WO2024016944A1 - 预防和保护脑缺血疾病的标志物及阿司匹林衍生物在预防和保护脑缺血疾病中的应用 - Google Patents

预防和保护脑缺血疾病的标志物及阿司匹林衍生物在预防和保护脑缺血疾病中的应用 Download PDF

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WO2024016944A1
WO2024016944A1 PCT/CN2023/102415 CN2023102415W WO2024016944A1 WO 2024016944 A1 WO2024016944 A1 WO 2024016944A1 CN 2023102415 W CN2023102415 W CN 2023102415W WO 2024016944 A1 WO2024016944 A1 WO 2024016944A1
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camk2α
aspirin
acetylation
site
biomarker
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French (fr)
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王碧含
韩威
殷东敏
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华东师范大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11017—Ca2+/Calmodulin-dependent protein kinase (2.7.11.17)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event

Definitions

  • the invention belongs to the field of medical technology, reveals a new molecular target for aspirin to prevent stroke, clarifies a new mechanism of aspirin's protective effect on cerebral ischemia that does not rely on anticoagulation, and on this basis, develops a new aspirin Derivatives can be safer and more effective in preventing stroke.
  • aspirin often also brings side effects such as bleeding risk and gastric mucosal damage during use. It has been reported in the literature that aspirin can achieve anti-platelet aggregation by inhibiting the activity of cyclooxygenase 1 (COX-1). Therefore, there is an urgent need to explore a new mechanism of aspirin's neuroprotective effect that does not rely on anticoagulation, and to develop aspirin derivatives through this new mechanism, so that they can exert a protective effect on cerebral ischemia while reducing the side effects of bleeding, thereby providing new treatments for cardio-cerebral stroke. Preventive medication offers a better approach.
  • the present invention proposes a new molecular target for the neuroprotective effect of aspirin on stroke for the first time in the process of studying the neuroprotective effect of aspirin on stroke.
  • Aspirin exerts a neuroprotective effect on stroke model mice by acetylating calcium ion-calmodulin-dependent protein kinase 2 ⁇ (Ca2 + /calmodulin-dependent kinase 2 ⁇ , CaMK2 ⁇ ) in nerve cells.
  • Ca2 + /calmodulin-dependent kinase 2 ⁇ , CaMK2 ⁇ calcium ion-calmodulin-dependent protein kinase 2 ⁇
  • the present invention also proposes a safe and effective new strategy for preventing stroke and cerebral ischemic diseases: by modifying aspirin to reduce its anticoagulant activity, but retaining its acetylated CaMK2 ⁇ effect, it can better prevent cerebral ischemia. Neuroprotective effects in stroke while reducing hemorrhagic side effects.
  • the invention provides a safer and more effective way to prevent stroke and cerebral ischemic diseases.
  • the present invention proposes a new strategy to improve aspirin and increase its benefit-risk ratio, and is not limited to aspirin derivatives such as acetylsalicylate.
  • the chemical formula of aspirin of the present invention is C 9 H 8 O 4 , the molecular structural formula is CH 3 COOC 6 H 4 COOH, the relative molecular mass is 180.16, and the CAS number is 50-78-2.
  • the present invention proposes a biomarker, which is an acetylation site of calcium ion-calmodulin-dependent protein kinase 2 ⁇ (CaMK2 ⁇ ) in nerve cells.
  • a biomarker which is an acetylation site of calcium ion-calmodulin-dependent protein kinase 2 ⁇ (CaMK2 ⁇ ) in nerve cells.
  • the amino acid sequence of CaMK2 ⁇ is shown in SEQ ID NO.1 below:
  • the nucleotide sequence is as shown in SEQ ID NO.2:
  • the acetylation sites are K42 and K286.
  • the present invention also proposes the application of the biomarker in preparing drugs for preventing and/or treating stroke disease and cerebral ischemic disease.
  • the drug is a derivative obtained by esterification of aspirin, including aspirin derivative acetylsalicylate.
  • the chemical formula of aspirin derivative acetylsalicylate ethyl ester of the present invention is C 11 H 12 O 4 , the molecular weight is 208.21100, and the product number is 529-68-0.
  • the aspirin increases the acetylation level of CaMK2 ⁇ by acetylating the calcium ion-calmodulin-dependent protein kinase 2 ⁇ (CaMK2 ⁇ ) in nerve cells, and inhibits the kinase activity (autophosphorylation and priming of CaMK2 ⁇ ) of CaMK2 ⁇ . phosphorylation), thereby affecting the downstream proteins of CaMK2 ⁇ to exert corresponding effects and producing direct neuroprotective effects.
  • CaMK2 ⁇ calcium ion-calmodulin-dependent protein kinase 2 ⁇
  • the aspirin increases the acetylation level of the K42 site of CaMK2 ⁇ in the brain by about six times (the acetylation modification level of the K42 site of CaMK2 ⁇ continues to increase from about 0.5% under normal conditions and stabilizes at 3% ).
  • the derivative acetylsalicylate obtained after esterification of aspirin increases the acetylation of CaMK2 ⁇ by acetylating calcium ion-calmodulin-dependent protein kinase 2 ⁇ (CaMK2 ⁇ ) in nerve cells. level, inhibiting the kinase activity of CaMK2 ⁇ (autophosphorylation and substrate phosphorylation), thereby affecting the downstream proteins of CaMK2 ⁇ to exert corresponding effects and producing direct neuroprotective effects.
  • CaMK2 ⁇ calcium ion-calmodulin-dependent protein kinase 2 ⁇
  • the calcium ion-calmodulin-dependent protein kinase 2 ⁇ (CaMK2 ⁇ ) is an upstream protein of the calmodulin kinase family that can regulate the activity and functional expression of multiple types of proteins. Its activity and neuroprotective function Related.
  • CaMK2 ⁇ according to the present invention is composed of 28 similar homologous isomers, which are derived from 4 genes ( ⁇ , ⁇ , ⁇ and ⁇ ). ⁇ - and ⁇ -subunits are the main subtypes in the brain, forming A dodecameric holoenzyme composed of one or two subunit types.
  • the action site of aspirin that can acetylate calcium ion-calmodulin-dependent protein kinase 2 ⁇ is the K42 (Lys42) site and the K286 (Lys286) site.
  • the derivative obtained after esterification of aspirin can acetylate calcium ion-calmodulin-dependent protein kinase 2 ⁇ (CaMK2 ⁇ ).
  • the action site is the K42 (Lys42) site.
  • the Lys42 refers to the lysine at position 42 labeled by the Ballesteros-Weinstein numbering method in the CaMK2 ⁇ amino acid sequence.
  • the Lys286 refers to the lysine at position 286 labeled by the Ballesteros-Weinstein numbering method in the CaMK2 ⁇ amino acid sequence.
  • the acetylation of lysine 42 inhibits the activity of CaMK2 ⁇ by interfering with the binding to ATP rather than the binding of calcium ions.
  • the change in CaMK2 ⁇ activity is measured using a kinase kit.
  • the acetylation modification of the K42 site of CaMK2 ⁇ is enriched in the cytoplasm (acetylated protein accounts for 12% of the total protein), while the proportion of acetylation modification in synapses is low (acetylation Protein accounts for only 2% of total protein).
  • the calcium ion-calmodulin-dependent protein kinase 2 ⁇ (CaMK2 ⁇ ) can undergo autophosphorylation and phosphorylation of substrates, and its activity is related to neuroprotective function.
  • the acetylation of lysine 42 inhibits the kinase activity of CaMK2 ⁇ by interfering with the binding to ATP rather than the binding of calcium ions.
  • the K42 site is the ATP binding site of calmodulin kinase, and the acetylation modification of the K42 site significantly damages the binding between CaMK2 ⁇ protein kinase and ATP.
  • the acetylation modification of the lysine K42 site does not affect the binding between CaMK2 ⁇ and Ca 2+ /CaM.
  • the acetylation of the K42 site of CaMK2 ⁇ can inhibit its autophosphorylation and phosphorylation of the substrate, but the acetylation of the K258 site does not affect the autophosphorylation of CaMK2 ⁇ and the phosphorylation of the substrate. Phosphorylation.
  • the application includes the use of derivatives obtained by esterification of aspirin in the preparation of drugs for preventing and/or treating stroke diseases and recurrent cerebral ischemia, and in the preparation of drugs that cannot be used/cannot be used in large doses.
  • the aspirin derivative acetylsalicylate retains the effectiveness of aspirin in preventing cerebral ischemia, reduces the bleeding risk of aspirin, and improves the safety of medication for preventing cerebral ischemia.
  • the effectiveness of the aspirin derivative acetylsalicylate as a drug for preventing/treating recurrent cerebral ischemia is compared with aspirin.
  • the basis is that aspirin proposed by the present invention is effective in preventing cerebral ischemia.
  • the direct mechanism of neuroprotection in blood-related diseases is evaluated and judged based on their biochemical experimental indicators.
  • the safety of the aspirin derivative acetylsalicylate as a drug for the prevention/treatment of recurrent cerebral ischemia is compared with aspirin, based on the coagulation function test of experimental model animals.
  • the present invention also provides a modulator of a biomarker, which modulator includes an agonist, inhibitor or antagonist of the biomarker.
  • the present invention also proposes a drug/pharmaceutical composition comprising the biomarker or a compound that can also bind or react with the biomarker.
  • the medicine/pharmaceutical composition includes derivatives obtained by esterification of aspirin.
  • the drug/pharmaceutical composition includes aspirin derivative acetylsalicylate.
  • the present invention also proposes a detection reagent/kit, which contains active ingredients that can be used to detect the aforementioned biomarkers and their acetylation levels.
  • the present invention also proposes a polyclonal antibody that specifically recognizes acetylated CaMK2 ⁇ at the K42 site, which is a synthetic modified polypeptide (C-QEYYAK Ac IINTKK) selected from amino acids No. 37 to 48 coupled with KLH (Keyhole Limpet Hemocyanin, hemocyanin). protein), obtained after immunizing rabbits; the antibody can specifically recognize the CaMK2 ⁇ protein acetylated at the K42 site, but cannot recognize the wild-type non-acetylated CaMK2 ⁇ protein, nor can it recognize the CaMK2 ⁇ protein acetylated at other sites such as K258.
  • C-QEYYAK Ac IINTKK synthetic modified polypeptide
  • the present invention also proposes a method for preparing an acetylation-modified polyclonal antibody that specifically recognizes CaMK2 ⁇ , including the following steps:
  • C-QEYYAK Ac IINTKK For the specific acetylation modification of the K42 site of CaMK2 ⁇ , select amino acids 37 to 48 to synthesize a modified polypeptide (C-QEYYAK Ac IINTKK), couple it to KLH (Keyhole Limpet Hemocyanin, hemocyanin), and then immunize After the rabbit is sacrificed, the antibody is purified from the serum and the control peptide (C-QEYYAKIINTKK) is synthesized for purification and enzyme-linked immunosorbent assay (ELISA) detection;
  • C-QEYYAK Ac IINTKK modified polypeptide
  • KLH Keyhole Limpet Hemocyanin, hemocyanin
  • ELISA enzyme-linked immunosorbent assay
  • the present invention also proposes a method for preparing acetylated CaMK2 ⁇ protein, including a protein modified by site-directed acetylation at all K42 sites (His-K42 Ac -CaMK2 ⁇ ) and a protein modified by site-directed acetylation at K258 site (His-K258 Ac -CaMK2 ⁇ ). .
  • the present invention also proposes a method for preparing acetylated CaMK2 ⁇ protein, which specifically includes the following steps: in vitro purification of site-directed acetylated proteins (His-K42 AC -CaMK2 ⁇ , His-K258 AC -CaMK2 ⁇ ) plasmid list:
  • pCDF-AcKR3-tRNACUA primer design information point mutation primer design
  • E. coli purified protein (His-K42 AC -CaMK2 ⁇ , His-K258 AC -CaMK2 ⁇ ):
  • Transformation Take 50-100 ⁇ l of the recovered competent cells in a tube, add 5 ⁇ l of mutant plasmid, and place on ice for 20-30 minutes;
  • Heat shock Heat shock in a 42°C water bath for 50-90 seconds, then immediately put it back on ice and let it stand for 2 minutes; add 900 ⁇ l to 1000 ⁇ l of antibiotic-free LB liquid culture medium, 37°C, 200 rpm, shake and culture on a constant temperature shaker for 1 hour to make the bacteria normal Growth; in order to increase the mutant transformation efficiency, centrifuge at low speed for 1 minute at 3000 rpm at room temperature, aspirate 600 ⁇ l of the culture medium in a sterile environment, slowly pipette and resuspend the cells, and pipet about 100 ⁇ l to coat the plate;
  • Small shake Use a bacteria-picking needle to pick single clone colonies from the culture medium, place the colonies directly into 5 ml of liquid culture medium (containing antibiotics corresponding to the plasmid vector) for culture, and cultivate overnight in a 37°C incubator at 200 rpm.
  • Da Yao When Xiao Yao's culture medium becomes turbid, it means that the bacteria have been reproduced and cultured on a large scale. Then, all the bacterial liquid from Xiao Yao is directly transferred to 500ml liquid culture medium for culture. 37 degree oven, 200 rpm, the density of the shaken bacteria needs to control the OD value between 0.4 and 0.6.
  • Induced expression When the OD value of the bacterial solution reaches the range of 0.4 to 0.6, take it out, add 0.5 mmol/L IPTG to the ultra-clean bench to induce expression, and put it back into the oven to induce expression for 6 hours. Due to the purification of site-specific acetylated proteins, an additional 1mM acetylated lysine was added, and the deacetylase inhibitor NAM was added after half an hour of culture, and the culture was continued for 6 hours.
  • Lysed protein After inducing expression for 6 hours, take out the bacterial liquid, centrifuge, remove the supernatant, and keep the bottom bacteria. The cells were lysed with cell lysis solution on ice and lysed by ultrasonic lysis. Each ultrasonic process lasted 10 seconds, with an interval of 5 minutes between two times. After ultrasonic for about 5 to 10 times, observe whether the bacteria become permeable. If not, it may be that too much bacteria has been added and the cells are not fully lysed. The number of times of ultrasound can be increased appropriately.
  • Purified protein Take out the lysed bacterial cells on ice, centrifuge at 12000g ⁇ 15min, retain the supernatant, and remove the precipitate in the lower layer.
  • the present invention also provides the modulator of the biomarker, the drug/pharmaceutical composition, the detection reagent/kit, or the antibody/protein for use in prediction, evaluation, prevention and/or Or its application in the treatment of stroke disease, cerebral ischemic disease, recurrent cerebral ischemic disease, its application in the preparation of drugs for patients who are unable to use/cannot use high-dose aspirin but are at risk of cerebral ischemia, or in the preparation of drugs Use of Aspirin in Alternative Therapeutics.
  • the regulator, drug/pharmaceutical composition, and detection reagent/kit can be used alone or in combination with other drugs.
  • the present invention also provides a drug modification idea for preventing/treating cerebral ischemic disease.
  • the acetylation of lysine at position 42 of CaMK2 ⁇ can be used as a new target for preventing/treating cerebral ischemic disease.
  • the present invention also provides the mechanism of action of the drug or drug combination in preventing/treating cerebral ischemic disease, and the measurement and evaluation of safety and effectiveness.
  • the present invention also provides a method for screening candidate drugs for preventing/treating cerebral ischemic diseases and cerebral ischemic diseases.
  • the method includes detecting the effect of the candidate drugs on the biomarkers described in the subject or the sample obtained from the subject.
  • the impact of the biological effect includes detecting the impact of the candidate drug on the acetylation level of lysine 42 of CaMK2 ⁇ in the subject or a sample obtained from the subject or the kinase activity of CaMK2 ⁇ or the biological effect downstream of its signal.
  • the present invention also provides a method for preventing or treating stroke disease and cerebral ischemia-related diseases in a subject in need, or whether the derivatives obtained by esterification of aspirin can be used to prevent/treat cerebral ischemia in the patient. Methods for prediction or prognosis assessment of stroke and cerebral ischemia-related diseases.
  • the method includes detecting the acetylation level of lysine 42 of CaMK2 ⁇ or the kinase activity of CaMK2 ⁇ as the aforementioned biomarker.
  • the aspirin derivative acetylsalicylate can reduce the risk of bleeding compared to aspirin.
  • the coagulation test detected and compared the coagulation time of mice fed aspirin and acetylsalicylate for 14 days, and found that aspirin increased the total coagulation time. , while acetylsalicylate significantly reduces clotting time.
  • the aspirin derivative acetylsalicylate maintains the ability to acetylate lysine 42 of the CaMK2 ⁇ protein, and compared with aspirin, acetylsalicylate can better increase the acetylation level of CaMK2 ⁇ .
  • the innovative point of the present invention is to propose a new mechanism by which aspirin exerts a neuroprotective effect on stroke that is independent of its anticoagulant function, and proves that aspirin derivative acetylsalicylate is more effective in preventing and protecting brain defects than aspirin. It has a better benefit-risk ratio in blood diseases, that is to say, the aspirin derivative acetylsalicylate has a smaller bleeding risk than aspirin, and its neuroprotective effect is equivalent to that of aspirin.
  • the beneficial effect of the present invention is that in the process of in-depth research on the neuroprotective effect of aspirin on stroke, the present invention proposes for the first time a new molecular target for the neuroprotective effect of aspirin on stroke.
  • Aspirin exerts neuroprotective effects on stroke model mice by acetylating calcium ions-calmodulin-dependent protein kinase 2 ⁇ in nerve cells. This finding elucidates a novel mechanism by which aspirin exerts its neuroprotective effects independent of its anticoagulant function.
  • the present invention can better exert its neuroprotective effect in preventing stroke while reducing the side effects of bleeding.
  • the invention provides a safer and more effective way for preventive medication of cerebral ischemic diseases, and has broad application prospects in the field of prevention/treatment of cerebral ischemic diseases.
  • Figure 1 shows the protein spectrum identifying acetylated proteins and lysine sites in the mouse cerebral cortex that are regulated by aspirin. Aspirin can directly increase the acetylation level of Camk2 ⁇ in a dose-dependent manner, and K42 and K258 are the main acetylation sites.
  • Figure 2 Preparation and verification of a rabbit polyclonal antibody that specifically recognizes the acetylation modification of lysine 42 of CaMK2 ⁇ .
  • FIG. 4 Acetylation of the K42 site inhibits CaMK2 ⁇ kinase activity by interfering with the binding to ATP rather than interfering with the binding to Ca 2+ /CaM.
  • Figure 7 The bleeding risk of acetylsalicylate is significantly lower than that of aspirin, and its neuroprotective effect is similar to that of aspirin.
  • mice 8-12 weeks old. All mice had free access to water and food, and were maintained in a 12h:12h light:dark constant temperature and humidity environment.
  • MCAO animal model construction The experimental process of MCAO animal model construction refers to the Longa modified suture method to establish a mouse focal cerebral ischemia model. This method has been recognized by the global academic community. Before surgery, mice were housed on a 12:12 light/dark cycle with free access to water and rodent chow. Surgical procedure: After weighing the mice, induce anesthesia with 4% isoflurane in the induction chamber, using 1.5-2% isoflurane in a mixed gas of 30% O2 and 70% N2 through a mask during the process Maintain anesthesia. Place the mouse in a prone position on a gauze pad placed on a temperature-controlled operating surface (heat blanket); The hair on the head was shaved.
  • lubricant eye ointment is applied to both eyes and the eyes are closed to prevent them from drying out.
  • the mouse's neck is placed on a soft pad and its nose is secured in the nose cone of the anesthesia device. Fix the mouse in a supine position, disinfect the neck skin with 75% alcohol, make a 1cm incision slightly to the right of the middle of the neck, bluntly separate the subcutaneous muscles and tissues layer by layer, separate the common carotid artery, internal carotid artery and external carotid artery, and ligate the common carotid artery Between the artery and the external carotid artery, make a small hole in the common carotid artery 1.5cm away from the bifurcation, and slowly insert the suture plug with the head end covered with silicone into the internal carotid artery until it encounters a slight resistance.
  • the insertion depth is about 9 to 10 mm (from the bifurcation of the inner and outer neck), causing obstruction of the right middle cerebral artery and cerebral ischemia. Fix the suture bolts and suture the muscles and skin layer by layer. After 1 hour of ischemia, the tethered sutures were pulled out and cerebral ischemia-reperfusion was performed. Finally the mice were removed from the surgical sheath and placed in a warm cage to prevent hypothermia and with ample, easily accessible soft food and water.
  • ELASA chemical titration was used to quantify the acetylation of CaMK2 ⁇ in mouse brain.
  • purified His-K42 Ac -CaMK2 ⁇ and normal His-CaMK2 ⁇ protein were mixed at different percentages (0.5%, 1%, 2%, 4%, 8%), and the CaMK2 ⁇ antibody was used to bind the protein in a 96-well plate.
  • CaMK2 ⁇ itself is one of the most abundant proteins in the brain, accounting for about one percent of the protein in the entire cerebral cortex, it is easier to calculate the overall protein amount in each well and ensure that the amount of protein loaded in each well is consistent.
  • the present invention selects amino acids No. 37 to 48 to synthesize a modified polypeptide (C-QEYYAK Ac IINTKK). After coupling to KLH, it is immunized into rabbits and the rabbits are sacrificed. , serum purified antibodies, and synthesized the control peptide (C-QEYYAKIINTKK) at the same time for purification and enzyme-linked immunosorbent assay (ELISA) detection;
  • a single plasmid vector capable of expressing aaRSs and tRNACUA orthogonal pair simultaneously was constructed and optimized: the aaRSs/tRNACUA orthogonal pair selected was the AcKR3/tRNACUA orthogonal pair because it has been proven to be able to express in mammalian cell lines. , effectively read through the amber codon UAG in the mouse forebrain, allowing the unnatural amino acid acetyl-lysine to be integrated into the target protein;
  • tRNACUA Places behind the U6 promoter and construct four copies of the U6-tRNACUA expression component. In order to increase the expression of tRNACUA, it is usually placed downstream of the U6 promoter to initiate the transcription of tRNACUA. In addition, increasing the copy number of tRNACUA can also increase the coding efficiency of the gene.
  • EF1a promoter was selected to initiate the transcription and expression of AcKR3.
  • EF1a is a strong eukaryotic promoter and can exist stably in mammalian cells and is not easily silenced.
  • EGFPamb reporter plasmid and four copies of tRNACUA's RS (tRNACUA) 4X expression plasmid were co-transfected into HEK293T cells, and the cells were cultured in media containing and without 1mM acetyl-lysine. If EGFPamb's RS (tRNACUA) 4X expression plasmid If the amb codon at position 39 is read through, full-length EGFP can be expressed, and green fluorescence can be seen under a fluorescence microscope, indicating that acetyl-lysine is integrated into the EGFP fluorescent protein;
  • COX-1 activity detection was performed using a commercially available kit (Abcam, Cat No. ab204699).
  • the sample reacts to produce prostaglandin F (PGF), and the fluorescence intensity emission is detected through fluorescence dynamics in a microplate reader.
  • PPF prostaglandin F
  • the K42 site is the ATP binding site of calmodulin kinase.
  • acetylation modification of the K42 site significantly damaged the binding of CaMK2 ⁇ protein kinase and ATP, as shown in the figure As shown in 4;
  • K42 was found to be expressed in the mouse forebrain.
  • the CaMK2 ⁇ protein modified by site-specific acetylation can play a direct neuroprotective effect in the mouse stroke model. That is, the acetylation modification of the K42 position of the CaMK2 ⁇ protein can fully exert the neuroprotective effect, as shown in the figure. 6 shown.
  • mice 15 Detect the bleeding time of mice after drinking normal water, water containing aspirin or acetylsalicylate (0.1mg/ml) for 14 days.
  • the bleeding time of mice drinking acetylsalicylate was significantly shorter than that of mice drinking the same dose of aspirin, as shown in Figure 7;
  • acetylsalicylate reduced the mortality rate 24 hours after stroke in mice, improved cognitive function, and reduced infarct size. It has a neuroprotective effect similar to the neuroprotective effect of aspirin. , as shown in Figure 7;
  • liver and kidney functions of mice after drinking normal water, water containing aspirin or acetylsalicylate (0.1mg/ml) for 14 days.
  • the liver and kidney function indicators of mice drinking acetylsalicylate were basically the same as those of control mice, and the indicators of alanine aminotransferase (ALT) and uric acid (UA) were better than those of aspirin, as shown in Figure 8.
  • the protection content of the present invention is not limited to the above aspirin derivatives. Any idea of reducing the inhibitory effect on COX-1 and the risk of bleeding by modifying the carboxyl group of aspirin while retaining the acetyl group of aspirin and the ability to acetylate calcium ion signaling proteins to exert a neuroprotective effect falls within the scope of the present invention. Without departing from the spirit and scope of the concept of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and are protected by the appended claims.

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Abstract

本发明公开了阿司匹林对脑卒中发挥神经保护作用的新的分子靶点。阿司匹林通过乙酰化神经细胞内的钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)发挥对脑卒中模型小鼠的神经保护作用。本发明阐明了阿司匹林发挥神经保护作用不依赖其抗凝血功能的一种新机制。本发明还公开阿司匹林衍生物也可以通过乙酰化CaMK2α对脑卒中模型小鼠发挥神经保护作用，但其抗凝血作用和出血风险要弱于阿司匹林。本发明还公开了一种安全有效的预防脑卒中的新策略：通过对阿司匹林进行改造，降低其抗凝血活性，但保留其乙酰化CaMK2α的作用，可以更好发挥预防脑卒中的神经保护作用，且降低出血的副作用。本发明给脑缺血疾病的预防性用药提供一种更加安全有效的途径。

Description

预防和保护脑缺血疾病的标志物及阿司匹林衍生物在预防和保护脑缺血疾病中的应用 技术领域
本发明属于医药技术领域，揭示了阿司匹林预防脑卒中的新的分子靶点，阐明了阿司匹林脑缺血保护作用的不依赖抗凝血的新机制，并在此基础之上，开发出新的阿司匹林衍生物，可以更加安全有效的预防脑卒中。
背景技术
全世界每4个人中就有1个人会发生脑卒中(俗称脑中风或脑缺血)，每6秒钟就有1个人死于卒中。脑卒中因其高患病率、高复发率、高致死和致残率，已经成为威胁人类生命健康和生活质量的“头号杀手”。根据我国心血管病中心发布的《中国心血管病健康和疾病报告2019》，我国心血管病患者人数高达3.30亿，其中脑卒中患者1300万例。世界卫生组织指出，每日口服阿司匹林可以通过抗凝血作用有效预防脑卒中的发生。阿司匹林治疗对脑卒中风险人群(如有脑中风病史的人群)很有帮助。但是阿司匹林在使用的过程中往往也会带来出血风险和胃黏膜损伤等副作用。已有文献报导，阿司匹林可以通过抑制环氧合酶1(COX-1)的活性从而达到抗血小板聚集的作用。因此亟需探究阿司匹林神经保护作用的不依赖抗凝血的新机制，并通过此新机制开发阿司匹林衍生物，使其发挥脑缺血保护作用的同时，降低出血的副作用，从而为心脑卒中的预防性用药提供一种更好的途径。
发明内容
为了克服现有技术存在的问题，本发明首次在研究阿司匹林对脑卒中发挥神经保护作用的过程中，首次提出了阿司匹林对脑卒中发挥神经保护作用的新的分子靶点。阿司匹林通过乙酰化神经细胞内的钙离子-钙调蛋白依赖的蛋白激酶2α(Ca2+/calmodulin-dependent kinase 2α,CaMK2α)发挥对脑卒中模型小鼠的神经保护作用。本发明首次阐明了阿司匹林发挥神经保护作用不依赖其抗凝血功能的一种新机制。
本发明还提出了一种安全有效的预防脑卒中、脑缺血疾病的新策略：通过对阿司匹林进行改造，降低其抗凝血活性，但保留其乙酰化CaMK2α的作用，可以更好发挥预防脑卒中的神经保护作用，同时降低出血的副作用。本发明给脑卒中、脑缺血疾病的预防性用药提供一种更加安全有效的途径。本发明提出改进阿司匹林提高其收益-风险比的新策略，并不局限于乙酰水杨酸乙酯这一种阿司匹林衍生物。其它阿司匹林衍生物只要符合下述两点要求：1)减弱与COX-1的结合力和降低抗凝功能；2)保留乙酰化脑内CaMK2α蛋白的能力；均可以起到类似乙酰水杨酸乙酯的作用。
本发明所述阿司匹林的化学式为C9H8O4，分子结构式为CH3COOC6H4COOH，分子相对质量为180.16，CAS号为50-78-2。
本发明提出了一种生物标志物，所述生物标志物为神经细胞内的钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)的乙酰化位点。
所述CaMK2α的氨基酸序列如下SEQ ID NO.1所示：
核苷酸序列如下SEQ ID NO.2所示：


所述乙酰化位点为K42、K286。
本发明还提出了所述的生物标志物在制备预防和/或治疗脑卒中疾病、脑缺血疾病的药物中的应用。
本发明所述应用中，所述药物为阿司匹林经酯化后得到的衍生物,包括阿司匹林衍生物乙酰水杨酸乙酯。
本发明所述阿司匹林衍生物乙酰水杨酸乙酯化学式为C11H12O4，分子量为208.21100，货号为529-68-0。
本发明所述应用中，所述阿司匹林通过乙酰化神经细胞内钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)，提高CaMK2α的乙酰化水平，抑制CaMK2α的激酶活性(自我磷酸化以及对底物磷酸化)，从而影响CaMK2α下游蛋白发挥相应的效应，产生直接的神经保护作用。
本发明所述应用中，所述阿司匹林提高脑内CaMK2α的K42位点乙酰化水平约六倍左右(CaMK2α的K42位点乙酰化修饰水平从正常情况的0.5％左右不断升高并稳定在3％)。
本发明所述应用中，所述阿司匹林经酯化后得到的衍生物乙酰水杨酸乙酯通过乙酰化神经细胞内钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)，提高CaMK2α的乙酰化水平，抑制CaMK2α的激酶活性(自我磷酸化以及对底物磷酸化)，从而影响CaMK2α下游蛋白发挥相应的效应，产生直接的神经保护作用。
本发明所述应用中，所述钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)是钙调蛋白激酶家族，能够调节多种类型蛋白活性与功能表达的上游蛋白，其活性与神经保护功能相关。
本发明所述的CaMK2α由28个相似的同源异构体组成，它们来自4个基因(α，β，γ和δ)，α-和β-亚基是大脑中主要的亚型，形成由一种或两种亚基类型组成的十二聚全酶。
本发明所述应用中，所述阿司匹林能够乙酰化钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)的作用部位为K42(Lys42)位点和K286(Lys286)位点。
本发明所述应用中，所述阿司匹林经酯化后得到的衍生物,能够乙酰化钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)的作用部位为K42(Lys42)位点。
所述Lys42指CaMK2α氨基酸序列中用Ballesteros-Weinstein numbering法标记的第42位赖氨酸。
所述Lys286指CaMK2α氨基酸序列中用Ballesteros-Weinstein numbering法标记的第286位赖氨酸。
本发明所述应用中，所述赖氨酸42号位点的乙酰化是通过干扰了和ATP的结合而不是钙离子结合抑制了CaMK2α活性。
本发明所述应用中，所述CaMK2α活性的变化的测定采用激酶试剂盒。
本发明所述应用中，所述CaMK2α的K42位点乙酰化修饰富集在胞浆中(乙酰化蛋白占总蛋白比例达12％)，而突触中乙酰化修饰的比例较低(乙酰化蛋白占总蛋白比例只有2％)。
本发明所述应用中，所述钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)能够发生自我磷酸化和对底物的磷酸化，其活性与神经保护功能相关。
本发明所述应用中，所述赖氨酸42号位点的乙酰化是通过干扰了和ATP的结合而不是钙离子结合抑制了CaMK2α的激酶活性。
本发明所述应用中，所述K42号位点是钙调蛋白激酶的ATP结合位点，K42位点的乙酰化修饰显著损伤了CaMK2α蛋白激酶和ATP的结合。
本发明所述应用中，所述赖氨酸K42位点乙酰化修饰的发生并不影响CaMK2α和Ca2+/CaM之间的结合。
本发明所述应用中，所述CaMK2α的K42位点的乙酰化可以抑制其自身磷酸化和对底物的磷酸化，但是K258位点的乙酰化不影响CaMK2α的自我磷酸化和对底物的磷酸化。
本发明所述应用中，所述应用包括阿司匹林经酯化后得到的衍生物在制备预防和/或治疗脑卒中疾病、复发性脑缺血药物中的应用，在制备针对无法使用/无法大剂量使用阿司匹林但同时存在脑缺血风险的患者的药物中的应用，或在制备阿司匹林的替代治疗药物中的应用。
所述阿司匹林衍生物乙酰水杨酸乙酯保留了阿司匹林在预防脑缺血上的效用，降低阿司匹林的出血风险，提高了脑缺血预防用药的安全性。
本发明所述应用中，所述阿司匹林衍生物乙酰水杨酸乙酯作为制备预防/治疗复发性脑缺血药物的有效性采用与阿司匹林对照的方法，依据为本发明提出的阿司匹林在预防脑缺血相关疾病上发挥神经保护作用的直接机制，根据二者的生化学实验指标进行评估判断。
本发明所述应用中，所述阿司匹林衍生物乙酰水杨酸乙酯作为制备预防/治疗复发性脑缺血药物的安全性采用与阿司匹林对照的方法，依据为实验模型动物凝血功能测试。
本发明还提出了一种生物标志物的调节剂，所述调节剂包括所述的生物标志物的激动剂、抑制剂或拮抗剂。
本发明还提出了一种药物/药物组合物，其包含所述的生物标志物，或包含可以也与所述生物标志物结合或反应的化合物。
其中，所述药物/药物组合物包括阿司匹林经酯化后得到的衍生物。
进一步地，所述药物/药物组合物包括司匹林衍生物乙酰水杨酸乙酯。
本发明还提出了一种检测试剂/试剂盒，其包含可用于检测如前所述的生物标志物及其乙酰化水平的有效成分。
本发明还提出了一种特异性识别K42位点乙酰化CaMK2α的多克隆抗体，其为选取第37～48号氨基酸合成修饰多肽(C-QEYYAKAcIINTKK)偶联KLH(Keyhole Limpet Hemocyanin，血蓝蛋白)，免疫兔子后获得；所述抗体能够特异性识别K42位点乙酰化的CaMK2α蛋白，不能识别野生型的非乙酰化的CaMK2α蛋白，也不能识别K258等其他位点发生乙酰化CaMK2α蛋白。
本发明还提出了一种特异性识别CaMK2α的乙酰化修饰的多克隆抗体的制备方法，包括以下步骤：
(1)针对CaMK2α的K42位点特异性的乙酰化修饰，选取第37～48号氨基酸合成修饰多肽(C-QEYYAKAcIINTKK)，偶联到KLH(Keyhole Limpet Hemocyanin，血蓝蛋白)后，免疫到兔子体内，牺牲掉兔子后，血清纯化抗体，同时合成对照肽(C-QEYYAKIINTKK)，进行纯化和酶联免疫吸附实验(ELISA)检测；
(2)利用非天然编码乙酰化赖氨酸技术直接体外合成并纯化了全K42位点定点乙酰化修饰的蛋白(His-K42Ac-CaMK2α)和K258位点定点乙酰化修饰的蛋白(His-K258Ac-CaMK2α)；
(3)通过考马斯亮蓝染色检测了蛋白质的纯度，然后通过利用的纯化的定点乙酰化化蛋白检测抗体在体外的特异性，通过不断优化条件和对抗体进行多次纯化。
本发明还提出了制备乙酰化CaMK2α蛋白的方法，包括全K42位点定点乙酰化修饰的蛋白(His-K42Ac-CaMK2α)和K258位点定点乙酰化修饰的蛋白(His-K258Ac-CaMK2α)。
本发明还提出了制备乙酰化CaMK2α蛋白的方法，具体包括以下步骤：体外纯化定点乙酰化蛋白(His-K42AC-CaMK2α、His-K258AC-CaMK2α)质粒列表：
pET-N-His-CaMK2α
pET-N-His-K42TAG-CaMK2αpET-N-His-K258TAG-CaMK2α
pCDF-AcKR3-tRNACUA引物设计信息：点突变引物设计
大肠杆菌纯化蛋白(His-K42AC-CaMK2α、His-K258AC-CaMK2α)：
(1)转化大肠杆菌感受态细胞
复苏：从-80℃冰箱中迅速取出(表达型感受态细胞)BL21，为了使细胞复苏在冰上放置10min；
转化：在一管复苏后的感受态细胞中取50-100μl加入5μl突变质粒，在冰上放置20-30min；
热击：42℃水浴锅中热击50-90s，然后立即放回冰上静置2min；加入900μl～1000μl无抗生素LB液体培养基，37℃，200rpm，恒温摇床振荡培养1h，使细菌正常生长；为增加突变体转化效率，常温下3000rpm低速离心1min，无菌环境下吸除600μl培养基，缓慢吹打重悬菌体，吸取100μl左右涂板；
将菌液涂布于含相应抗性的LB固体培养基平板上，37℃恒温培养箱倒置培养过夜。
(2)转化大肠杆菌感受态细胞
小摇：从培养基中用挑菌针头挑单克隆菌落，将菌落直接放入含有5ml液体培养基(含有质粒载体对应的抗生素)中进行培养，37℃培养箱200转过夜培养。
大摇：当小摇的培养基变浑浊时，说明菌体已经进行了大规模的繁殖和培养，然后将小摇的菌液，全部直接转入500ml的液体培养基中进行培养。37度烘箱，200转，大摇的菌体的密度需要控制OD值处在0.4～0.6之间。
诱导表达：当大摇的菌液OD值达到0.4～0.6的区间后取出，在超净台中加入0.5mmol/L的IPTG诱导表达，重新放入烘箱中诱导表达6h。由于纯化定点乙酰化蛋白，则额外加入1mM的乙酰化赖氨酸，并在培养半小时后加入去乙酰化酶的抑制剂NAM，持续培养6h。
裂解蛋白：诱导表达6h后，将菌液取出，离心，去上清，保留底部菌体。将菌体用菌体裂解液放冰上裂解，通过超声裂解，每次超声过程持续10s时间，两次之间间隔5min。超声大约5到10次后，观察菌体是否变得通透，如果没有通透则可能菌体加入的量过多，没有裂解充分，可适当增加超声次数。
纯化蛋白：冰上裂解的菌体取出，离心12000g×15min后，保留上清，去除下层的沉淀。
通过BCA法测定蛋白质浓度，每管取5～10mg蛋白的菌体裂解液，用PBS稀释至1ml，然后每管中加入100ul的His-beads磁珠，震荡混悬后，放入旋转混悬仪上进行4℃过夜混悬。次日，取出混悬的管子，离心1500g×3min后去上清保留下层磁珠，使用PBS进行洗脱，连续1500g×3min洗脱6次。洗脱后，每管磁珠中加入200ul的蛋白洗脱液吹打混悬60s，再放入离心机10000g×10min离心，保留上层洗脱液，去掉下层磁珠。通过分光度计进行蛋白质浓度测定。分装后放入-80℃冰箱保存。
本发明还提出了所述的生物标志物的调节剂、所述的药物/药物组合物、所述的检测试剂/试剂盒、或所述的抗体/蛋白在用于预测、评估、预防和/或治疗脑卒中疾病、脑缺血疾病、复发性脑缺血疾病中的应用，在制备针对无法使用/无法大剂量使用阿司匹林但同时存在脑缺血风险的患者的药物中的应用，或在制备阿司匹林的替代治疗药物中的应用。
本发明所述应用中，所述调节剂、药物/药物组合物、检测试剂/试剂盒可以单独使用，也可以与其他药物联合使用。
本发明还提供了一种用于预防/治疗脑缺血疾病的药物改造思路，CaMK2α的第42位赖氨酸的乙酰化可作为脑缺血疾病的预防/治疗的一种新靶标。
本发明还提供了所述药物或药物组合在预防/治疗脑缺血疾病的作用机制，和安全有效性的测量评估。
本发明还提供了一种筛选预防/治疗脑缺血疾病、脑缺血疾病的候选药物的方法，所述方法包括检测候选药物对对象或获自对象的样本中所述的生物标志物的生物学效应的影响，包括检测候选药物对对象或获自对象的样本中CaMK2α的第42位赖氨酸乙酰化水平或者CaMK2α的激酶活性或其信号下游的生物学效应的影响。
本发明还提供了一种对有需要的受试者预防或治疗脑卒中疾病、脑缺血相关疾病的方法，或对所述患者是否适用阿司匹林经酯化后得到的衍生物来预防/治疗脑卒中疾病、脑缺血相关疾病进行预测或预后评估的方法。
所述方法包括检测如前所述的生物标志物的CaMK2α的第42位赖氨酸的乙酰化水平或者CaMK2α的激酶活性。
所述阿司匹林衍生物乙酰水杨酸乙酯能够相对于阿司匹林降低出血风险上的作用，凝血实验检测并对比喂食阿司匹林和乙酰水杨酸乙酯14天后的老鼠凝血时间，发现阿司匹林增加了总凝血时间，而乙酰水杨酸乙酯明显使凝血时间减少。
所述阿司匹林衍生物乙酰水杨酸乙酯保持了乙酰化CaMK2α蛋白第42位赖氨酸的能力，并且和阿司匹林相比，乙酰水杨酸乙酯能更好地提高CaMK2α的乙酰化水平。
阿司匹林和阿司匹林衍生物乙酰水杨酸乙酯都能不依赖其抗凝血功能而发挥神经保护作用。
本发明的创新点在于提出了阿司匹林对脑卒中发挥神经保护作用的不依赖于其抗凝血功能的新机制，而且证明阿司匹林衍生物乙酰水杨酸乙酯相比于阿司匹林在预防和保护脑缺血疾病上具有更好的收益-风险比，也就是说，阿司匹林衍生物乙酰水杨酸乙酯的出血风险比阿司匹林小，神经保护作用和阿司匹林相当。
本发明的有益效果在于：本发明在深入研究阿司匹林对脑卒中发挥神经保护作用的过程中，首次提出了阿司匹林对脑卒中发挥神经保护作用的新的分子靶点。阿司匹林通过乙酰化神经细胞内的钙离子-钙调蛋白依赖的蛋白激酶2α发挥对脑卒中模型小鼠的神经保护作用。这一发现阐明了阿司匹林发挥神经保护作用不依赖其抗凝血功能的一种新机制。本发明通过对阿司匹林进行改造，降低其抗凝血活性，但保留其乙酰化CaMK2α的作用，可以更好发挥预防脑卒中的神经保护作用，同时降低出血的副作用。本发明给脑缺血疾病的预防性用药提供一种更加安全有效的途径，在脑缺血疾病的预防/治疗领域具有广泛的应用前景。
附图说明
图1为蛋白质谱鉴定出受阿司匹林调控的小鼠大脑皮层乙酰化蛋白质和赖氨酸位点。阿司匹林可以剂量依赖性直接提高Camk2α的乙酰化水平，且K42和K258是主要的乙酰化位点。
图2制备并验证特异性识别CaMK2α第42位赖氨酸乙酰化修饰的兔多克隆抗体。
图3小鼠脑内蛋白质乙酰化修饰发生的定量。
图4K42位点的乙酰化是通过干扰了和ATP的结合而不是干扰与Ca2+/CaM结合来抑制CaMK2α激酶活性。
图5干扰CaMK2α蛋白K42位点的乙酰化削弱阿司匹林对缺血型脑卒中小鼠模型的保护作用。
图6在真核系统中表达K42位点乙酰化CaMK2α，并且小鼠脑内表达CaMK2α的K42位点乙酰化蛋白具有直接的神经保护作用。
图7乙酰水杨酸乙酯的出血风险显著低于阿司匹林，神经保护作用和阿司匹林类似。
图8长期引用含乙酰水杨酸乙酯的水，不损害小鼠的肝肾功能。
具体实施方式
结合以下具体实施例和附图，对本发明作进一步的详细说明。实施本发明的过程、条件、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。
实施例1
(1)体内实验
1)实验动物：清洁级雄性野生型小鼠(C57BL/6)，72只，8-12周龄；Camk2α-K42R品系的C57BL/6的小鼠，6只，8-12周龄；Camk2α-knock out品系的C57BL/6的小鼠，6只，
8-12周龄。所有小鼠自由饮水饮食，并维持在12h：12h光：暗的恒温恒湿环境中。小鼠分组:①对照组：清洁级雄性野生型小鼠饲喂超纯水；②阿司匹林组：清洁级雄性野生型小鼠饲喂含有一定浓度阿司匹林的水(Asp，0.1mg/mL)；③乙酰水杨酸乙酯组：清洁级雄性野生型小鼠饲喂含有一定浓度乙酰水杨酸乙酯组的水(0.1mg/mL)；
2)MCAO动物模型构建：MCAO动物模型构建实验流程参考Longa改良线栓法建立小鼠局灶性脑缺血模型，该方法已得到全球学术界公认。手术前，小鼠被安排在12:12光照/黑暗循环的环境中，可以自由获得水和啮齿动物食物。手术过程：称量小鼠体重后，在诱导室中用4％异氟醚诱导麻醉，在过程中用1.5-2％异氟醚在30％O2和70％N2的混合气体中通过面罩维持麻醉。将小鼠以俯卧的姿势放置在置于温控操作表面(热毯)上的纱布垫上； 头上的毛被剃光。在手术过程中，双眼涂抹润滑剂眼膏，闭眼以防止眼睛干燥。小鼠的脖子被放在一个软垫上，它的鼻子被固定在麻醉装置的鼻锥中。将小鼠仰卧位固定，75％酒精消毒颈部皮肤，于颈正中稍偏右1cm切口，逐层钝性分离皮下肌肉及组织，分离颈总动脉、颈内动脉和颈外动脉，结扎颈总动脉与颈外动脉，在颈总动脉距分叉处1.5cm处开1个小孔，将头端包有硅胶的线栓缓慢插入颈内动脉至稍遇阻力时停止。插入深度约为9～10mm(距颈内与颈外分叉处)，造成大脑右侧中动脉阻塞导致脑缺血。固定线栓，逐层缝合肌肉和皮肤。缺血1h后，将拴线拔出，进行脑缺血再灌注。最后小鼠被从手术套中取出，放在一个温暖的笼子里，以防止体温过低，并有足量的、容易获得的软食物和水。
3)给药：定期更换小鼠饮水瓶中的含有药物的水，防止其中的药物发生降解；
4)样本收集：根据不同实验处理结束后，将小鼠头部剪下，剪开头部皮肤并清理组织暴露颅骨；从后脑往前揭开头皮骨，暴露完整的脑，用精细弯镊小心托出全脑，将其至于生理盐水中冲洗干净，于-80℃冰箱保存，用于蛋白表达和免疫染色检测。
5)Western-blot法检测小鼠脑组织中Camk2α、Camk2α-K42等蛋白的表达。
6)ELASA化学滴定进行小鼠脑内CaMK2α的乙酰化定量。首先使用纯化的His-K42Ac-CaMK2α和正常的His-CaMK2α蛋白在进行不同百分比(0.5％，1％，2％，4％，8％)的混合，在96孔板中通过CaMK2α抗体结合蛋白，然后通过Anti-K42Ac-CaMK2α检测，上机读取OD450数值，绘制乙酰化比例和OD450的化学标准曲线y＝0.056x。因为CaMK2α本身是脑内含量最多的蛋白质之一，约占整个大脑皮层的蛋白的百分之一左右，能够比较容易计算出每孔总体的蛋白量，确保每孔蛋白上样量一致后，将不同时间的样本的OD450值同标准曲线进行比对，根据标注曲线求出不同时间点CaMK2α的K42位点乙酰化修饰的化学定量曲线(n＝6)，通过化学定量实验计算得出每个时间点乙酰化的CaMK2α所对应的百分比数值。
(2)体外实验
1、行为学实验：自主运动、前肢伸展运动、网屏实验、触觉反应、悬尾实验等。各项行为学实验开始之前，为了减轻C57BL/6小鼠(饲水以及饲阿司匹林或乙酰水杨酸乙酯的清洁级雄性野生型小鼠)的焦虑情绪，实验操作者会在实验前对C57BL/6小鼠进行碰触抚摸，使其慢慢适应实验者的触碰，尽量减缓其紧张焦虑情绪。
1)自主运动具体步骤：①实验时将C57BL/6小鼠依次放入新的动物笼盒(长27cm×宽27cm×高38cm)；②应用Anymaze追踪记录软件记录C57BL/6小鼠5min内活动情况并记录小鼠碰壁次数按标准进行打分；③结束后将C57BL/6小鼠取出并放回原饲养笼盒中， 清理活动检测盒中C57BL/6小鼠留下的尿液和粪便，用10％酒精除去C57BL/6小鼠的气味后再将下一只C57BL/6小鼠放入检测盒中。
2)前肢伸展运动具体实验步骤：①从饲养笼盒中取出一只C57BL/6小鼠，将其放在一块白板(具有足够大的面积)上，用手将其尾巴提起使小鼠只有前肢能触碰在白板上，使其自然爬行；②应用Anymaze追踪记录软件记录C57BL/6小鼠在白板上前肢爬行时的前肢伸展情况并按标准进行打分；③结束后将C57BL/6小鼠取出并放回原饲养笼盒中，清理活动检测盒中C57BL/6小鼠留下的尿液和粪便，用10％酒精除去C57BL/6小鼠的气味后再将下一只C57BL/6小鼠放在白板上。
3)网屏实验具体实验步骤：①准备一个呈45°倾斜的铁制网屏(具有足够大的面积)；
②从饲养笼盒中取出一只C57BL/6小鼠，放在倾斜的网屏中央；③应用Anymaze追踪记录软件记录C57BL/6小鼠在网屏上的运动状况及攀爬情况并按标准进行打分；④结束后将C57BL/6小鼠取出并放回原饲养笼盒中，清理活动检测盒中C57BL/6小鼠留下的尿液和粪便，用10％酒精除去C57BL/6小鼠的气味后再将下一只C57BL/6小鼠放在网屏上。
1)触觉反应具体实验步骤：①实验时将C57BL/6小鼠依次放入新的动物笼盒(长27cm×宽27cm×高38cm)用棉签轻轻分别触碰小鼠的两侧胡须和两侧身体，观察小鼠反应并感受两侧反应是否具有差异；②应用Anymaze追踪记录软件记录C57BL/6小鼠两侧反应差异情况并按标准进行打分；③结束后将C57BL/6小鼠取出并放回原饲养笼盒中，清理活动检测盒中C57BL/6小鼠留下的尿液和粪便，用10％酒精除去C57BL/6小鼠的气味后再将下一只C57BL/6小鼠放入检测盒中。
2)悬尾实验具体实验步骤：①从饲养笼盒中取出一只C57BL/6小鼠，用手提起小鼠尾巴使其悬空，以一块白板(具有足够大的面积)作为背景使追踪记录成像清晰，观察小鼠四肢活动的体态对称性；②应用Anymaze追踪记录软件记录C57BL/6小鼠在悬尾时的四肢运动状况根据体态对称性按标准进行打分；③悬尾3～7秒将C57BL/6小鼠放回原饲养笼盒中，用10％酒精除去C57BL/6小鼠的气味后再对下一只小鼠进行实验。尸体的处理：修剪剩下的脑块和动物尸体一起放到脑功能所指定放动物尸体的冰柜中，统一处理。
2、制备特异性识别CaMK2α的乙酰化修饰的兔多克隆抗体：
1)针对CaMK2α的K42位点特异性的乙酰化修饰，本发明选取第37～48号氨基酸合成修饰多肽(C-QEYYAKAcIINTKK)，偶联到KLH后，免疫到兔子体内，牺牲掉兔子后，血清纯化抗体，同时合成对照肽(C-QEYYAKIINTKK)，进行纯化和酶联免疫吸附实验(ELISA)检测；
2)利用非天然编码乙酰化赖氨酸技术直接体外合成并纯化了全K42位点定点乙酰化修饰的蛋白(His-K42Ac-CaMK2α)和K258位点定点乙酰化修饰的蛋白(His-K258Ac-CaMK2α)；
3)通过考马斯亮蓝染色检测了蛋白质的纯度，然后通过利用的纯化的定点乙酰化化蛋白检测抗体在体外的特异性，通过不断优化条件和对抗体进行多次纯化；
4)通过基因敲除和点突变的小鼠在体验证抗体的特异性。
3、构建真核细胞系统表达定点乙酰化CaMK2α蛋白：
1)首先构建并优化了能够同时表达aaRSs和tRNACUA正交对的单质粒载体：选择的aaRSs/tRNACUA正交对是AcKR3/tRNACUA正交对，因为其已经被证明了能够在哺乳动物的细胞系、小鼠前脑内有效地通读琥珀密码子UAG，使非天然氨基酸acetyl-lysine整合到目的蛋白上；
2)将tRNACUA放在U6启动子后，并构建四个拷贝U6-tRNACUA表达组件。为了增加tRNACUA的表达，通常将其放在U6启动子下游，启动tRNACUA的转录，另外增加tRNACUA的拷贝数也可以增加基因的编码效率。
3)为了使AcKR3在小鼠的脑内可以高效表达，选择EF1a启动子来启动AcKR3的转录和表达。实验证实EF1a是真核强启动子，且能在哺乳动物细胞内稳定存在不易沉默。
4)将四个拷贝U6-tRNACUA表达组件克隆到AcKR3表达质粒上，成功构建了四个拷贝U6-tRNACUA和AcKR3表达的单质粒表达系统(RS(tRNACUA)4X)。为了确定和验证RS(tRNACUA)4X的表达，同时在EGFP基因的39位引入琥珀密码子(amb)，构建了EGFPamb单荧光报告分子；
5)将EGFPamb报告质粒和四个拷贝数的tRNACUA的RS(tRNACUA)4X表达质粒分别共同转染到HEK293T细胞，且分别用含有和不含有1mM的acetyl-lysine的培养基培养细胞，如果EGFPamb的39位amb密码子被通读，则能够表达全长的EGFP，可以在荧光显微镜下看到绿色荧光，表明acetyl-lysine被整合到了EGFP荧光蛋白中；
6)在细胞转染24h后，只要在1mM的acetyl-lysine培养基培养的细胞中，观察到了绿色荧光蛋白的表达，表明acetyl-lysine已经被整合到了EGFP蛋白中；
7)检测RS(tRNACUA)4X质粒在真核细胞内的表达效率。
4、COX-1活性检测
COX-1的活性检测是使用市面上的试剂盒(Abcam，Cat No.ab204699)进行的。
1)通过试剂盒绘制线性范围的标准曲线，样品对比寻找线性范围内的上样量。
2)在每个反应中添加一定量的血小板蛋白，确保蛋白量是在COX-1活性的线性范围内。在每次检测中加入COX-1抑制剂SC560以验证该方法的特异性。
3)将不同组小鼠的血小板裂解液与反应缓冲液和COX-1底物花生四烯酸混合。
4)样品反应产生前列腺素F(PGF)，在微孔板阅读器中通过荧光动力学进行检测荧光强度发放。
5)每个样品中的COX-1含量由ELISA试剂盒(上海科顺生物有限公司)进行检测。
6)将活性和蛋白表达量相除，做归一化处理，计算单位质量的活性值。
(3)统计：研究结果数据采用Prism 5.0软件进行统计分析，实验数据结果以均值±标准误(mean±SEM)表示。用双尾非配对t检验(two-tailed unpaired t-tests)Student’s t-test进行两组间统计，多组间计量统计采用单因素方差分析(one-wayANOVA)。p<0.05为有统计学差异。
(4)过程性结论：
1)根据质谱结果发现，饲喂阿司匹林14天后使小鼠脑内大部分蛋白质的乙酰化修饰水平升高，其中Camk2α的赖氨酸42位点和258位点最为突出，如图1所示；
2)在体外条件下，阿司匹林可以直接提高Camk2α的乙酰化水平，且具有剂量依赖特点，并且通过LC/MS质谱检测，发现K42和K258是主要的乙酰化位点，如图1、图2所示；
3)用ELASA化学滴定的形式进行乙酰化CaMK2α的定量发现，阿司匹林给药可以时间依赖地提高CaMK2α的K42位点乙酰化水平，并在稳定后提高六倍左右(CaMK2α的K42位点乙酰化修饰水平从正常情况的0.52％左右不断升高并稳定在3.01％)，如图3所示；
4)采用细胞亚组分分馏技术发现，CaMK2α的K42位点乙酰化修饰主要富集在胞浆中，而突触中乙酰化修饰的比例较低(胞浆中乙酰化的比例达到了13％，而在突触中CaMK2α的K42位点乙酰化修饰占总蛋白中的比例低，只有2％)，如图2所示；
5)K42位点乙酰化修饰的发生并没有影响CaMK2α和Ca2+/CaM之间的结合，如图4所示；
6)K42号位点是钙调蛋白激酶的ATP结合位点，采用计算结构模拟和ATP免疫沉淀两种方法发现，K42位点的乙酰化修饰显著损伤了CaMK2α蛋白激酶和ATP的结合，如图4所示；
7)通过体外自我磷酸化实验发现，K42位点的乙酰化损伤了CaMK2α的T286位点的自我磷酸化，但是K258位点的乙酰化对CaMK2α的自我磷酸化没有影响；
8)采用激酶试剂盒，进行底物磷酸化实验发现，CaMK2α的K42位点的乙酰化可以抑制其对底物的磷酸化，但是K258位点的乙酰化不影响CaMK2α对底物的磷酸化功能，且模拟13％的K42位点乙酰化修饰的比例可以显著抑制其自身的过度激活，对CaMK2α在Ca2+/CaM/ATP和Ca2+/CaM/H2O2两种条件下的过度激活都具有显著的抑制作用，如图4所示；
9)通过激酶实验发现，阿司匹林不是CaMK2α的直接抑制剂而抑制其激酶活性，如图5所示；
10)干扰CaMK2α蛋白K42位点的乙酰化削弱阿司匹林对缺血型脑卒中小鼠模型的保护作用，如图5所示；
11)通过脑卒中模型小鼠的构建、抑制肽脑室注射和Carcia score法认知行为学实验，从死亡率，认知功能评分，梗死面积三个层面进行分析和统计，发现阿司匹林对于脑卒中模型的小鼠具有比较好的神经保护作用注射抑制肽降低K42位点的乙酰化修饰后，削弱了阿司匹林在这三个层面的神经保护作用，证明CaMK2α蛋白K42位点的乙酰化修饰对于阿司匹林的神经保护作用而言是必要的，如图5所示；
12)通过构建真核细胞系统表达定点乙酰化CaMK2α蛋白，发现小鼠前脑内表达K42
位点定点乙酰化修饰的CaMK2α蛋白就能在小鼠的脑卒中模型中起到直接的神经保护作用，即CaMK2α蛋白K42位点的乙酰化修饰可以充分发挥神经保护作用发挥神经保护作用，如图6所示。
13)小分子模拟阿司匹林或乙酰水杨酸乙酯与COX-1结合的结构，并计算结合的自由能。乙酰水杨酸乙酯结合COX-1的自由能要高于阿司匹林，说明乙酰水杨酸乙酯和COX-1的亲和力要低于阿司匹林，如图7所示；
14)检测饮用正常水、含阿司匹林或乙酰水杨酸乙酯(0.1mg/ml)的水14天后的小鼠血小板的COX-1活性。乙酰水杨酸乙酯对COX-1活性的抑制作用显著低于相同剂量的阿司匹林，如图7所示；
15)检测饮用正常水、含阿司匹林或乙酰水杨酸乙酯(0.1mg/ml)的水14天后的小鼠的出血时间。饮用乙酰水杨酸乙酯小鼠的出血时间明显短于饮用相同剂量阿司匹林的小鼠，如图7所示；
16)阿司匹林的改造物乙酰水杨酸乙酯，保持了乙酰化CaMK2α蛋白K42位点的能力，并且和阿司匹林乙酰化CaMK2α的作用类似，如图7所示；
17)和对照组相比，乙酰水杨酸乙酯降低了小鼠脑卒中后24h的死亡率，改善了认知功能，并减少了梗死面积，具有神经保护作用，和阿司匹林的神经保护作用类似，如图7所示；
18)检测饮用正常水、含阿司匹林或乙酰水杨酸乙酯(0.1mg/ml)的水14天后的小鼠的肝肾功能。饮用乙酰水杨酸乙酯小鼠的肝肾功能指标和对照小鼠基本一致，谷丙转氨酶(ALT)和尿酸(UA)的指标比阿司匹林好，如图8所示。
本发明的保护内容不局限于以上一种阿司匹林衍生物。凡是通过改造阿司匹林的羧基，来降低对COX-1的抑制作用和出血风险；同时保留阿司匹林的乙酰基团和乙酰化钙离子信号蛋白的能力，发挥神经保护作用的思路都属于本发明的范畴。在不背离本发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。

Claims (15)

  1. 一种生物标志物，其特征在于，所述生物标志物为神经细胞内的钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)的乙酰化位点；所述CaMK2α的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示；所述乙酰化位点为K42、K286。
  2. 如权利要求1所述的生物标志物在制备预防和/或治疗脑卒中疾病、脑缺血疾病的药物中的应用，其特征在于，所述药物为阿司匹林经酯化后得到的衍生物。
  3. 如权利要求2所述的应用，其特征在于，所述药物包括阿司匹林衍生物乙酰水杨酸乙酯。
  4. 如权利要求2所述的应用，其特征在于，所述阿司匹林经酯化后得到的衍生物通过乙酰化神经细胞内钙离子-钙调蛋白依赖的蛋白激酶2α(CaMK2α)，提高CaMK2α的乙酰化水平，抑制CaMK2α的激酶活性；所述阿司匹林经酯化后得到的衍生物对CaMK2α的乙酰化位点包括K42、K286。
  5. 如权利要求2所述的应用，其特征在于，所述CaMK2α的K42位点的乙酰化修饰富集在胞浆中；所述CaMK2α的K42位点的乙酰化抑制其自身磷酸化和对底物的磷酸化，但不影响CaMK2α和Ca2+/CaM之间的结合。
  6. 如权利要求2所述的应用，其特征在于，所述应用包括阿司匹林经酯化后得到的衍生物在制备预防和/或治疗脑卒中疾病、复发性脑缺血药物中的应用，在制备针对无法使用/无法大剂量使用阿司匹林但同时存在脑缺血风险的患者的药物中的应用，或在制备阿司匹林的替代治疗药物中的应用。
  7. 一种生物标志物的调节剂，其特征在于，所述调节剂包括如权利要求1所述的生物标志物的激动剂、抑制剂或拮抗剂。
  8. 一种药物/药物组合物，其特征在于，其包含如权利要求1所述的生物标志物，或包含可以也与如权利要求1所述生物标志物结合或反应的化合物。
  9. 如权利要求8所述的药物/药物组合物，其特征在于，其包括阿司匹林经酯化后得到的衍生物。
  10. 一种检测试剂/试剂盒，其特征在于，其包含可用于检测如权利要求1所述的生物标志物及其乙酰化水平的有效成分。
  11. 一种特异性识别K42位点乙酰化CaMK2α的多克隆抗体，其特征在于，其为选取第37～48号氨基酸合成修饰多肽(C-QEYYAKAcIINTKK)偶联KLH，免疫兔子后获得；所述抗体能够特异性识别K42位点乙酰化的CaMK2α蛋白，不能识别野生型的非乙酰化的CaMK2α蛋白，也不能识别K258等其他位点发生乙酰化的CaMK2α蛋白。
  12. 如权利要求7所述的生物标志物的调节剂、如权利要求8所述的药物/药物组合物、如权利要求10所述的检测试剂/试剂盒、或如权利要求11所述的抗体在用于预测、评估、预 防和/或治疗脑卒中疾病、脑缺血疾病、复发性脑缺血疾病中的应用，在制备针对无法使用/无法大剂量使用阿司匹林但同时存在脑缺血风险的患者的药物中的应用，或在制备阿司匹林的替代治疗药物中的应用。
  13. 如权利要求12所述的应用，其特征在于，所述调节剂、药物/药物组合物、检测试剂/试剂盒可以单独使用，也可以与其他药物联合使用。
  14. 一种筛选预防和/或治疗脑卒中疾病、脑缺血疾病的候选药物的方法，其特征在于，所述方法包括检测所述候选药物对对象或获自对象的样品中如权利要求1所述的生物标志物的生物学效应的影响，包括检测候选药物对对象或获自对象的样本中CaMK2α的K42位点的乙酰化水平或者CaMK2α的激酶活性。
  15. 一种对有需要的受试者预防和/或治疗脑卒中疾病、脑缺血疾病的方法，或对所述患者是否适用阿司匹林经酯化后得到的衍生物来预防和/或治疗脑卒中疾病、脑缺血疾病进行预测和/或预后评估的方法，其特征在于，所述方法包括检测如权利要求1所述的生物标志物的CaMK2α的K42位点的乙酰化水平或者CaMK2α的激酶活性。
PCT/CN2023/102415 2022-07-18 2023-06-26 预防和保护脑缺血疾病的标志物及阿司匹林衍生物在预防和保护脑缺血疾病中的应用 WO2024016944A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011157905A1 (en) * 2010-06-18 2011-12-22 Helsingin Yliopisto Polyclonal antibody binding to acetylated hmgb1
CN103900893A (zh) * 2013-05-20 2014-07-02 上海华盈生物医药科技有限公司 富集乙酰化修饰的蛋白质的试剂盒及方法和应用
CN110325106A (zh) * 2017-02-20 2019-10-11 加利福尼亚大学董事会 无症状性脑缺血的血清学测定
CN110456035A (zh) * 2012-04-02 2019-11-15 博格有限责任公司 基于细胞的探询式分析及其应用
CN110526843A (zh) * 2018-03-28 2019-12-03 中央研究院 抑制钙/钙调蛋白依赖性蛋白激酶ⅱ的化合物及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011157905A1 (en) * 2010-06-18 2011-12-22 Helsingin Yliopisto Polyclonal antibody binding to acetylated hmgb1
CN110456035A (zh) * 2012-04-02 2019-11-15 博格有限责任公司 基于细胞的探询式分析及其应用
CN103900893A (zh) * 2013-05-20 2014-07-02 上海华盈生物医药科技有限公司 富集乙酰化修饰的蛋白质的试剂盒及方法和应用
CN110325106A (zh) * 2017-02-20 2019-10-11 加利福尼亚大学董事会 无症状性脑缺血的血清学测定
CN110526843A (zh) * 2018-03-28 2019-12-03 中央研究院 抑制钙/钙调蛋白依赖性蛋白激酶ⅱ的化合物及其应用

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