Application of SPP1 in preparation of medicine for preventing and treating cerebral arterial thrombosis

Abstract

The invention discloses application of SPP1 in preparation of a medicine for preventing and treating cerebral arterial thrombosis, and relates to the technical field of biological medicines. Comprising the following steps: S1: experimental animals and materials: S11: ensuring that 80 healthy male SPF-level Sprague-Dawley rats which are 7-8 weeks old and 250-280 g are selected; and S12, preparing various reagents and materials required by the experiment, wherein the reagents and materials comprise an RIPA lysate, a BCA protein concentration determination kit, skim milk powder, a chemiluminescence reagent, an SDS-PAGE sample loading buffer solution, an SDS-PAGE gel preparation kit, an MCAO suture, an SPP1 antibody, an MAG antibody, an MBP antibody and a beta-actin antibody. The invention directly regulates and controls the key protein SPP1, has strong pertinence and a clear research target, and provides a basis for deeply understanding the specific effect of SPP1 in cerebral apoplexy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of SPP1 in preparing drugs for preventing and treating ischemic stroke. Background Art

[0002] Ischemic stroke is an acute cerebrovascular disease caused by impaired cerebral blood supply, which leads to ischemic hypoxic necrosis of brain tissue. It is characterized by high morbidity, high mortality and high disability rate. At present, the main clinical treatment methods are thrombolysis, antiplatelet aggregation, lipid-lowering and blood pressure-lowering, but the treatment effect is limited and there are certain side effects. Therefore, it is of great significance to develop new drugs for the prevention and treatment of ischemic stroke.

[0003] After searching, the application scheme of Chinese patent application number CN202110627046.6 discloses a gaseous neuroprotectant, which is an argon-oxygen mixture. The research method of the inhibitory effect of gaseous neuroprotectants on NLRP3 inflammasomes includes the following steps: S1. Establishing animal and cell models of ischemic stroke; S2. Observing the effects and molecular mechanisms of argon on the inflammatory activation of primary microglia stimulated by lipopolysaccharide in cell models; S3. Explore the role of argon in regulating NLRP3 inflammasomes in microglia activation in neuroprotection of cerebral ischemia-reperfusion injury. The gaseous neuroprotectants in the above patents have the following shortcomings: it is relatively difficult to obtain a sufficient number of high-quality cells for clinical applications, the proportion of maintaining a specific phenotype may be low, and a complex screening and culture process is required, which needs to be improved. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose the use of SPP1 in the preparation of drugs for preventing and treating ischemic stroke.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The application of SPP1 in the preparation of drugs for preventing and treating ischemic stroke includes:

[0007] S1: Experimental animals and materials:

[0008] S11: Ensure that 80 healthy male SPF Sprague-Dawley rats are selected, 7-8 weeks old, 250-280 g;

[0009] S12: Prepare various reagents and materials required for the experiment, including RIPA lysis buffer, BCA protein concentration determination kit, skim milk powder, chemiluminescence reagent, SDS-PAGE loading buffer, SDS-PAGE gel preparation kit, MCAO plug, SPP1 antibody, MAG antibody, MBP antibody, β-actin antibody, HRP-labeled goat anti-mouse secondary antibody, HRP-labeled goat anti-rabbit secondary antibody, PVDF membrane and adeno-associated virus, etc.;

[0010] S2: Stereotaxic injection of adeno-associated virus into the brain:

[0011] S21: Twenty-one days before modeling, carefully prepare AAV-OE-SPP1 and AAV-eGFP (about 1×1013 infectious units / ml);

[0012] S22: Slowly inject 5 μL of AAV-OE-SPP1 or AAV-eGFP into the lateral ventricle;

[0013] S23: After injection, the syringe should be left in place for at least 5 minutes before removal to prevent backflow and ensure complete dispersion of the virus;

[0014] S3: Establishment of animal model:

[0015] S31: I / R model was constructed using the line plug method;

[0016] S32: Rats were anesthetized with 2% sodium pentobarbital (50 mg / kg) by intraperitoneal injection;

[0017] S33: The rats were fixed on the animal surgery board, and a midline incision was made in the neck after skin preparation;

[0018] S34: Expose the left common carotid artery, external carotid artery, and internal carotid artery;

[0019] S35: The proximal end of the left common carotid artery was ligated, a small incision was made between the two ends and a silicone suture was inserted to cause focal ischemia;

[0020] S36: After 120 minutes of I / R, the suture plug was removed to complete reperfusion after vascular occlusion;

[0021] S37: The surgical procedure of rats in the sham operation group was the same as before, and the nylon thread was only inserted about 6 mm;

[0022] S4: Vagus nerve stimulation:

[0023] S41: Use Huatuo brand electronic acupuncture instrument SDZ-V;

[0024] S42: After 30 min of ischemic occlusion in rats, the left vagus nerve was carefully isolated;

[0025] S43: Place the VNS stimulation electrode on the left vagus nerve and give repeated electrical stimulation for 60 minutes;

[0026] S44: Stimulation parameters are: 0.5 mA square wave, wave width 0.5 ms, frequency 20 Hz, continuous 30 s at a time, once every 5 minutes;

[0027] S45: The rats were then placed on an electric heating blanket with body temperature maintained at 37°C until they woke up;

[0028] S46: After waking up, the models were placed in separate cages and allowed to eat and drink freely;

[0029] S47: After 3 days of continuous intervention, samples were collected for testing of various indicators.

[0030] Preferably, in S1:

[0031] RIPA Lysis Buffer (Strong): Used to extract proteins from cells; choose a formula that is suitable for cells and tissues, and make sure it contains ingredients that effectively inhibit protease and phosphatase activity to maintain protein integrity;

[0032] BCA Protein Concentration Assay Kit: used to determine the concentration of protein in the sample in order to adjust the sample loading amount before protein electrophoresis;

[0033] Skim milk powder: used for membrane blocking in Western Blot to reduce nonspecific antigen binding;

[0034] Chemiluminescent reagent: used in Western Blot detection, it generates signals after binding with specific antibodies to visualize protein bands on the membrane;

[0035] SDS-PAGE loading buffer: used to prepare SDS-PAGE electrophoresis samples, containing SDS to denature proteins, and adding reducing agents such as β-mercaptoethanol to destroy disulfide bonds within proteins;

[0036] SDS-PAGE Gel Preparation Kit: Contains all necessary chemicals and buffers for preparing polyacrylamide gels for separating proteins of different molecular weights;

[0037] MCAO suture: used for surgical introduction into the rat carotid artery to trigger focal cerebral ischemia. The suture with appropriate diameter and length is selected to fit the vascular size of the rat.

[0038] SPP1 antibody, MAG antibody, MBP antibody: targeting secretory phosphoprotein 1 (SPP1), myelin-associated glycoprotein (MAG), and myelin basic protein (MBP), respectively, used to detect and quantify the expression of these proteins;

[0039] β-actin antibody: used as an internal control for Western Blot to determine the consistency of sample loading;

[0040] HRP-labeled goat anti-mouse and goat anti-rabbit secondary antibodies: Following primary antibody binding, use HRP-labeled secondary antibodies corresponding to the species of the primary antibody for immunodetection;

[0041] PVDF membrane: used to transfer and fix proteins in Western Blot for antigen-antibody reaction and chemiluminescence detection;

[0042] Adeno-associated virus: Customize AAV vectors containing SPP1 overexpression constructs or control eGFP according to the experimental design and determine the appropriate virus titer and purity.

[0043] Preferably, in S21, AAV-OE-SPP1 and AAV-eGFP are prepared as follows:

[0044] Design and construct an adeno-associated virus vector containing the SPP1 overexpression sequence, and prepare a control vector encoding eGFP;

[0045] Choose a suitable viral backbone, such as AAV9;

[0046] Viruses are amplified and purified in a GMP-grade laboratory to ensure sterility and non-pathogenicity;

[0047] The virus titer was determined by quantitative PCR to ensure that the infectious unit (IU) reached 1×10 13 IU / mL;

[0048] Before injection, the virus was stored in a -80°C freezer and gradually thawed onto ice when used.

[0049] Preferably, in S36, the suture plug is removed to complete the reperfusion after the blood vessel is blocked, as follows:

[0050] Confirm that the ischemic time has reached 120 minutes;

[0051] The silicone plug was removed promptly but carefully under sterile conditions to restore blood flow and avoid further tissue damage;

[0052] Ligate the distal end of the common carotid artery to ensure blood reperfusion and reduce the risk of bleeding;

[0053] The incision was sutured, treated with antibiotics to prevent possible infection, and the rat was placed in a warm environment to recover;

[0054] The behavioral and physiological indicators of the rats, including neurological function scores and body temperature, were monitored to ensure their health status and evaluate the successful establishment of the model.

[0055] Preferably: the skimmed milk powder is used for membrane blocking in Western Blot to reduce non-specific antigen binding, as follows:

[0056] Preparation: First, prepare an appropriate amount of TBST buffer (TBS containing 0.1% Tween-20), which is the basic solution for diluting skim milk powder and later washing the membrane;

[0057] Prepare blocking solution: weigh an appropriate amount of skim milk powder (usually 5% w / v), dissolve it in TBST and mix well to ensure complete dissolution without particles;

[0058] Blocking membrane: Place the PVDF membrane (proteins have been separated by SDS-PAGE and transferred to the membrane in advance) into the blocking solution, ensuring that the membrane is completely covered; then shake slowly on a shaker and block at room temperature for 1 hour or at 4°C overnight to optimize the blocking effect;

[0059] Washing the membrane: After blocking, wash the membrane three times with TBST buffer for 5-10 minutes each time to remove excess blocking solution and prepare for subsequent antibody incubation.

[0060] Preferably, the SDS-PAGE loading buffer is used to prepare the SDS-PAGE electrophoresis sample, specifically as follows:

[0061] Preparation of loading buffer: First, prepare a 4X concentration of loading buffer stock solution, which includes 200 mM Tris-HCl (pH 6.8), 8% SDS, 40% glycerol, 0.4% bromophenol blue and 20% β-mercaptoethanol (as a reducing agent);

[0062] Sample treatment: Mix the protein sample with an equal volume of 4X SDS-PAGE loading buffer and heat at 95-100°C for 5-10 minutes to completely denature and reduce the protein;

[0063] Cooling and centrifugation: The heated samples need to be cooled on ice, and then the condensed water on the lid is collected to the bottom of the tube by brief centrifugation;

[0064] Sample loading and electrophoresis: The processed samples are directly used for SDS-PAGE electrophoresis. Before loading, ensure that the sample loading amount in each well is consistent.

[0065] Preferably, the SPP1 antibody, MAG antibody, and MBP antibody are used to detect and quantify protein expression, as follows:

[0066] Choose the right antibodies: Select antibodies with high specificity and affinity as primary antibodies for each target protein, such as anti-SPP1, anti-MAG, and anti-MBP monoclonal or polyclonal antibodies;

[0067] Antibody incubation: The blocked membrane was incubated with primary antibodies (such as SPP1, MAG or MBP antibodies) at 4°C overnight;

[0068] Washing and secondary antibody incubation: After primary antibody incubation, wash the membrane thoroughly with TBST and then incubate with the corresponding HRP-labeled secondary antibody (such as goat anti-mouse or goat anti-rabbit) at room temperature for 1-2 hours;

[0069] Chemiluminescent detection: Wash the membrane again to remove unbound secondary antibody, use chemiluminescent substrate for color reaction, and capture the signal by imaging system to perform qualitative and quantitative analysis of protein expression.

[0070] The beneficial effects of the present invention are:

[0071] 1. The present invention directly regulates the key protein SPP1, has strong pertinence and clear research objectives, and provides a basis for in-depth understanding of the specific role of SPP1 in stroke.

[0072] 2. Compared with traditional drug therapy, gene therapy of the present invention is more precise, can reduce damage to normal tissues, and reduce possible side effects during treatment.

[0073] 3. The present invention provides a new molecular target and method for the treatment of ischemic stroke through AAV-mediated SPP1 overexpression, which helps to expand new areas of scientific research and clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is a comparison chart of the difference in neurological function scores between the SPP1 overexpression group and the SPP1 empty load group in the present invention;

[0075] Figure 2 This is a comparison chart of the difference in neurological function score between the SPP1 overexpression group and the SPP1 empty load group in the present invention;

[0076] Figure 3 It is a comparison diagram of the infarct volume of rats in the SPP1 overexpression group and the SPP1 empty group in the present invention;

[0077] Figure 4 This is a comparison of the myelin-related protein expressions of the SPP1 overexpression group and the SPP1 empty group in the present invention;

[0078] Figure 5 This is a comparison diagram of myelin-associated glycoprotein expression between the SPP1 overexpression group and the SPP1 empty group in the present invention;

[0079] Figure 6 It is a statistical graph of the protein expression of SPP1 in the SPP1 overexpression group and the SPP1 empty load group in the present invention;

[0080] Figure 7 This is a comparison chart of SPP1 protein expression between the SPP1 overexpression group and the SPP1 empty vector group in the present invention. Detailed implementation manners

[0081] The technical solutions of the present invention will be further described in detail below in conjunction with the specific implementation manners.

[0082] Example 1:

[0083] The application of SPP1 in the preparation of drugs for preventing and treating ischemic stroke includes:

[0084] Materials and methods:

[0085] 1.1 Experimental animals

[0086] 80 healthy male SPF-grade Sprague-Dawley rats, 7 - 8 weeks old, weighing 250 - 280 g, were purchased from the Experimental Animal Center of Chongqing Medical University [Experimental Animal Production License Number: SCXK(YU)2022 - 0010]. The animals were housed in an SPF-grade animal room at a room temperature of (23 ± 1) °C, with a light-dark cycle of 12 h, and free access to food and water. The experiment followed the relevant regulations on the ethics of experimental animals of Chongqing Medical University. After 1 week of adaptive feeding, the experiment was carried out.

[0087] 1.2 Experimental materials

[0088] The reagents used in this study were: RIPA lysis buffer (strong), BCA protein concentration assay kit, skim milk powder, chemiluminescence reagent (Beyotime Biotechnology Company), SDS-PAGE loading buffer, SDS-PAGE gel preparation kit (Yamei Biotechnology Company), MCAO thread embolism (Beijing Xinuo Technology Co., Ltd.), SPP1 antibody (Abcam Company, USA), MAG antibody (CST Company, USA), MBP antibody (Abcam Company, USA), β-actin antibody, HRP-labeled goat anti-mouse secondary antibody, HRP-labeled goat anti-rabbit secondary antibody (Wuhan Sanying Company), PVDF membrane (Millipore Company, USA), adeno-associated virus (Heyuan Biotechnology).

[0089] 1.3 Stereotaxic injection of adeno-associated virus into the brain

[0090] Stereotaxic injection of adeno-associated virus AAV-OE-SPP1 was purchased from Heyuan Biotechnology Co., Ltd. (Shanghai, China). AAV encoding GFP was used as a negative control (AAV-eGFP). Twenty-one days before modeling, 5 μL of AAV-OE-SPP1 or AAV-eGFP (about 1×1013 infectious units / mL) was slowly injected into the lateral ventricle (anterior to the anterior bregma - 0.8 mm, medial to the lateral side - 1.5 mm, 3.5 mm deep from the surface) (after injection, the syringe was left in place for at least 5 minutes before being removed to prevent backflow and ensure complete dispersion of the virus)

[0091] 1.4 Establishment of animal model

[0092] The experiment was conducted 21 days after the rats were stereotaxically injected with adeno-associated virus. The I / R model was constructed by the thread plug method, and the specific method was as follows: 2% sodium pentobarbital (50 mg / kg) was used for intraperitoneal anesthesia, and the rats were fixed on the animal surgery board. After the skin of the rat neck was prepared, a midline incision was made to expose the left common carotid artery, external carotid artery, and internal carotid artery. The proximal end of the left common carotid artery was ligated, a small incision was made between the two ends, and a silicone thread plug was inserted to cause focal ischemia. The thread plug was removed 120 minutes after I / R to complete the reperfusion after vascular occlusion. The surgical operation of the sham group rats was the same as before, and the nylon thread was only inserted about 6 mm.

[0093] 1.5 Vagus nerve stimulation

[0094] Using Huatuo brand electronic acupuncture instrument SDZ-V, after 30 minutes of ischemia and blockage of rats, the left vagus nerve was carefully separated, and the VNS stimulation electrode was placed on the left vagus nerve for 60 minutes of repeated electrical stimulation. The stimulation parameters were as follows: 0.5mA square wave, wave width 0.5ms, frequency 20HZ, once continuously 30s, once every 5 minutes. The rats were then placed on an electric blanket and the body temperature was maintained at 37°C until they woke up. After waking up, the models were separated into cages and free to eat and drink. After 3 days of continuous intervention, the samples were collected for testing of various indicators.

[0095] Results reference Figure 1-5 .

[0096] Figure 1 Compared with the SPP1 empty group, the neurological function score in the SPP1 overexpression group was worse;

[0097] Figure 2 Compared with the SPP1 empty group, the SPP1 overexpression group had poor neurological function scores, shorter rod-staying time, and poor motor coordination, suggesting that overexpression of SPP1 could reverse the protective effect of VNS;

[0098] Figure 3The infarct volume (white area) shown by TTC staining after I / R; the results showed that the infarct volume of rats in the SPP1 overexpression group was significantly increased compared with that in the SPP1 empty group;

[0099] Figure 4 Compared with the SPP1 empty group, the expression of myelin-related protein (MBP) in the SPP1 overexpression group was decreased;

[0100] Figure 5 Compared with the SPP1 empty group, the expression of myelin-associated glycoprotein (MAG) in the SPP1 overexpression group was decreased.

[0101] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Application of SPP1 in the preparation of a drug for preventing and treating ischemic stroke, characterized in that: include: S1: Experimental animals and materials: S11: Select healthy male SPF rats; S12: Prepare various reagents and materials required for the experiment, including RIPA lysis buffer, BCA protein concentration determination kit, skim milk powder, chemiluminescence reagent, SDS-PAGE loading buffer, SDS-PAGE gel preparation kit, MCAO plug, SPP1 antibody, MAG antibody, MBP antibody, β-actin antibody, HRP-labeled goat anti-mouse secondary antibody, HRP-labeled goat anti-rabbit secondary antibody, PVDF membrane and adeno-associated virus; S2: Stereotaxic injection of adeno-associated virus into the brain: S21: Twenty-one days before modeling, prepare AAV-OE-SPP1 and AAV-eGFP; S22: Slowly inject 5 μL of AAV-OE-SPP1 or AAV-eGFP into the lateral ventricle; S3: Establishment of animal model: S31: I / R model was constructed using the line plug method; S32: Rats were anesthetized with 2% sodium pentobarbital injected intraperitoneally; S33: The rats were fixed on the animal surgery board, and a midline incision was made in the neck after skin preparation; S34: Expose the left common carotid artery, external carotid artery, and internal carotid artery; S35: The proximal end of the left common carotid artery was ligated, a small incision was made between the two ends and a silicone suture was inserted to cause focal ischemia; S36: After 120 minutes of I / R, the suture plug was removed to complete reperfusion after vascular occlusion; S37: The surgical procedure of rats in the sham operation group was the same as before, and the nylon thread was only inserted 6 mm; S4: Vagus nerve stimulation: S41: Use of electronic acupuncture device; S42: After 30 min of ischemic occlusion in rats, the left vagus nerve was isolated; S43: Place the VNS stimulation electrode on the left vagus nerve and give repeated electrical stimulation for 60 minutes; S44: The rat's body temperature was maintained at 37°C until it woke up; S45: After waking up, the models were placed in separate cages and allowed to eat and drink freely; S46: After 3 days of continuous intervention, samples were collected for testing of various indicators.

2. The use of SPP1 according to claim 1 in preparing a drug for preventing and treating ischemic stroke, characterized in that: In the S11, healthy male SPF-grade Sprague-Dawley rats, 7-8 weeks old, weighing 250-280 g, were selected.

3. The use of SPP1 according to claim 1 in preparing a drug for preventing and treating ischemic stroke, characterized in that: In S22, after injection, the syringe is left in place for at least 5 minutes before being removed to prevent backflow and ensure complete dispersion of the virus.

4. The use of SPP1 according to claim 1 in preparing a drug for preventing and treating ischemic stroke, characterized in that: In the S32, rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital at 50 mg / kg.

5. The use of SPP1 according to claim 1 in preparing a drug for preventing and treating ischemic stroke, characterized in that: In the above S43, the stimulation parameters of the electrical stimulation are: 0.5 mA square wave, wave width 0.5 ms, frequency 20 Hz, continuous for 30 s at a time, once every 5 minutes.

6. The use of SPP1 according to claim 1 in preparing a drug for preventing and treating ischemic stroke, characterized in that: In S1: RIPA lysis buffer: used to extract proteins from cells; choose a formula that is appropriate for the cells and tissues, and make sure it contains ingredients that effectively inhibit protease and phosphatase activity to maintain protein integrity; BCA protein concentration assay kit: used to determine the concentration of protein in samples; Skim milk powder: used for membrane blocking in Western Blot to reduce nonspecific antigen binding; Chemiluminescent reagent: used in Western Blot detection, it generates signals after binding with specific antibodies and is used to visualize protein bands on the membrane; SDS-PAGE loading buffer: used to prepare SDS-PAGE electrophoresis samples, containing SDS to denature proteins, and adding reducing agents such as β-mercaptoethanol to destroy disulfide bonds within proteins; SDS-PAGE Gel Preparation Kit: Contains all necessary chemicals and buffers for preparing polyacrylamide gels for separating proteins of different molecular weights; MCAO suture: used for surgical introduction into the rat carotid artery to trigger focal cerebral ischemia; SPP1 antibody, MAG antibody, MBP antibody: targeting secretory phosphoprotein 1, myelin-associated glycoprotein, and myelin basic protein, respectively, used to detect and quantify the expression of these proteins; β-actin antibody: used as internal control for Western Blot; HRP-labeled goat anti-mouse and goat anti-rabbit secondary antibodies: Following primary antibody binding, use HRP-labeled secondary antibodies corresponding to the species of the primary antibody for immunodetection; PVDF membrane: used for transferring and fixing proteins in Western Blot; Adeno-associated virus: Customize AAV vectors containing SPP1 overexpression constructs or control eGFP according to the experimental design and determine the appropriate virus titer and purity.

7. The use of SPP1 according to claim 6 in preparing a drug for preventing and treating ischemic stroke, characterized in that: In S21, AAV-OE-SPP1 and AAV-eGFP are prepared as follows: Design and construct an adeno-associated virus vector containing the SPP1 overexpression sequence, and prepare a control vector encoding eGFP; Choose the appropriate viral backbone; Viruses are amplified and purified in a GMP-grade laboratory to ensure sterility and non-pathogenicity; The virus titer was determined by quantitative PCR to ensure that the infectious unit (IU) reached 1 × 10 13 IU / mL; Before injection, the virus was stored in a -80°C freezer and gradually thawed onto ice when used.

8. The use of SPP1 according to claim 6 in preparing a drug for preventing and treating ischemic stroke, characterized in that: The skimmed milk powder is used for membrane blocking in Western Blot, specifically as follows: Preparation: First, prepare an appropriate amount of TBST buffer, TBS containing 0.1% Tween-20, which is used to dilute skim milk powder and the basic solution for washing the membrane later; Prepare blocking solution: weigh 5% w / v skim milk powder, dissolve it in TBST and mix well to ensure complete dissolution without particles; Blocking membrane: Place the PVDF membrane in the blocking solution to ensure that the membrane is completely covered; then shake slowly on a shaker and block at room temperature for 1 hour or at 4°C overnight; Washing the membrane: After blocking, wash the membrane three times with TBST buffer for 5-10 minutes each time to remove excess blocking solution and prepare for subsequent antibody incubation.

9. The use of SPP1 according to claim 6 in preparing a drug for preventing and treating ischemic stroke, characterized in that: The SDS-PAGE loading buffer is used to prepare SDS-PAGE electrophoresis samples, specifically as follows: Prepare loading buffer: First, prepare a 4X loading buffer stock solution, which includes 200 mM Tris-HCl at pH 6.8, 8% SDS, 40% glycerol, 0.4% bromophenol blue, and 20% β-mercaptoethanol; Sample treatment: Mix the protein sample with an equal volume of 4X SDS-PAGE loading buffer and heat at 95-100°C for 5-10 minutes to completely denature and reduce the protein; Cooling and centrifugation: The heated samples need to be cooled on ice, and then the condensed water on the lid is collected to the bottom of the tube by brief centrifugation; Sample loading and electrophoresis: The processed samples are directly used for SDS-PAGE electrophoresis. Before loading, ensure that the sample amount in each well is consistent.

10. The use of SPP1 according to claim 6 in preparing a drug for preventing and treating ischemic stroke, characterized in that: The SPP1 antibody, MAG antibody, and MBP antibody are used to detect and quantify protein expression, as follows: Choose the right antibody: Choose an antibody with high specificity and affinity for each target protein as the primary antibody; Antibody incubation: The blocked membrane was incubated with primary antibodies at 4°C overnight; Washing and secondary antibody incubation: After primary antibody incubation, the membrane was washed thoroughly with TBST and then incubated with the corresponding HRP-conjugated secondary antibody at room temperature for 1-2 hours; Chemiluminescent detection: Wash the membrane again to remove unbound secondary antibody, use chemiluminescent substrate for color reaction, and capture the signal by imaging system to perform qualitative and quantitative analysis of protein expression.

Images