Method, system and equipment for evaluating treatment effect of RNF14 accelerant on cerebral infarction based on Notch1

Abstract

The invention discloses a method, system and equipment for evaluating the treatment effect of an RNF14 accelerant on cerebral infarction based on Notch1, it is found for the first time that RNF14 can treat cerebral infarction by adjusting Notch1 protein, and an efficient method is provided for evaluating the treatment effect of the RNF14 accelerant on cerebral infarction. An efficient, accurate and rapid screening method is provided for research and development of new drugs for treating cerebral infarction, and the method has very wide application prospects in the aspects of cerebral infarction drug evaluation and new drug screening.

Description

Technical Field

[0001] The present invention belongs to the field of bioinformatics, and specifically relates to a method, system and device for evaluating the therapeutic effect of an RNF14 promoter on cerebral infarction based on Notch1, and a method, system and device for screening drugs for treating cerebral infarction based on Notch1 and RNF14. Background Art

[0002] Cerebral infarction, also known as ischemic stroke, is one of the most common cerebrovascular diseases. Data surveys show that the incidence, disability and mortality of cerebral infarction are very high, which has a serious impact on the quality of life of patients. According to statistics from the World Health Organization in 2015, on average, one person dies from cerebral infarction every 225 seconds worldwide. Even in the United States, where medical care is relatively advanced, deaths from cerebral infarction account for about 5.6% of all deaths. Cerebral infarction not only seriously affects the quality of life of patients, but also brings huge burdens and pressures to society and families. Therefore, it is very important to develop drugs that can treat cerebral infarction.

[0003] RNF14 is a key E3 ubiquitin protein ligase that participates in the translation quality control pathway and is associated with the ubiquitination and degradation of multiple translation factors and ribosomal proteins. Notch1 is a member of the Notch family. As a cell surface receptor, it plays a regulatory role in cell proliferation, apoptosis, differentiation and other processes, and its signaling pathway is of great significance in embryonic development and tumorigenesis. Although both play an important role in biological functions, according to current information, the relationship between the two has not been made public, and there is no report on the correlation between the two and cerebral infarction. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the purpose of the present invention is to provide a method, system and device for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1, and a method, system and device for screening drugs for treating cerebral infarction based on Notch1 and RNF14.

[0005] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1.

[0006] Further, the method comprises the following steps: Acquiring data 1: Acquiring the Notch1 expression level in brain tissue cells of patients with cerebral infarction; Acquiring data 2: Acquiring the Notch1 expression level of brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them; Processed data: Comparison of Notch1 expression levels in brain tissue cells of patients with cerebral infarction and Notch1 expression levels in brain tissue cells of patients with cerebral infarction after administration of RNF14 promoter; Output result: If the Notch1 expression level of brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them is lower than the Notch1 expression level of brain tissue cells in patients with cerebral infarction, then the result that the RNF14 promoter has a therapeutic effect on cerebral infarction is output.

[0007] Furthermore, the patient includes humans and / or mammals.

[0008] Furthermore, the brain tissue cells include neurons, astrocytes, microglia, and vascular epithelial cells.

[0009] The second aspect of the present invention provides a system for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1.

[0010] Further, the system comprises: Data acquisition unit: acquiring the Notch1 expression level of brain tissue cells in patients with cerebral infarction, and acquiring the Notch1 expression level of brain tissue cells in patients with cerebral infarction after the RNF14 promoter is administered to them; Data analysis unit: Compare the Notch1 expression level in brain tissue cells of patients with cerebral infarction with the Notch1 expression level in brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them; Result output unit: if the Notch1 expression level of brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them is lower than the Notch1 expression level of brain tissue cells in patients with cerebral infarction, then the result that the RNF14 promoter has a therapeutic effect on cerebral infarction is output.

[0011] The third aspect of the present invention provides a method for screening drugs for treating cerebral infarction based on Notch1 and RNF14.

[0012] Further, the method comprises the following steps: Obtaining a system containing a protein complex formed by Notch1 and RNF14; adding a candidate substance into the system; The interaction between Notch1 and RNF14 proteins in the protein complex is analyzed before and after the addition of the candidate substance. If the candidate substance can promote the interaction between Notch1 and RNF14 proteins in the complex, it indicates that the candidate substance is a potential drug for treating cerebral infarction.

[0013] Furthermore, the system includes a cerebral infarction cell system, a cerebral infarction tissue system, a cerebral infarction organ system or a cerebral infarction animal model system.

[0014] The fourth aspect of the present invention provides a system for screening drugs for treating cerebral infarction based on Notch1 and RNF14.

[0015] Further, the system comprises: Data acquisition unit: acquiring a system containing a protein complex formed by Notch1 and RNF14; Data analysis unit: adding the candidate substance to the system, and analyzing the interaction between Notch1 and RNF14 proteins in the protein complex before and after the addition of the candidate substance; Drug screening unit: If the candidate substance can promote the interaction between Notch1 and RNF14 proteins in the complex, it indicates that the candidate substance is a potential drug for treating cerebral infarction.

[0016] A fifth aspect of the present invention provides a computer device and a readable storage medium.

[0017] Further, the device comprises: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction described in the first aspect of the present invention or the method for screening drugs for treating cerebral infarction described in the third aspect of the present invention is implemented.

[0018] Furthermore, the computer-readable storage medium stores a computer program, which, when executed by the processor, implements the method for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction described in the first aspect of the present invention or implements the method for screening drugs for treating cerebral infarction described in the third aspect of the present invention.

[0019] The sixth aspect of the present invention provides an isolated protein-complex, which includes: Notch1 and RNF14 protein, and the two interact with each other.

[0020] The seventh aspect of the present invention provides a method for regulating Notch1 expression.

[0021] Furthermore, the method includes negatively regulating the expression of Notch1 in cerebral infarction tissue by RNF14; by adjusting the expression level of RNF14, the regulation of Notch1 expression in cerebral infarction tissue can be achieved.

[0022] Furthermore, the method also includes that when the expression of RNF14 is up-regulated, the expression level of Notch1 decreases; when the expression of RNF14 is inhibited, the expression level of Notch1 increases.

[0023] Furthermore, the upregulation of RNF14 expression is achieved through gene overexpression technology.

[0024] Furthermore, the gene overexpression vector includes a eukaryotic expression vector and a viral vector.

[0025] The beneficial effects of the present invention are as follows: The present invention discovers for the first time that RNF14 protein in cerebral infarction cells can play a role in treating cerebral infarction by regulating the expression level of Notch1 protein. Based on this, the present invention provides a method, system and device for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1 in the field, as well as a method, system and device for screening drugs for treating cerebral infarction based on Notch1 and RNF14, which have very broad application prospects in the evaluation of cerebral infarction drugs and the screening of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 A schematic flow chart of a method for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1 provided in an embodiment of the present invention; Figure 2 A schematic diagram of a system for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1 provided in an embodiment of the present invention; Figure 3 A schematic flow chart of a method for screening drugs for treating cerebral infarction based on Notch1 and RNF14 provided in an embodiment of the present invention; Figure 4 A schematic diagram of a system for screening drugs for treating cerebral infarction based on Notch1 and RNF14 provided in an embodiment of the present invention; Figure 5 A schematic diagram of a device for evaluating the therapeutic effect of an RNF14 promoter on cerebral infarction based on Notch1 or a device for screening drugs for treating cerebral infarction based on Notch1 and RNF14 provided in an embodiment of the present invention; Figure 6 The results of ROS production in the astrocyte control group and the RNF14 overexpression group are shown in Figure A. The results of ROS detection by fluorescence microscopy, and Figure B is the results of quantitative analysis of ROS. Figure 7This is the result of detecting the cell survival rate of the glial cell control group and RNF14 overexpression group by CCK8; Figure 8 The results of TTC staining of brain tissue of the control group and RNF14 overexpression group after MCAO in mice; Fig. 9 The results of cerebral infarction volume measurement in the control group and RNF14 overexpression group after MCAO surgery in mice; Fig.10 The docking model of RNF14 and Notch1 protein and the surface map of their interface residues; Fig.11 This is the result diagram corresponding to the significant correlation between RNF14 and Notch1. Fig.11 A in the figure is the CO-IP mass spectrometry analysis verification diagram. Fig.11 B in the figure is a Western blot verification picture. DETAILED DESCRIPTION

[0028] In order to make up for the shortcomings of the prior art, the overall idea of ​​the technical solution provided in this application is as follows: by overexpressing RNF14 in the mouse cerebral infarction MCAO model and OGD / R astrocyte model, it was found that RNF14 plays an important role in the treatment of cerebral infarction; then, through CO-IP mass spectrometry analysis, it was found that RNF14 protein and Notch1 protein can interact with each other, and further verified by Western blot, it was found that in cerebral infarction tissue, RNF14 protein negatively regulates Notch1 protein; based on this, we propose a method, system and equipment for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1, and a method, system and equipment for screening drugs for treating cerebral infarction based on Notch1 and RNF14.

[0029] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0030] In some of the processes described in the specification and claims of the present invention and the above-mentioned drawings, multiple operations appearing in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this document or may be executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel.

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0032] Example 1 like Figure 1 As shown, Example 1 of the present invention provides a method for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1. Specifically, the method comprises the following steps: 101: Obtain the Notch1 expression level in brain tissue cells of patients with cerebral infarction; In one embodiment, the patient can be human or non-human, and can include, for example, animal strains or species used as a "model system" for research purposes, such as the mouse model used in this application. Likewise, patients can include adults or adolescents (e.g., children). In addition, patients can refer to any living organism that can benefit from the administration of the RNF14 promoter herein, preferably mammals (e.g., humans or non-humans). Examples of mammals include, but are not limited to, any member of the mammal class: humans, non-human primates (e.g., chimpanzees), and other apes and monkeys; livestock, such as cattle, horses, sheep, goats, pigs; livestock, such as rabbits, dogs, and cats; experimental animals include rodents, such as rats, mice, and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment provided herein, the mammal is a mouse.

[0033] In one embodiment, the brain tissue cells include neurons, astrocytes, microglia, and vascular epithelial cells; in a specific embodiment of the present invention, the brain tissue cells are astrocytes.

[0034] In one embodiment, the method for obtaining brain tissue from a patient with cerebral infarction includes preparing an animal model of cerebral infarction.

[0035] In some embodiments, methods for preparing cerebral infarction models include but are not limited to: embolic cerebral infarction animal model: using methods such as injecting microembolic suspension, multiple regular emboli and intravascular injection of thrombin, for example, using dog venous blood mixed with thrombin and then injecting it through carotid artery catheterization to form a middle cerebral artery occlusion (MCAO) model; spontaneous cerebral infarction model: based on hypertensive animals, such as spontaneously hypertensive rats (SHR) and their stroke-prone subtypes, simulating the incidence of human cerebral infarction; gerbil middle cerebral artery ischemia model: by clamping one side of the gerbil carotid artery to cause ischemia on the ipsilateral hemisphere; acute rat middle cerebral artery occlusion global brain ischemia model: surgically expose the middle cerebral artery, separate the surrounding pia mater and arachnoid membrane, and free the middle artery; large-area cerebral infarction animal model: including the carotid-vertebral artery four-vessel occlusion model, bilateral carotid artery permanent occlusion method, etc.

[0036] In a specific embodiment of the present invention, the cerebral infarction model is a mouse MCAO model, and its construction method is as follows: a male C57BL / 6 mouse of about 25 g is taken, and 0.1 mL of 1.25% avertin is injected intraperitoneally. After successful anesthesia, the mouse is fixed on the operating table; routine skin preparation and disinfection are performed, hair is removed, a midline incision is made in the neck, and the surrounding tissues are separated. The common carotid artery, the external carotid artery and the internal carotid artery are exposed and separated under a microscope; the pterygomandibular artery is separated and ligated, and the internal carotid artery is kept open; a 15 mm long silicone-coated wire plug of an appropriate size is inserted from the external carotid artery into the internal carotid artery about 10 mm, and stops at the beginning of the middle cerebral artery; after 1 hour, the wire plug is pulled out to complete reperfusion, and the mouse is placed on a heating pad. When the mouse wakes up, symptoms of hemiplegia can be observed. After waking up, the mouse is kept in an environment with suitable temperature and given sufficient feed and water; the corresponding samples are collected 24 hours after the operation.

[0037] In some embodiments, Notch1 expression level data can be obtained by measuring the amount of Notch1 RNA, mRNA or any other RNA species using methods well known in the art to obtain expression at the nucleic acid level, including digital PCR and real-time (RT) quantitative or semi-quantitative PCR, fluorescence activated cell sorting (FACS) and in situ hybridization.

[0038] In other embodiments, the expression level data of Notch1 can be obtained by measuring the expression level at the Notch1 protein level, including mass spectrometry-based quantitative proteomics technology, immunoassay, Western blotting, spectrophotometry, enzyme assay, ultraviolet assay, kinetic assay, electrochemical assay, colorimetric assay, turbidimetric assay, atomic absorption assay, flow cytometry, mass spectrometry flow cytometry, or any combination thereof.

[0039] In a specific embodiment of the present invention, the step of acquiring Notch1 expression level data includes Western blotting: brain tissue and astrocytes are homogenized in RIPA lysis buffer (Beyotime) containing a mixture of protease and phosphatase inhibitors (Beyotime), and centrifuged at 14,000×g for 30 minutes at 4°C, the supernatant is transferred to a fresh test tube, and the concentration is determined by a BCA kit (Beyotime), equal amounts of protein are separated by 4-15% SDS-PAGE, and electroblotted onto a PVDF membrane; after blocking, the membrane is incubated with the primary antibody overnight at 4°C; after incubation with an appropriate secondary antibody conjugated to horseradish peroxidase (anti-rabbit antibody or anti-mouse antibody, 1:1000–1:2000) at room temperature for 1 hour, the protein bands are observed using a fluorescent secondary antibody, and developed using an Odyssey developer; the density of the bands is measured using ImageJ; and the changes in target protein expression are normalized using control protein expression.

[0040] 102: Obtain the Notch1 expression level of brain tissue cells in patients with cerebral infarction after the RNF14 promoter is administered; In one embodiment, the RNF14 promoter refers to any substance that can increase the activity of RNF14 protein, improve the stability of RNF14 gene or protein, upregulate the expression of RNF14 protein, increase the effective action time of RNF14 protein, or promote the transcription and translation of RNF14 gene, and these substances can be used in the present invention. For example, the promoter includes a vector that promotes overexpression of RNF14.

[0041] In a specific embodiment of the present invention, the method of administering an RNF14 promoter to patients with cerebral infarction includes: overexpressing RNF14 in a mouse MCAO model, using adeno-associated virus for stereotactic brain injection, recombinant PHP.B type adeno-associated virus (AAV-PHP.B) is used to construct an RNF14 gene overexpression virus (Ji Kai Gene, China), and injecting the adeno-associated virus into the striatum area of ​​the mouse by stereotactic brain injection.

[0042] 103: Compare the Notch1 expression levels in brain tissue cells of patients with cerebral infarction and the Notch1 expression levels in brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered; In one embodiment, the present invention studies and analyzes the relationship between RNF14 and cerebral infarction. Specifically, a mouse MCAO model and an astrocyte oxygen glucose deprivation / reoxygenation / re-glucose model were first constructed, and then the changes in ROS levels and cell viability after overexpression of RNF14 in the astrocyte oxygen glucose deprivation / reoxygenation / re-glucose model (OGD / R) were detected. The results are as follows: Figure 7 , 8As shown. In the OGD / R astrocyte model, compared with the control group, overexpression of RNF14 significantly decreased the ROS production of OGD / R glial cells ( Figure 7 ), while the cell survival rate increased significantly ( Figure 8 In addition, we observed that after overexpression of RNF14 in the mouse MCAO model, the color of the mouse brain tissue returned to normal and turned red ( Fig. 9 ), and compared with the control group, the cerebral infarction volume was significantly reduced after overexpression of RNF14 ( Fig.10 ). These results indicate that RNF14 can play a role in treating cerebral infarction.

[0043] In one embodiment, we observed the association between Notch1 protein and RNF14 protein in cerebral infarction tissue. First, we found that Notch1 protein and RNF14 protein interacted with each other through CO-IP mass spectrometry analysis. Then we extracted brain tissue from the mouse MCAO model and performed Western blot analysis to verify this result ( Figure 7 Specifically, the expression of Notch1 protein increased in cerebral infarction tissue, while the expression of Notch1 protein decreased after overexpression of RNF14 protein in the mouse MCAO model, indicating that RNF14 plays a role in treating cerebral infarction by downregulating the expression of Notch1 protein.

[0044] 104: Output the evaluation result.

[0045] In one embodiment, if the Notch1 expression level of brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them is lower than that of brain tissue cells in patients with cerebral infarction, the result that the RNF14 promoter has a therapeutic effect on cerebral infarction is outputted.

[0046] Example 2 like Figure 2 As shown, Example 2 of the present invention provides a system for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1. Specifically, the system comprises: Data acquisition unit: acquiring the Notch1 expression level of brain tissue cells in patients with cerebral infarction, and acquiring the Notch1 expression level of brain tissue cells in patients with cerebral infarction after the RNF14 promoter is administered to them; Data analysis unit: Compare the Notch1 expression level in brain tissue cells of patients with cerebral infarction with the Notch1 expression level in brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them; Result output unit: if the Notch1 expression level of brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them is lower than the Notch1 expression level of brain tissue cells in patients with cerebral infarction, then the result that the RNF14 promoter has a therapeutic effect on cerebral infarction is output.

[0047] In one embodiment, the system may be a user's electronic device or a computer system remotely located relative to the electronic device.

[0048] Example 3 like Figure 3 As shown, Example 3 of the present invention provides a method for screening drugs for treating cerebral infarction based on Notch1 and RNF14. Specifically, the method comprises: 301: Obtaining a system containing a protein complex formed by Notch1 and RNF14; In some embodiments, the system includes a cerebral infarction cell system, a cerebral infarction tissue system, a cerebral infarction organ system or a cerebral infarction animal model system.

[0049] In one embodiment, Notch1 protein is a cell surface receptor and a key component of the Notch signaling pathway, which can bind to a variety of ligands. The Notch1 protein described in the present invention can be obtained through conventional purchasing channels known to those skilled in the art.

[0050] In one embodiment, the RNF14 protein is an E3 ubiquitin ligase having a RING zinc finger structure and is involved in protein-protein interaction. The RNF14 protein of the present invention can be obtained through conventional purchasing channels known to those skilled in the art.

[0051] 302: adding a candidate substance into the system; In one embodiment, the candidate substance can be selected from small molecule compounds, wherein the source of the small molecule compound is selected from: newly synthesized or existing databases; wherein the existing databases include but are not limited to general natural product databases (COCONUT, Super Natural II, NPASS), plant natural product databases (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), Chinese medicine natural product databases (TCM@Taiwan, CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), microbial natural product databases (StreptomeDB, NP Altas, ProCarDB, PAMDB, LichenDatabase), marine natural product databases (MNPD, SWMD), natural product databases of different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB), food natural product databases (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet database), toxic natural product databases (Exposome-Explorer, T3DB, Snake Neurotoxin Database, TPPT), natural product industry catalogs (Greenpharma, AnalytiCon Discovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol), databases for deduplicating MS data (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), databases for deduplicating NMR data (NMRShiftDB, NAPROC-13), etc.

[0052] 303: Analyze the interaction between Notch1 and RNF14 proteins in the protein complex. If the candidate substance can promote the interaction between Notch1 and RNF14 proteins in the complex, it indicates that the candidate substance is a potential drug for treating cerebral infarction.

[0053] In the present invention, the interaction between proteins and the strength of the interaction can be detected by various techniques well known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation experiment, yeast two-hybrid system or immunoprecipitation technology.

[0054] In one embodiment, the method for screening drugs for treating cerebral infarction based on Notch1 and RNF14 further comprises: Obtain cerebral infarction cell model and / or cerebral infarction animal model; The cerebral infarction cell model and / or cerebral infarction animal model are treated with the screened cerebral infarction treatment drug to verify the effectiveness of the cerebral infarction treatment drug.

[0055] In one embodiment, the cerebral infarction cell model is treated with the screened cerebral infarction treatment drug to verify the effectiveness of the cerebral infarction treatment drug.

[0056] Furthermore, the cerebral infarction cell model is treated with the screened therapeutic drug for cerebral infarction to verify the effectiveness of the therapeutic drug for cerebral infarction. Specifically, the cerebral infarction cell model is cultured in a cell culture medium suitable for the growth of the cerebral infarction cell model, a certain concentration of the screened therapeutic drug for cerebral infarction is added to the cell culture medium, and the changes in cell viability and ROS in the cerebral infarction cell model after the intervention of the screened therapeutic drug for cerebral infarction are observed.

[0057] Furthermore, if after the intervention of the screened drug for treating cerebral infarction, the cell viability and ROS concentration in the cerebral infarction cell model are significantly lower than those before the intervention, then the drug is an effective drug for treating cerebral infarction.

[0058] In one embodiment, the cerebral infarction cell model of the present invention can be obtained through conventional purchasing channels known to those skilled in the art, or can be constructed by conventional cerebral infarction cell model construction methods known to those skilled in the art.

[0059] Furthermore, the cerebral infarction cell model includes, but is not limited to, a cerebral infarction astrocyte model and a cerebral infarction microglia model.

[0060] In one embodiment, the cerebral infarction animal model is treated with the screened drug for treating cerebral infarction to verify the effectiveness of the drug for treating cerebral infarction.

[0061] Furthermore, the cerebral infarction animal model is treated with the screened therapeutic drug for cerebral infarction to verify the effectiveness of the therapeutic drug for cerebral infarction. Specifically, a certain concentration of the screened therapeutic drug for cerebral infarction is administered to the cerebral infarction animal model, and the changes in brain tissue color and cerebral infarction volume in the cerebral infarction animal model after the administration of the screened therapeutic drug for cerebral infarction are observed.

[0062] Furthermore, if after the screened drug for treating cerebral infarction is administered, the brain tissue color of the cerebral infarction animal model obviously turns red, and the volume of cerebral infarction is significantly lower than before administration, then the drug is an effective drug for treating cerebral infarction.

[0063] In one embodiment, the cerebral infarction animal model of the present invention can be obtained through conventional purchasing channels known to those skilled in the art, or can be constructed by conventional cerebral infarction animal model construction methods known to those skilled in the art.

[0064] Furthermore, the animals include but are not limited to: mice, rats, gerbils, dogs and other animals.

[0065] Example 4 like Figure 4 As shown, Example 4 of the present invention provides a system for screening drugs for treating cerebral infarction based on Notch1 and RNF14. Specifically, the system comprises: Data acquisition unit: acquiring a system containing a protein complex formed by Notch1 and RNF14; Data analysis unit: adding the candidate substance to the system, and analyzing the interaction between Notch1 and RNF14 proteins in the protein complex before and after the addition of the candidate substance; Drug screening unit: If the candidate substance can promote the interaction between Notch1 and RNF14 proteins in the complex, it indicates that the candidate substance is a potential drug for treating cerebral infarction.

[0066] In one embodiment, the system may be a user's electronic device or a computer system remotely located relative to the electronic device.

[0067] Example 5

[0068] like Figure 5 As shown, Embodiment 5 of the present invention provides a computer device and a readable storage medium.

[0069] The computer device 500 includes a processor 501 and a memory 502 coupled to the processor 501, wherein the memory 502 stores program instructions. When the program instructions are executed by the processor 501, the processor 501 executes the steps of evaluating the therapeutic effect of the RNF14 promoter on cerebral infarction as described in the above-mentioned Example 1 or screening drugs for treating cerebral infarction as described in the above-mentioned Example 3.

[0070] The processor 501 may also be referred to as a CPU (Central Processing Unit). The processor 501 may be an integrated circuit chip having the ability to process signals. The processor 501 may also be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components. The general-purpose processor may be a microprocessor or the processor may also be any conventional processor, etc.

[0071] Computer device 500 may be a mobile electronic device.

[0072] The storage medium of the embodiment of the present invention stores program instructions that can implement the above-mentioned method for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction or the method for screening drugs for treating cerebral infarction, wherein the program instructions can be stored in the above-mentioned storage medium in the form of a software product, including several instructions for enabling a computer device (which can be a personal computer, server, or network device, etc.) or a processor (processor) to perform all or part of the steps of the method described in each embodiment of the present application. The aforementioned storage medium includes: various media that can store program codes, such as USB flash drives, mobile hard disks, read-only memories (ROM, Read-Only Memory), random access memories (RAM, Random Access Memory), magnetic disks or optical disks, or computer devices such as computers, servers, mobile phones, and tablets.

[0073] It should be understood that the systems, devices and methods described in the present invention can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the modules is only a logical function division. There may be other division methods in actual implementation, such as multiple modules or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or modules, which can be electrical, mechanical or other forms.

[0074] The modules described as separate components may or may not be physically separated, and the components shown as modules may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0075] In addition, each functional module in each embodiment of the present invention may be integrated into one processing module, each module may exist physically separately, or two or more modules may be integrated into one module. The above integrated modules may be implemented in the form of hardware or software functional modules.

[0076] Example 6

[0077] 1. Experimental methods (1) Construction of mouse MCAO model: Male C57BL / 6 mice weighing about 25 g (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were weighed and injected intraperitoneally with 0.1 mL of 1.25% avertin. After successful anesthesia, the mice were fixed on the operating table. After routine skin preparation and disinfection, hair removal was performed. A midline incision was made in the neck to separate the surrounding tissues. The common carotid artery, external carotid artery, and internal carotid artery were exposed and separated under a microscope. The pterygopalatine artery was separated and ligated to keep the internal carotid artery open. A 15 mm long silicone-coated suture of appropriate size was inserted from the external carotid artery into the internal carotid artery for about 10 mm and stopped at the beginning of the middle cerebral artery. After 1 hour, the suture was pulled out to complete reperfusion. The mice were placed on a heating pad. When the mice woke up, symptoms of hemiplegia were observed. After waking up, they were kept in an environment with suitable temperature and given sufficient feed and water. The corresponding samples were collected 24 hours after surgery.

[0078] (2) Primary astrocyte culture: Cortices were isolated from P1-3 C57BL / 6J mice and digested with trypsin (Gibco) at 37°C for 15 min; the dissociated cells were rinsed and resuspended in DMEM (Gibco) containing 10% fetal bovine serum and 1% antibiotic-antimycotic (Gibco). Subsequently, live cells (2×10 6 Astrocytes were seeded in T75 culture flasks. The cultures were maintained in a cell culture incubator at 37°C and 5% CO2, and the medium was changed every 3 days during the entire study. When the confluence reached 80%-90%, astrocytes were seeded in 60 mm culture dishes for subsequent studies.

[0079] (3) Construction of cellular oxygen-glucose deprivation / reoxygenation-reglycogenation (OGD / R) model: The original culture medium of the astrocyte OGD / R group was discarded, and the cells were rinsed once with an appropriate amount of PBS. An equal volume of sugar-free DMEM culture medium was added to the cells. The cells were cultured in a cell culture incubator pre-set with 95% nitrogen and 5% carbon dioxide at 37°C for 6 hours, and then replaced with normal culture medium. The cells were placed in a normal cell culture incubator for a further 24 hours to complete the reoxygenation-reglycogenation process.

[0080] (4) Overexpression of RNF14 in the mouse MCAO model: Adeno-associated virus was used for stereotaxic injection. Recombinant adeno-associated virus type PHP.B (AAV-PHP.B) was used to construct the RNF14 gene overexpression virus (Gene Gene, China). The adeno-associated virus was injected into the striatum of mice by stereotaxic injection.

[0081] (5) Astrocyte transfection: One day before transfection, cells were plated in 500 μL of growth medium without antibiotics to achieve a confluency of 30% to 50% at the time of transfection. For each transfection sample, polyplus complexes were prepared as follows: 50 nmol of siRNA or DNA plasmid was dissolved in 200 μL jetPRIME buffer and gently mixed; 4 μL jetPRIME was added, gently mixed and incubated at room temperature for 10 minutes; the mixture was added to each well containing cells and serum-containing medium, and gently mixed by shaking the plate back and forth; the medium was replaced after 24 hours, and the cells were incubated at 37°C in a CO2 incubator for 72 hours.

[0082] (6) ROS measurement: ROS measurement was performed using the non-fluorescent cell-permeable compound 2'-7'-dichlorofluorescein diacetate (DCFH-DA) according to the manufacturer's instructions (Biyuntian). DCFH-DA is hydrolyzed by intracellular esterases to dichlorofluorescein (DCFH), which is trapped inside the cell; this non-fluorescent molecule is then oxidized to fluorescent dichlorofluorescein by the action of cellular oxidants; primary astrocytes treated under different conditions were washed twice with PBS 1× solution and loaded with 5 μM DCFH-DA diluted in serum-free medium at 37°C for 30 minutes; cells were washed three times with PBS, and fluorescence images were captured using a Nikon inverted epifluorescence microscope equipped with a 20× objective.

[0083] (7) CCK8 method for measuring cell survival rate: Collect cells in good growth state, inoculate them in 96-well plates (density 5000 / well), and treat them according to experimental groups, with 6 replicate wells in each group; wash with PBS twice and replace with new culture medium, then add 10 μL CCK8 solution to each well, incubate at 37℃ for 4 hours, set zero wells and control wells; adjust the microplate reader to 450 nm wavelength, and measure the absorbance value (OD value) of each well. Calculation: relative cell survival rate % = (OD value of experimental group) / (OD value of solvent group) × 100%, OD value of experimental group = OD value of experimental group - OD value of zero well, OD value of solvent group = OD value of solvent group - OD value of zero well.

[0084] (8) TTC staining and determination of cerebral infarction volume: Mice were stained with 2,3,5-triphenylterazolium chloride (TTC) and the percentage of cerebral infarction volume was determined one day after MCAO surgery. The brains of the experimental animals in each group were removed and placed in the groove of the brain mold. The front and back ends were removed. The coronal sections were cut from the front to the back with a thickness of 2 mm, and the sections were immersed in 2% TTC staining solution at 37°C in the dark for 10 minutes. The color changes were observed and the sections were turned over and immersed for another 10 minutes. The brain slices were arranged neatly in the front-to-back order of the brain tissue. The normal brain tissue was red, and the cerebral infarction area was not stained (white). After taking pictures and collecting images, the volume of the white area and the total volume of the brain tissue were measured using Image J image analysis software. The percentage of infarct volume was calculated according to the formula [(B1+B2+…+Bn)x2 / (A1+A2…+An)x2]×100%, where B is the volume of the infarct area (white) and A is the total volume of brain tissue.

[0085] (9) CO-IP steps: ① Sample preparation: Remove the culture medium and sample 1×10 5 150 μL of cells were washed twice with 1×PBS (pH7.4); cells were scraped off with a cell scraper and collected into a 1.5 mL EP tube. 5 Add cell lysis buffer at a ratio of 20-30 μL of cells and add protease inhibitors at the same time, mix well and place on ice for 10 minutes; centrifuge (4℃, 14000 g, 10 min), collect the supernatant and place on ice for later use.

[0086] ② Magnetic bead pretreatment: fully suspend the magnetic beads, take 25-50 μL of magnetic beads, place them in a 1.5 mL EP tube, add 400 μL of binding / washing buffer, fully suspend, place on a magnetic stand, perform magnetic separation, and discard the supernatant; repeat the above washing steps twice.

[0087] ③ Antibody binding to magnetic beads: Antibody Pretreatment: Dilute the antibody to a final concentration of 5-50 μg / mL in Binding / Wash Buffer.

[0088] Antibody-magnetic bead binding: Add 400 μL of diluted antibody to the magnetic beads treated in step ②, suspend thoroughly, incubate in a flip mixer (room temperature, 30 min; 4°C, 2 hours), magnetically separate, collect the magnetic beads, and collect the supernatant in a new EP tube for subsequent use.

[0089] Washing: Add 400 μL binding / washing buffer, fully suspend the magnetic beads, perform magnetic separation, and discard the supernatant; repeat washing 4 times.

[0090] ④ Binding of antigen and antibody-magnetic bead complex: Antigen-antibody-magnetic bead complex binding: Add 400 μL of the antigen sample prepared in step ①, suspend thoroughly, incubate in a flip mixer (room temperature, 30 min; 4°C, 2 hours), separate magnetically, and discard the supernatant.

[0091] Washing: Use 400 μL of binding / washing buffer to fully resuspend the magnetic beads, magnetically separate, and discard the supernatant; repeat washing 4 times.

[0092] ⑤ Antigen elution: Separate the magnetic beads, discard the supernatant, add 25-50 μL 1×SDS-PAGE Loading Buffer to the magnetic beads, mix well, and heat at 95℃ for 5 min. Separate the magnetic beads, collect the supernatant, and perform SDS-PAGE detection.

[0093] (11) Immunoblotting: Brain tissue and astrocytes were homogenized in RIPA lysis buffer (Beyotime) containing a mixture of protease and phosphatase inhibitors (Beyotime) and centrifuged at 14,000 × g for 30 min at 4°C; the supernatant was transferred to a fresh tube and the concentration was determined by a BCA kit (Beyotime). Equal amounts of protein were separated by 4-15% SDS-PAGE and electroblotted onto a PVDF membrane; after blocking, the membrane was incubated with the primary antibody overnight at 4°C. After incubation with an appropriate secondary antibody conjugated to horseradish peroxidase (anti-rabbit antibody or anti-mouse antibody, 1:1000-1:2000) at room temperature for 1 hour, the protein bands were observed using a fluorescent secondary antibody, developed using an Odyssey developer, and the density of the bands was measured using ImageJ; changes in target protein expression were normalized using control protein expression.

[0094] 2. Experimental results (1) ROS detection results: The ROS detection results of the astrocyte control group and the RNF14 overexpression group are as follows Figure 6 As shown in the OGD / R glial cell model, overexpression of RNF14 significantly decreased the ROS production in OGD / R glial cells.

[0095] (2) CCK8 detection results: In the OGD / R glial cell model, overexpression of RNF14 significantly increased the cell survival rate ( Figure 7 ).

[0096] (3) TTC staining results: TTC staining results are as follows Figure 8As shown, the brain tissue of mice turned white after MCAO surgery, while after overexpression of RNF14, the color of the brain tissue of mice after MCAO surgery returned to normal and turned red.

[0097] (4) Measurement results of cerebral infarction tissue volume: The measurement results of cerebral infarction volume are as follows: Fig. 9 As shown, in the mouse MCAO model, overexpression of RNF14 significantly reduced the cerebral infarct volume compared with the control group.

[0098] (5) Molecular docking results: Fig.10 As shown, molecular docking revealed that RNF14 interacts with Notch1 protein. The red area represents RNF14 and the green area represents Notch1.

[0099] (6) CO-IP results: Fig.11 As shown in A, RNF14 interacts with Notch1 protein in astrocytes and mouse brain tissue.

[0100] (7) Western blot results: Fig.11 As shown in B, Notch1 was downregulated when RNF14 was overexpressed after MCAO in mice.

[0101] The above are only implementation methods of the present application, and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1, characterized in that: The method comprises the following steps: Acquiring data 1: Acquiring the Notch1 expression level in brain tissue cells of patients with cerebral infarction; Acquiring data 2: Acquiring the Notch1 expression level of brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them; Processed data: Comparison of Notch1 expression levels in brain tissue cells of patients with cerebral infarction and Notch1 expression levels in brain tissue cells of patients with cerebral infarction after administration of RNF14 promoter; Output result: If the Notch1 expression level of brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them is lower than the Notch1 expression level of brain tissue cells in patients with cerebral infarction, then the result that the RNF14 promoter has a therapeutic effect on cerebral infarction is output.

2. The method according to claim 1, characterized in that The patients include humans and / or mammals; the brain tissue cells include neurons, astrocytes, microglia, and vascular epithelial cells.

3. A system for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction based on Notch1, characterized in that: The system comprises: Data acquisition unit: acquiring the Notch1 expression level of brain tissue cells in patients with cerebral infarction, and acquiring the Notch1 expression level of brain tissue cells in patients with cerebral infarction after the RNF14 promoter is administered to them; Data analysis unit: Compare the Notch1 expression level in brain tissue cells of patients with cerebral infarction with the Notch1 expression level in brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them; Result output unit: if the Notch1 expression level of brain tissue cells of patients with cerebral infarction after the RNF14 promoter is administered to them is lower than the Notch1 expression level of brain tissue cells in patients with cerebral infarction, then the result that the RNF14 promoter has a therapeutic effect on cerebral infarction is output.

4. A method for screening drugs for treating cerebral infarction based on Notch1 and RNF14, characterized in that: The method comprises the following steps: Obtaining a system containing a protein complex formed by Notch1 and RNF14; adding a candidate substance into the system; The interaction between Notch1 and RNF14 proteins in the protein complex is analyzed before and after the addition of the candidate substance. If the candidate substance can promote the interaction between Notch1 and RNF14 proteins in the complex, it indicates that the candidate substance is a potential drug for treating cerebral infarction.

5. The method according to claim 4, characterized in that The system includes a cerebral infarction cell system, a cerebral infarction tissue system, a cerebral infarction organ system or a cerebral infarction animal model system.

6. A system for screening drugs for treating cerebral infarction based on Notch1 and RNF14, characterized in that: The system comprises: Data acquisition unit: acquiring a system containing a protein complex formed by Notch1 and RNF14; Data analysis unit: adding the candidate substance to the system, and analyzing the interaction between Notch1 and RNF14 proteins in the protein complex before and after the addition of the candidate substance; Drug screening unit: If the candidate substance can promote the interaction between Notch1 and RNF14 proteins in the complex, it indicates that the candidate substance is a potential drug for treating cerebral infarction.

7. A computer device and a readable storage medium, characterized in that: The device comprises: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction described in claims 1-2 or the method for screening drugs for treating cerebral infarction described in claims 4-5 is implemented; The computer-readable storage medium stores a computer program, which, when executed by a processor, implements the method for evaluating the therapeutic effect of RNF14 promoter on cerebral infarction described in claims 1-2 or the method for screening drugs for treating cerebral infarction described in claims 4-5.

8. An isolated protein-complex comprising: Notch1, as well as RNF14 proteins, interact with each other.

9. A method for regulating Notch1 expression, characterized in that: RNF14 negatively regulates the expression of Notch1 in cerebral infarction tissue; by regulating the expression level of RNF14, the regulation of Notch1 expression in cerebral infarction tissue can be achieved.

10. The method according to claim 9, characterized in that Up-regulating RNF14 expression decreased Notch1 expression; inhibiting RNF14 expression increased Notch1 expression; Preferably, the upregulation of RNF14 expression is achieved by gene overexpression technology; Preferably, the gene overexpression vector includes a eukaryotic expression vector and a viral vector.

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