the reagent comprises primers specific for amplifying the spp1 gene.

[0010] In some embodiments of the present application, the sequence of the primers specific for amplifying the spp1 gene is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0011] In a second aspect of the present application, a product for assisting in the diagnosis or diagnosis of cerebral infarction is provided, which comprises primers specific for amplifying the spp1 gene, and the sequence of the primers is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0012] The application is constructed by constructing a mouse brain infarction model and performing single cell data analysis, it is found that in the brain tissue of the mouse brain infarction group, the spp1 gene as a specific up-regulated differential gene shows a significant up-regulation trend in many cell types, and further collection of clinical samples is verified, it is proved that the spp1 gene in the peripheral blood samples of the brain infarction patients presents significant up-regulation, and has high diagnostic efficiency, which provides a new direction for diagnosing brain infarction, and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The mouse brain TTC staining pictures and the statistical result graphs of the cerebral infarction area percentage obtained in Example 1 are shown, wherein the A graph is the TTC staining graph of the control group, the B graph is the TTC staining graph of the tMCAO group, and the C graph is the statistical result graph of the cerebral infarction area percentage of the control group and the tMCAO group;

[0014] Figure 2 The single cell data analysis result graphs of the mouse brain infarction in Example 2 are shown, wherein the A graph is the mouse brain single cell clustering graph, the B graph is the marker gene expression graph of defining 11 groups of cells, and the C graph is the expression level graph of the spp1 gene in different cell groups;

[0015] Figure 3 The mRNA expression level graphs of spp1 in human and mouse peripheral blood are shown, wherein the A graph is the gene expression graph of human peripheral blood spp1, the B graph is the spp1 gene expression level of mouse peripheral blood, and the C graph is the correlation analysis result graph of the spp1 gene expression level of mouse peripheral blood and the infarction area. DETAILED DESCRIPTION

[0016] The application is not limited by the following specific examples.

[0017] The application helps to solve the problems of not being able to detect brain infarction early and not being able to grade the degree of neurological impairment of patients in clinical practice, and can accurately detect brain infarction, so as to properly intervene in patients with effective therapy.

[0018] Example 1

[0019] Mouse brain infarction model (tMCAO) group: male BL6J mice were used, the mice were anesthetized with avertin (10 mg / kg, i.p.), neck surgery was performed, the right carotid artery was separated, and the blood supply of the brain was interrupted by blocking with a thread plug, the thread plug was pulled out after 1 hour, and the mice were analyzed for behavior after 24 hours, and the results are shown in Table 1.

[0020] Control group (Ctrl): male BL6J mice, without any treatment.

[0021] Then the brain tissues of the mice were collected for TTC staining to evaluate the infarction area, and the results are shown in Figure 1 Table 1. According to Table 1, the mice in the cerebral infarction group had a higher behavioral score. According to Figure 1 Table 1, the TTC staining infarction area of the mice in the cerebral infarction group was larger and significantly higher than that of the control group. This indicates that the mouse cerebral infarction model was successfully constructed.

[0022] Table 1. According to Table 1, the mice in the cerebral infarction group had a higher behavioral score. According to

[0023] Mouse No. Group Score (0-5) 1 Control 0 2 Control 0 3 Control 0 4 tMCAO cerebral infarction model 2 5 tMCAO cerebral infarction model 2 6 tMCAO cerebral infarction model 3 7 tMCAO cerebral infarction model 3 8 tMCAO cerebral infarction model 2.5 9 tMCAO cerebral infarction model 3

[0024] Note: According to the Clark scoring system, the Bederson score is divided into five functional levels, 0 points: no neurological deficit symptoms; 1 point: unable to fully extend the contralateral forelimb; 2 points: contralateral persistent rotation; 3 points: paralysis or very active; 4 points: death.

[0025] Example 2: Single-cell data analysis of mouse cerebral infarction model

[0026] The brains of the mice in the cerebral infarction model (tMCAO) group and the control group (Ctrl) collected in Example 1 were subjected to single-cell sequencing, and the single-cell data was analyzed using R studio. The analysis results are shown in Figure 2 Figure A is a mouse brain single-cell clustering diagram, Figure B is a marker gene expression diagram defining 11 groups of cells, and Figure C is an expression level diagram of spp1 gene in different cell groups, showing that spp1 gene is highly expressed in all cells.

[0027] Example 3: Detection of spp1 expression level in human peripheral blood

[0028] Peripheral blood (CI) from cerebral infarction patients was collected from the Affiliated Hospital of Nantong University, and peripheral blood from the same age group undergoing physical examination was collected as the control group (Ctrl). The expression level of spp1 in the peripheral blood was detected by RT-PCR.

[0029] Peripheral blood leukocyte extraction: 3-5 ml of venous blood was taken, anticoagulated with EDTA, and centrifuged at 2500 rpm for 10 min; the upper plasma was carefully aspirated and divided into 3 0.5 ml centrifuge tubes; 3 times the volume of hemolytic solution was added to the blood cells, shaken well, and ice-bathed for 15 min; centrifuged at 2500 rpm for 10 min, and the supernatant was discarded; 10 ml of hemolytic solution was added, shaken well, and ice-bathed for 15 min; centrifuged at 3000 rpm for 10 min, and the supernatant was discarded; the centrifuge tube was inverted to remove the residual liquid; the leukocytes were obtained and stored at -80°C to avoid repeated freezing and thawing; the interval between blood collection and leukocyte separation should not exceed 2 h at room temperature or 5 h at 4°C to prevent leukocyte autolysis.

[0030] RNA extraction: after the frozen leukocytes were thawed, 1 ml of sterile PBS was added for resuspension, and centrifuged at 1000 rpm at 4°C for 1 min; after the supernatant was discarded, 1 ml of TRIzol was added to lyse the cells, and the tip was mixed evenly by blowing; it was placed at room temperature for 5 min, and the supernatant was transferred to a 1.5 ml EP tube; centrifuged at 12000 rpm for 5 min, and the supernatant was taken, chloroform was added, and after mixing evenly, it was placed at room temperature for 15 min to allow it to separate naturally; centrifuged at 12000 rpm at 4°C for 15 min, and the sample was divided into three layers, the yellow organic layer, the middle layer and the upper layer were colorless aqueous phase, and the RNA was mainly in the aqueous phase; the upper aqueous phase was carefully aspirated into a new 1.5 ml EP tube, an equal volume of ice-cold isopropanol was added, and it was placed at -20°C for 1 h; centrifuged at 12000 rpm at 4°C for 10 min; the supernatant was discarded, 1 ml of 75% ethanol was added, the EP tube was gently shaken, and the precipitate was suspended; centrifuged at 8000 rpm at 4°C for 5 min, the supernatant was discarded, and it was air-dried at room temperature for 5-10 min; the RNA precipitate was dissolved with 50 μl of DEPC water, the RNA concentration was detected, and it was stored at -80°C for standby.

[0031] cDNA synthesis reaction: 4.0 μl of 5×Prime Script Buffer, 1.0 μg of Total RNA, 20 ul of RNase Free ddH2O were gently mixed, reacted at 37°C for 15 min, 85°C for 5 s, and gradually cooled to 4°C.

[0032] RT-qPCR reaction: 10.0 μl of SYBR Green Premix Ex Taq II, 1.0 μl of Forward Primer (SEQ ID NO: 1: 5'-AGTTTCGTACAA-3'), 1.0 μl of Reverse Primer (SEQ ID NO: 2: 5'-TTCGCCATATAC-3'), 2.0 μl of cDNA, 20 ul of Rnase Free ddH2O were mixed to prepare a reaction solution, and then 95°C was pre-denatured for 30 s; 95°C was denatured for 5 s, 60°C was annealed for 30-34 s, and 40 cycles were performed.

[0033] (6) The Ct value of the reference gene GAPDH was standardized to process the Ct value of the target gene, and the 2-△△Ct method was used for relative quantitative analysis of the expression difference of the sample gene, that is, the mRNA expression level of the spp1 gene was obtained, and the results were as follows Figure 3The expression of spp1 gene in peripheral blood of human and mouse is shown in the figure. In the figure, A is the gene expression chart of human peripheral blood spp1, and it can be seen that the expression level of spp1 gene in peripheral blood of cerebral infarction patients is obviously higher than that of normal people, which indicates that the increase of expression level of spp1 gene is closely related to the occurrence and development of cerebral infarction. B is the expression level of spp1 gene in peripheral blood of mouse, and C is the correlation analysis result chart of expression level of spp1 gene in peripheral blood of mouse and infarction area. It can be found that the infarction area (the marker of cerebral infarction degree) of tMCAO group mouse and the expression level of spp1 gene in peripheral blood have a significant positive correlation, that is, with the increase of expression level of spp1 gene of sample, the reaction degree of cerebral infarction in vivo is also enhanced, which reflects that the higher the cerebral infarction degree is, the higher the cerebral infarction degree is.

[0034] The above merely illustrates the preferred embodiments of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can make equivalent replacements or changes according to the technical scheme and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. Use of a reagent for detecting the expression level of spp1 gene in a sample in the manufacture of a product for aiding diagnosis or diagnosis of cerebral infarction, wherein, The expression level of the spp1 gene is significantly up-regulated in samples taken from patients with cerebral infarction.

2. Use according to claim 1, characterized in that, The sample is selected from blood.

3. Use according to claim 1, characterized in that, The expression level is the mRNA expression level of the spp1 gene.

4. Use according to claim 1, characterized in that, The reagent comprises primers specific for amplifying the spp1 gene.

5. Use according to claim 4, characterized in that, The sequence of the primers specific for amplifying the spp1 gene is shown in SEQ ID NO: 1 and SEQ ID NO:

2.

6. A product for aiding in the diagnosis or diagnosis of cerebral infarction, characterized by, The product comprises primers specific for amplifying the spp1 gene, and the sequence of the primers is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

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