Preparation Method and Application of Protein-Conjugated Microspheres

Abstract

The present invention discloses a method for preparing protein-coupled microspheres and their application. The method uses carboxyl polystyrene microspheres as carriers and chemically couples fibrinogen to the surface of the microspheres to prepare the protein-coupled microspheres. The prepared protein-coupled microspheres are then used as embolic agents in an animal model of coronary artery microembolism. Electron microscopy confirmed that the protein-coupled microspheres produced by the present invention not only exhibit physical obstruction of the microcirculation in the new model, but also activated fibrinogen on the surface of the microspheres to convert into fibrin, which aggregates platelets and forms microthrombi, a more realistic disease state. This addresses the technical problem that existing embolic agents fail to exert their biological function of promoting microthrombosis and cannot replicate the biological damage mechanism of microembolism.

Description

Technical Field

[0001] The present invention relates to the technical field of embolic agents for simulating coronary artery microembolism in biomedicine, and more specifically to a method for preparing protein-coupled microspheres and their application. Background Art

[0002] Coronary microembolization (CME) is a common and challenging complication of coronary intervention, often leading to "no-reflow" and closely associated with adverse short- and long-term prognosis and a significant increase in major adverse cardiovascular events. However, effective treatments are currently lacking. Therefore, CME remains a clinical challenge that urgently needs to be addressed.

[0003] Research into the pathophysiological mechanisms and preventive measures of CME relies on reliable animal models. Ideally, these models should closely replicate the disease process and mimic the same pathophysiological phenomena. However, the myocardial injury mechanisms of CME are complex, stemming not only from mechanical obstruction of the microcirculation but also from multiple pathological responses induced by the biological functions of microthrombi.

[0004] Traditional CME animal models (traditional models) are established by tracheotomy with ventilator support followed by intracoronary injection of inert polystyrene microspheres. As research in this field expands and deepens, traditional models have numerous drawbacks. For example, the emboli only physically obstruct the blood vessels and fail to promote microthrombosis, thus failing to replicate the biological mechanisms of microembolism. Furthermore, the tracheotomy causes additional damage, compromising model quality.

[0005] Therefore, it is of great significance to prepare an embolic agent that is conducive to restoring the real disease process and simulating the biological function-induced pathological damage phenomenon, and to promote the establishment of a new coronary artery microembolism model (new model). Summary of the Invention

[0006] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0007] The present invention provides a method for preparing protein-coupled microspheres, which aims to prepare embolic agents by covalently coupling the carboxyl groups attached to the surface of the microspheres with the amino groups on fibrinogen by a chemical method, thereby solving the problem that the existing embolic agents cannot promote microthrombosis and exert biological functions.

[0008] In order to achieve these objects and other advantages of the present invention, a method for preparing protein-coupled microspheres is provided, wherein carboxyl polystyrene microspheres are used as carriers and fibrinogen is covalently coupled to the surface of the carboxyl polystyrene microspheres by a chemical method.

[0009] Preferably, the particle size of the carboxypolystyrene microspheres is 45 μm.

[0010] Preferably, the weight ratio of carboxyl polystyrene microspheres to fibrinogen is 4×10 4 Pieces: 200-600μg.

[0011] Preferably, the steps include:

[0012] 1) Centrifuge the carboxylated polystyrene microspheres and discard the supernatant;

[0013] 2) Add coupling buffer to resuspend and wash the microspheres, centrifuge and remove the supernatant, and repeat 1-3 times;

[0014] 3) Add coupling buffer, EDC solution, and Sulfo-NHS solution to the microsphere precipitate, mix well, incubate at room temperature for 10-20 minutes, centrifuge, and discard the supernatant; wherein the EDC solution is a solution prepared at a concentration of 50 mg / mL with coupling buffer, and the Sulfo-NHS solution is a solution prepared at a concentration of 50 mg / mL with coupling buffer. The volume ratio of the added coupling buffer: EDC solution: Sulfo-NHS solution is 60:20:20;

[0015] 4) Wash the microspheres with coupling buffer, vortex mix, centrifuge and discard the supernatant to obtain the carboxyl polystyrene microspheres to be coupled.

[0016] Preferably, the method further comprises the following steps:

[0017] 5) Add fibrinogen to 0.9% sodium chloride solution at 37°C to prepare a fibrinogen solution, wherein the weight-to-volume ratio of fibrinogen to sodium chloride solution is 5 mg:1 mL;

[0018] 6) Add coupling buffer and fibrinogen solution to the carboxylated polystyrene microspheres to be coupled, vortex to mix, and incubate at room temperature with continuous shaking for 50-70 minutes; wherein the volume ratio of coupling buffer to fibrinogen solution is 100:40-120;

[0019] 7) Centrifuge and remove the supernatant protein solution for subsequent quality control;

[0020] 8) adding quenching buffer to the microsphere pellet from step 7), vortexing to mix, centrifuging, and discarding the supernatant;

[0021] 9) adding quenching buffer to the microsphere pellet from step 8), incubating at room temperature for 60-70 minutes, centrifuging, and discarding the supernatant;

[0022] 10) adding quenching buffer to the microsphere precipitate from step 9), vortexing to mix, centrifuging, and discarding the supernatant;

[0023] 11) Add storage buffer to the microsphere precipitate from step 10), vortex mix, and store at 2-8° C. to obtain protein-coupled microspheres.

[0024] Preferably, the centrifugal speed is 2000 r / min, and the centrifugation is for 1 minute; the coupling buffer is 50 mmol / LMES with a pH of 6.0, containing 0.01% Triton X-100; the quenching buffer is TBS solution; and the storage buffer is TBS or PBS solution containing 0.01% Triton X-100.

[0025] Preferably, it further includes 2.5×10 4 The protein-coupled microspheres were prepared into 200 μL protein-coupled microsphere embolic agent with 0.9% sodium chloride solution.

[0026] Also provided is the use of the protein-coupled microspheres as embolic agents in constructing an animal model of coronary artery microembolism.

[0027] The present invention has at least the following beneficial effects:

[0028] The present invention uses a chemical method to covalently couple fibrinogen to the surface of carboxyl polystyrene microspheres as an embolic agent. Electron microscopy confirms that while the new model physically obstructs the microcirculation, the fibrinogen on the surface of the embolic agent is activated to become fibrin, which aggregates platelets and forms microthrombi, which is closer to the actual disease state.

[0029] Comparison confirmed that under the same conditions of ambient temperature, reagent dosage, operation steps, and animal gender and age, the new CME model constructed using the protein-coupled microspheres prepared by the present invention as embolic agents has significant advantages over the traditional CME model in simulating disease-induced myocardial damage and pathological phenotypic characteristics. The cardiac function of the new model decreased significantly; the results of immunofluorescence, protein blotting, and TUNEL staining of apoptotic proteins showed that the apoptotic damage in the improved model was more obvious.

[0030] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a comparison chart of immunofluorescence microscopy of the protein-coupled microspheres of the present invention;

[0032] Figure 2 This is a bar graph showing the BCA calculation of the coupling amount of the protein-coupled microspheres of the present invention;

[0033] Figure 3 This is a scanning electron microscopic comparison image of the protein-coupled microspheres of the present invention;

[0034] Figure 4 This is a light microscopic image of the myocardial microcirculation of the new model of the present invention;

[0035] Figure 5 This is an external view of the heart of the new model of the present invention;

[0036] Figure 6 This is an electron microscopic image of the surface of protein-coupled microspheres in the myocardial tissue of the new model rat of the present invention;

[0037] Figure 7 This is an ultrasound image of the heart of the new model of the present invention;

[0038] Figure 8 A comparison diagram of cardiac ultrasound between the new model of the present invention and the traditional model;

[0039] Figure 9 Comparison chart of the grayscale values of WB and immunofluorescence results of the new model of the present invention and the traditional model

[0040] Figure 10 This is a bar graph comparing the WB and immunofluorescence results of the new model of the present invention and the traditional model;

[0041] Figure 11 This is a comparison chart of the apoptosis status of the new model of the present invention and the traditional model by immunofluorescence;

[0042] Figure 12 This is a comparison chart showing the cell apoptosis status of the new model of the present invention and the traditional model by TUNEL experiment;

[0043] Figure 13 This is the technical route for preparing protein-coupled microsphere embolic agents of the present invention;

[0044] Figure 14 Create a technology roadmap for CME modeling. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments so that those skilled in the art can implement the invention with reference to the description.

[0046] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0047] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0048] 1. Preformed fibrinogen chemically coupled carboxyl polystyrene microspheres (hereinafter referred to as protein-coupled microspheres) embolic agents

[0049] <Example>

[0050] 1. Technical route of prefabricated protein-coupled microsphere embolic agents Figure 13 shown.

[0051] 2. Preparation of Protein-coupled Microspheres

[0052] 1) Vortex and resuspend the carboxyl polystyrene microspheres. 4 Carboxyl polystyrene microspheres with a particle size of 45 μm were added to a 1.5 mL EP tube, centrifuged (2000 r, 1 minute) and the supernatant was discarded.

[0053] 2) Add 200 μL of coupling buffer (50 mmol / LMES, pH 6.0, 0.01% Triton X-100) to resuspend and wash the microspheres, centrifuge and remove the supernatant, and repeat 3 times.

[0054] 3) Add 60ul coupling buffer and 20μL EDC solution (C8H 17 N3, prepared with coupling buffer to a concentration of 50 mg / mL) and 20 μL of Sulfo-NHS solution (C4H4NNaO6S, prepared with coupling buffer to a concentration of 50 mg / mL), mix well, incubate at room temperature for 15 minutes, centrifuge and discard the supernatant.

[0055] 4) Wash the microspheres with 200 μL of coupling buffer. Vortex to mix, centrifuge, and discard the supernatant to obtain the microsphere pellet to be coupled.

[0056] 5) Add 1 mL of 0.9% sodium chloride solution at 37°C to 5 mg of fibrinogen to prepare a fibrinogen solution.

[0057] 6) Add 100 μL of coupling buffer and 120 μL of fibrinogen solution (containing 600 μg of fibrinogen for the experimental group) to the microsphere pellet to be coupled (additional solutions containing 200 μg and 400 μg of fibrinogen were prepared for other groups). Vortex to mix and incubate at room temperature with continuous shaking for 60 minutes.

[0058] 7) Centrifuge and aspirate the supernatant residual protein solution (containing unbound protein for subsequent quality control).

[0059] 8) Add 250 μL of quenching buffer (TBS) to the microsphere pellet, vortex for 20 seconds, centrifuge and discard the supernatant.

[0060] 9) Add 500 μL of quenching buffer to the microsphere pellet, incubate at room temperature for 60 minutes, centrifuge and discard the supernatant.

[0061] 10) Add 250 μL of quenching buffer to the microspheres, vortex vigorously for 20 seconds, centrifuge and discard the supernatant to obtain the precipitate to obtain the protein-coupled microspheres.

[0062] 11) Add 100 μL of storage buffer (TBS or PBS solution containing 0.01% Triton X-100), vortex to mix and store at 2-8° C. to obtain the prefabricated protein-coupled microsphere storage solution.

[0063] 12) Before modeling, take 62.5 μL of pre-made protein-coupled microsphere storage solution (containing 2.5×10 4 Protein-coupled microspheres) were prepared with 0.9% sodium chloride solution to prepare 200 μL protein-coupled microsphere embolic agent for use.

[0064] The centrifugal speed was 2000 r / min for 1 minute. The coupling buffer was 50 mmol / L MES at pH 6.0 containing 0.01% Triton X-100. The quenching buffer was TBS. The storage buffer was TBS or PBS containing 0.01% Triton X-100.

[0065] 3. Quality Control of Protein-Coupled Microspheres

[0066] 3-1. Protein-coupled microsphere immunofluorescence assay

[0067] 1) Take 20 μL of protein-coupled microsphere solution into a 1.5 mL EP tube, centrifuge and discard the supernatant (2000 r, 1 minute), add 100 μL of deionized water, and repeat 3 times.

[0068] 2) Add 100 μL of 5% BSA and incubate at room temperature for 30 minutes. Centrifuge and discard the supernatant (2000 r, 1 minute).

[0069] 3) Add 100 μL of deionized water, centrifuge and discard the supernatant (2000 r, 1 minute), and repeat 3 times.

[0070] 4) Dilute the primary fluorescent antibody against fibrinogen with 1% BSA in the dark (IF: 1:250).

[0071] 5) Add 100 μL of diluted antibody to the microspheres, incubate at room temperature for 30 minutes, and centrifuge to discard the supernatant.

[0072] 6) Add 100 μL of deionized water (2000 r, 1 minute) and observe the fluorescence coverage of the protein-coupled microspheres under a fluorescence microscope.

[0073] 3-2. Determination of microsphere coupling amount (BCA method)

[0074] The operation was performed according to the instructions of the BCA protein concentration determination kit (Biyuntian, Cat No. P0011).

[0075] 1) Prepare BCA working solution. Dissolve protein standard in deionized water. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of standard to a 96-well plate, and dilute to 20 μL with PBS.

[0076] 2) Add 20 μL of supernatant residual protein solution to a 96-well plate, add 200 μL of BCA working solution to each well, and incubate at 37°C for 30 min;

[0077] 3) Measure the absorbance at a wavelength of 562 nm using a microplate reader. Draw a standard curve based on the absorbance of the standard. Then, determine the concentration of the residual protein solution in the supernatant based on the absorbance of the residual protein solution in the supernatant in combination with the standard curve. The amount of fibrinogen coupled to the protein-coupled microspheres = the amount of fibrinogen added for coupling - the amount of fibrinogen contained in the residual protein solution in the supernatant.

[0078] 3-3. Scanning electron microscopy observation of the coupling of protein-coupled microspheres

[0079] 1) Place 20 μL of protein-coupled microsphere solution in a 1.5 mL EP tube, centrifuge, and discard the supernatant. Add 1 mL of glutaraldehyde phosphate buffer to the microsphere pellet and fix at room temperature for 24 hours. Centrifuge and discard the supernatant.

[0080] 2) Add 1 mL of deionized water, centrifuge and discard the supernatant, repeat three times, vacuum dry for 6 hours, and spray with gold;

[0081] 3) Observe the fibrinogen coupled to the microsphere surface under a scanning electron microscope.

[0082] 3-4. Results Analysis

[0083] 1) Immunofluorescence assay of protein-coupled microspheres: The surface of the protein-coupled microspheres in the experimental group (600 μg Fib group) showed significant fluorescence intensity, indicating that fibrinogen was attached, indicating that the protein-coupled microspheres were successfully coupled to fibrinogen; while the surface of the protein-coupled microspheres in the control group (steps 2 to 4 were skipped in the preparation, the other steps were the same as the preparation of the embodiment, and the amount of added protein was 600 μg) had no obvious fluorescence, indicating that the protein-coupled microspheres in the control group failed to couple to fibrinogen ( Figure 1 )(×100, scale bar: 100 μm).

[0084] 2) BCA measurement of the coupling amount of protein-coupled microspheres: The coupling amount of the 200ugFib, 400ugFib, and 600ugFib groups showed a gradual upward trend, among which the coupling amount of the 600ugFib group was relatively stable and had good consistency ( Figure 2 ).

[0085] 3) Scanning electron microscopy of protein-coupled microspheres: Under a scanning electron microscope magnification of 1000 times, fibrinogen (red arrow) can be seen attached to the surface of the protein-coupled microspheres in the experimental group (600ugFib group), confirming that the fibrinogen coupling of the microspheres was successful; while the surface of the protein-coupled microspheres in the control group (key steps 2 to 4 were skipped in the preparation, the other steps were the same as the preparation of the example, and the amount of added protein was 600ug) was smoother and no fibrinogen was attached, indicating that the protein-coupled microspheres in the control group failed to couple with fibrinogen ( Figure 3 )(Scale bar: 10 μm).

[0086] 2. Preparation of rat coronary microembolism (CME) model

[0087] 1. CME modeling technology route such as Figure 14 shown.

[0088] 2. Construction of a new CME model using the protein-coupled microsphere embolic agent prepared in the example (experimental group)

[0089] SD rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30-40 mg / kg). After confirming that the rats were in an anesthetized state, they were fixed on the small animal operating table. The rat trachea was inserted through the mouth and connected to the small animal ventilator. The ventilator setting parameters were: inspiration-expiration ratio 1:1, respiratory rate 70 times / min, tidal volume 20 mL / kg, and air pressure 1.5 kPa. Connect the electrocardiogram monitor, perform routine disinfection, and lay a drape to expose only the surgical area. The skin was incised along the 3rd-4th intercostal space of the left sternum, and the fascia, muscle and soft tissue were bluntly separated and separated into the chest cavity. The thoracotomy was fixed with a thoracotomy device to expose the surgical area, the embolic agent was resuspended, the ascending aorta was freed and clamped for about 10 seconds (about 20 cardiac cycles), and the embolic agent (containing 2.5×10 45μm protein-coupled microspheres) was injected into the left ventricle through the apex of the heart. 4 After the clamping time reaches 10 seconds, release the artery clamp.

[0090] After the pleural fluid was aspirated, the chest was closed layer by layer. After the operation, the rats were placed on a constant temperature blanket at 36.5°C. After spontaneous breathing was restored and the anesthesia was awakened, the endotracheal tube was removed and the rats were returned to the cage for routine clean feeding.

[0091] 3. CME model detection

[0092] 3-1. Hematoxylin-eosin staining (HE staining): Under a 200x magnification microscope, protein-coupled microspheres can be seen in the myocardial microcirculation of the new model and in situ thrombosis is formed, confirming the pathological success of the new model ( Figure 4 )(Scale bar: 50 μm).

[0093] 3-2. Cardiac appearance: The surface of the rat heart in the new model showed pale, diffuse and uniform embolic lesions (red arrows), indicating that the new model was successful in terms of gross appearance ( Figure 5 ).

[0094] 3-3. Scanning electron microscopy: Under a 1000x magnification electron microscope, the surface of the protein-coupled microspheres in the myocardial tissue of the new model rats was covered with crisscrossing fibrin, concave disc-shaped red blood cells, and irregularly shaped granular platelets. The results indicate that the new model successfully produces in situ thrombosis ( Figure 6 )(Scale bar: 20 μm).

[0095] 3-4. Cardiac ultrasound: The average LVEF of the new model rats was about 49%, and the cardiac function level was significantly reduced ( Figure 7 ).

[0096] 3-5. ELISA test: The myocardial injury biomarker cTnI (about 408 pg / mL) was significantly increased in the new model rats.

[0097] 4. Construction of a traditional CME model using commercially available polystyrene microsphere embolic agents

[0098] SD rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30-40 mg / kg). After confirming that the rats were in an anesthetized state, they were fixed on the small animal operating table. The rat trachea was inserted through the mouth and connected to the small animal ventilator. The ventilator setting parameters were: inspiration-expiration ratio 1:1, respiratory rate 70 times / min, tidal volume 20 mL / kg, and air pressure 1.5 kPa. Connect the electrocardiogram monitor, perform routine disinfection, and lay a drape to expose only the surgical area. The skin was incised along the 3rd-4th intercostal space of the left sternum, and the fascia, muscle and soft tissue were bluntly separated and separated into the chest cavity. The thoracotomy was fixed with a thoracotomy device to expose the surgical area, the embolic agent was resuspended, the ascending aorta was freed and clamped for about 10 seconds (about 20 cardiac cycles), and the embolic agent (containing 2.5×10 45μm polystyrene microspheres) was injected into the left ventricle through the apex of the heart. 4 After the clamping time reaches 10 seconds, release the artery clamp.

[0099] After the pleural fluid was aspirated, the chest was closed layer by layer. After the operation, the rats were placed on a constant temperature blanket at 36.5°C. After spontaneous breathing was restored and the anesthesia was awakened, the endotracheal tube was removed and the rats were returned to the cage for routine clean feeding.

[0100] It is particularly noted that the environmental temperature, reagent dosage, operation steps, animal gender and age, etc. for constructing the traditional CME model and the new model are the same.

[0101] 5. Testing of traditional and new CME models (using conventional testing methods)

[0102] 5-1.Western Blotting

[0103] 5-1-1. Tissue protein extraction

[0104] 1) Prepare protein lysis solution: 1000 μl high-efficiency RIPA lysis buffer (Solarbio, Cat#R0010), 10 μl phenylmethylsulfonyl fluoride (PMSF);

[0105] 2) Weigh the myocardial tissue, cut it into pieces and place it in an Eppendorf tube. Keep the work on ice.

[0106] 3) Add 1000 μl of pre-chilled protein lysis solution to every 100 mg of tissue and grind in a cryogenic grinder;

[0107] 4) After lysis on ice for 15 minutes, centrifuge at 13,000 x g for 30 minutes at 4°C.

[0108] 5) Take the supernatant, discard the precipitate, and store at -80℃ for later use.

[0109] 5-1-2. The protein concentration determination (BCA method) is the same as the microsphere coupling amount determination.

[0110] 5-1-3. SDS-PAGE gel electrophoresis

[0111] 1) Sample preparation: Take 25 μg of the protein sample to be tested, add 5× loading buffer according to the volume ratio, boil at 100°C for 10 min to complete protein denaturation, and store at -80°C until use;

[0112] 2) Installing a Bio-Rad vertical electrophoresis apparatus;

[0113] 3) Preparation of 12% SDS-PAGE separation gel:

[0114] Table 1: 12% SDS-PAGE separation gel formula (per 5 mL, unit: mL)

[0115]

[0116] 4) Preparation of 5% SDS-PAGE stacking gel:

[0117] Table 2: 5% SDS-PAGE stacking gel recipe (per 2 mL, unit: mL)

[0118]

[0119] 5) Prepare electrophoresis buffer (10×, per 1000 mL) according to the following table:

[0120] Table 3: 10× electrophoresis buffer system

[0121]

[0122] Dilute to 1× with deionized water before use.

[0123] 6) After the gel solidifies, load the protein sample, 25 μg per well, and perform electrophoresis at a constant voltage of 80 V for 1.5-2 hours;

[0124] 7) Stop electrophoresis when the bromophenol blue indicator of the marker reaches about 1.5 cm below the bottom edge of the glass plate.

[0125] 5-1-4. Wet transfer

[0126] 1) Prepare the transfer buffer according to the table below:

[0127] Table 4: 1× Transfer Buffer Configuration

[0128]

[0129] 2) Cut a piece of filter paper slightly larger than the expected gel and soak it in transfer buffer. Cut a piece of PVDF membrane of the same size as the expected gel, cut off the upper left corner to mark it, soak it in anhydrous formaldehyde for 30 seconds to activate the PVDF membrane, and then transfer it to transfer buffer.

[0130] 3) After electrophoresis is complete, remove the glass plate, carefully remove the glass plate, scrape off the stacking gel, completely peel off the separation gel, cut the gel according to the molecular weight of the target protein, transfer the gel plate to a large culture dish and completely immerse it in transfer buffer for about 5 minutes.

[0131] 4) Place the transfer apparatus (sandwich) in the tray containing the transfer solution in the following order: negative electrode (black plate) in the transfer apparatus, fiber mat, three layers of filter paper, gel, PVDF membrane, three layers of filter paper, fiber mat, and positive electrode in the transfer apparatus. Ensure each layer is tightly packed and use a glass rod to remove any air bubbles.

[0132] 5) Place the prepared sandwich into the transfer tank, making sure the black side of the sandwich corresponds to the black side of the tank, and the white side corresponds to the red side of the tank. Transfer the film at 300mA constant current for 35 minutes.

[0133] 5-1-5. Blocking and Antibody Incubation

[0134] 1) After transfer, wash the PVDF membrane with TBST three times on a shaker for 5 minutes each time.

[0135] 2) Place the PVDF membrane in a 5% skim milk solution and incubate on a shaker at room temperature for 1 hour to block nonspecific binding. After blocking, wash the membrane three times with TBST on a shaker for 5 minutes each.

[0136] 3) Dilute the primary antibody in primary antibody diluent. Dilute the antibody according to the recommended dilution range in the manufacturer's instructions. For target protein antibodies (Bcl-2, cleaved caspase-3), the dilution ratio is 1:1000, for Bax, the dilution ratio is 1:2000, and for GAPDH, the dilution ratio is 1:10000.

[0137] 4) Completely immerse the PVDF membrane in the required diluted primary antibody solution. Decolorize the membrane by incubating on a shaker at room temperature for approximately 1 hour and then overnight at 4°C. Wash the membrane three times with TBST for 5 minutes each.

[0138] 5) Dilute the HRP-labeled secondary antibody in TBST at a dilution ratio of 1:10,000.

[0139] 6) Immerse the PVDF membrane completely in the secondary antibody solution, incubate at room temperature for 1 hour, and wash the membrane three times with TBST on a decolorizing shaker for 5 minutes each time.

[0140] 5-1-6. Scanning and result analysis

[0141] The membrane was scanned according to the Odyssey infrared fluorescence scanning imaging system operating instructions. The grayscale value of each band was analyzed using ImageJ software, and the relative expression level of the target protein was expressed as the grayscale value of the target band divided by the grayscale value of the internal reference GAPDH.

[0142] 5-2. Immunofluorescence detection

[0143] 1) Bake at 60℃ for 2 hours;

[0144] 2) Dewaxing: Dewaxing in xylene for 15 minutes, three times;

[0145] 3) Hydration: Wash with anhydrous ethanol, 95%, 90%, 80%, and 70% for 5 minutes each, distilled water for 5 minutes, and PBS for 5 minutes, three times;

[0146] 4) Antigen retrieval: Boil citrate buffer (approximately 92-95°C) in a boiling water bath for 15 minutes, then cool to room temperature. Wash with PBS for 5 minutes three times.

[0147] 5) Block with normal goat serum (BSA) at 37°C for 20 minutes, then wash with PBS for 5 minutes three times;

[0148] 6) Incubate with primary antibody dilution (1:250 in BSA) at 37°C for 1.5 hours, then wash with PBS for 10 minutes three times;

[0149] 7) Counterstain the cell nuclei with DAPI at room temperature for 5 minutes, then wash with PBS for 5 minutes three times;

[0150] 8) Seal the slides with anti-fading fluorescent mounting medium and observe under a microscope.

[0151] 5-3. Tunel staining

[0152] 1) Bake at 60℃ for 2 hours;

[0153] 2) Dewaxing: Dewaxing in xylene for 15 minutes, three times;

[0154] 3) Hydration: ethanol, 95%, 90%, 80%, 70%, 5 minutes each, and distilled water for 5 minutes;

[0155] 4) Add 20 μg / mL DNase-free proteinase K dropwise and incubate at 37°C for 30 minutes. Wash with PBS three times for 5 minutes each.

[0156] 5) Prepare TUNEL assay solution according to the following table:

[0157] Table 5: TUNEL detection solution configuration

[0158] name 1 sample TdT enzyme 5μl Fluorescent labeling solution 45 μl TUNEL detection solution 50 μl

[0159] 6) Circle the sample area with a PAP Pen, add 50 μl of TUNEL detection solution to each sample, incubate at 37°C in the dark for 60 minutes, and wash with PBS three times, 5 minutes each time.

[0160] 7) Counterstain the cell nuclei with DAPI at room temperature for 5 minutes, then wash with PBS for 5 minutes three times;

[0161] 8) Seal the slides with anti-fading fluorescent mounting medium and observe under a microscope.

[0162] 6. Results analysis:

[0163] 1) Cardiac ultrasound: Compared with the traditional model, the LVEF value of the new model rats was significantly decreased (p<0.01) ( Figure 8 ).

[0164] 2) Apoptosis damage: WB confirmed that the translation level of Bax in the new model was significantly increased (p<0.001) and the translation level of Bcl-2 was significantly decreased (p<0.05) compared with the traditional model. WB and immunofluorescence confirmed that the translation level of cleaved caspase-3 was significantly increased (p<0.05) compared with the traditional model ( Figure 9 、 Figure 10 , Figure 11 ). TUNEL confirmed that the number of TUNEL-positive cells in the new model was significantly higher than that in the traditional model (p<0.05) ( Figure 12 )(×200, scale bar 100 μm).

[0165] Comparison confirmed that under the same conditions of ambient temperature, reagent dosage, operation steps, and animal gender and age, the new CME model constructed using the protein-coupled microspheres prepared by the present invention as embolic agents has significant advantages over the traditional CME model in simulating disease-induced myocardial damage and pathological phenotypic characteristics. The cardiac function of the new model decreased significantly; the results of immunofluorescence, protein blotting, and TUNEL staining of apoptotic proteins showed that the apoptotic damage in the improved model was more obvious.

[0166] The protein-coupled microspheres prepared by the present invention are used as embolic agents to construct an animal model of coronary microembolism. The surface fibrinogen is activated to become fibrin, which aggregates platelets and forms microthrombi, which is closer to the real disease state and provides a more valuable research tool for medical research on the pathophysiological mechanism of coronary microcirculation and clinical prevention and treatment.

[0167] The number of devices and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be readily apparent to those skilled in the art.

[0168] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A method for preparing protein-coupled microspheres, characterized in that: Carboxyl polystyrene microspheres with a particle size of 45 μm were used as carriers, and the weight ratio of carboxyl polystyrene microspheres to fibrinogen was 4×10 4 200-600 μg of fibrinogen is covalently coupled to the surface of the carboxyl polystyrene microspheres by a chemical method, comprising the following steps: 1) Centrifuge the carboxylated polystyrene microspheres and discard the supernatant; 2) Add coupling buffer to resuspend and wash the microspheres, centrifuge and remove the supernatant, and repeat 1-3 times; 3) Add coupling buffer, EDC solution, and Sulfo-NHS solution to the microsphere precipitate, mix well, incubate at room temperature for 10-20 minutes, centrifuge, and discard the supernatant; wherein the EDC solution is a solution prepared with coupling buffer to a concentration of 50 mg / mL, and the Sulfo-NHS solution is a solution prepared with coupling buffer to a concentration of 50 mg / mL. The volume ratio of the added coupling buffer: EDC solution: Sulfo-NHS solution is 60:20:20; 4) Wash the microspheres with coupling buffer, vortex to mix, centrifuge and discard the supernatant to obtain the carboxylated polystyrene microspheres to be coupled; 5) Add fibrinogen to 0.9% sodium chloride solution at 37°C to prepare a fibrinogen solution, where the weight-to-volume ratio of fibrinogen to sodium chloride solution is 5 mg:1 mL. 6) Add coupling buffer and fibrinogen solution to the carboxylated polystyrene microspheres to be coupled, vortex to mix, and incubate at room temperature with continuous shaking for 50-70 minutes. The volume ratio of coupling buffer to fibrinogen solution is 100:40-120. 7) Centrifuge and remove the residual protein in the supernatant for subsequent quality control; 8) Add quenching buffer to the microsphere pellet from step 7), vortex to mix, centrifuge and discard the supernatant; 9) Add quenching buffer to the microsphere pellet from step 8), incubate at room temperature for 60-70 minutes, centrifuge, and discard the supernatant; 10) Add quenching buffer to the microsphere precipitate from step 9), vortex to mix, centrifuge and discard the supernatant to obtain the protein-coupled microspheres; 11) Add storage buffer to the microsphere pellet from step 10), vortex to mix, and store at 2-8°C to obtain the prefabricated protein-coupled microsphere storage solution.

2. The method for preparing protein-coupled microspheres according to claim 1, wherein The centrifugal speed is 2000 r / min, and the centrifugation is 1 minute; the coupling buffer is 50 mmol / L MES with a pH of 6.0, containing 0.01% Triton X-100; the quenching buffer is TBS solution; and the storage buffer is TBS or PBS solution containing 0.01% Triton X-100.

3. A protein-coupled microsphere prepared by the method according to claim 1 or 2.

4. An embolic agent, characterized in that: Includes 2.5×10 4 The protein-coupled microspheres as claimed in claim 3 are prepared into 200 μL of protein-coupled microsphere embolic agent with 0.9% sodium chloride solution.

5. Application of protein-coupled microspheres, characterized in that, Use of the protein-coupled microspheres as claimed in claim 3 as an embolic agent in constructing an animal model of coronary microembolism.

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