CN109553685A - A kind of micromolecule polypeptide and its application in preparation prevention and treatment cerebral arterial thrombosis drug - Google Patents

A kind of micromolecule polypeptide and its application in preparation prevention and treatment cerebral arterial thrombosis drug Download PDF

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CN109553685A
CN109553685A CN201710875498.XA CN201710875498A CN109553685A CN 109553685 A CN109553685 A CN 109553685A CN 201710875498 A CN201710875498 A CN 201710875498A CN 109553685 A CN109553685 A CN 109553685A
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cdk5
tat
ctm
arg
arterial thrombosis
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CN109553685B (en
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朱铃强
周雅帆
刘丹
汪晶
邓曼菲
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Huazhong University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

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Abstract

The invention discloses micromolecule polypeptide TAT-CDK5-CTM and its applications in preparation prevention or treatment cerebral arterial thrombosis drug.Pass through synthesis TAT protein transduction structural domain, the fusion protein TAT-CDK5-CTM of the polypeptide and autophagocytosis original part CTM three that can combine and inhibit CDK5, CDK5 polypeptide is carried using TAT and penetrates blood-brain barrier via blood transportation, and by neuronal uptake.The polypeptide is applied in vitro and in the cerebral arterial thrombosis model of body, the NR2B subunit that it blocks N- methylaspartic acid receptor (NMDAR) and the effect that Cyclin dependent kinase 5 (CDK5) is combined can effectively be played, and the peptide molecule for being combined with CDK5 albumen is mediated by CTM, into lysosomal degradation, the Neuron Apoptosis and necrosis for inhibiting the downstream CDK5 to cause, cerebral injury after reducing cerebral arterial thrombosis, the drug for exploitation treatment cerebral arterial thrombosis provide molecular target.

Description

A kind of micromolecule polypeptide and its application in preparation prevention and treatment cerebral arterial thrombosis drug
Technical field
The invention belongs to field of medicaments, are related to micromolecule polypeptide and its medical applications, and in particular to a kind of artificial synthesized Micromolecule polypeptide TAT-CDK5-CTM and micromolecule polypeptide TAT-CDK5-CTM is in preparation prevention or treatment ischemic cerebral apoplexy Application in middle drug.
Background technique
Cerebral arterial thrombosis is to cause thrombosis or embolism since brain blood flow is interrupted, lead to the one of cerebral ischemia The serious neurological disease of class, patient will appear the even death of paralysis, aphasis, visual loss.Because its high incidence, height are dead The features such as dying rate and high disability rate, it is considered to be seriously endanger the common refractory disease of human health and life security.According to Cerebral apoplexy epidemiology report in 2016, existing 70,000,000 people of patients with cerebral apoplexy in China, annual new hair 2,000,000 people of cerebral apoplexy, often 1,650,000 people of year stroke death toll has a people to die of cerebral apoplexy in every 21 seconds, every year as soon as there is within every 12 seconds the raw cerebral apoplexy of human hair Dead number accounts for the 22.45% of all death tolls due to cerebral apoplexy, and with the aggravation of aging of population, this data Still it is being gradually increasing.Not only the death rate is high for cerebral apoplexy, but also poor prognosis, and 70% patient work's ability is by different degrees of Damage, 30% minimal invasive treatment can not take care of oneself, this all causes serious financial burden to patient home and entire society And stress.However, extremely limited for the treatment means of cerebral arterial thrombosis at present, the only effective treatment is used Tissue plasminogen activator (tPA) carries out thromboembolism treatment, but therapeutic time window is extremely narrow (4.5 hours), therefore most of patients can only Symptomatic treatment.So far, the whole world surpasses more than 1000 for the small molecule compound of cerebral arterial thrombosis exploitation, and carries out About 200 clinical tests, but end in failure.Therefore, new treatment method is probed into, confrontation cerebral arterial thrombosis causes Cerebral injury, the death for reducing neuron are of crucial importance.
Cerebral apoplexy cause the molecular mechanism of cell death have excititoxic, oxygen/nitridation stress, mitochondrial function barrier Hinder with calcium overload etc..In the case where ischaemic, lacking sugar anoxic can cause glutamic acid to be excessively gathered in cynapse, activate cynapse Nmda receptor and ampa receptor afterwards, wherein nmda receptor plays a crucial role.A large amount of Ca2+It is logical by nmda receptor Road flows into the cell, causes Ca2+It is super.The Duan Keyu intracellular protein intracellular of NMDA receptor interacts, and activates a series of Protease, ribozyme and esterase cause neuronal death to cause fatal downstream reaction.Since glutamate receptor is normal Neuron activity kind play important physiological action, therefore directly blocked glutamic acid receptor to treatment cerebral arterial thrombosis simultaneously It is improper.The interaction for causing the albumen of neuronal death by blocking nmda receptor and downstream is inhibiting neurotrosis It does not influence the normal physiological activity of neuron simultaneously, is more safely and effectively therapeutic strategy.
TAT is cell-penetrating peptide (cell penetrating peptides), is that one kind can be with penetrating cell film and nuclear membrane Novel high-efficiency transport carrier.TAT can carry polypeptide, protein and DNA molecular etc. and is actively transported into carefully by receptor Cytoplasm and nucleus, so that the molecule carried be made to play corresponding biological effect.Internal and external test shows HIV-TAT at present All histocytes including nerve cell can be passed through, and without obvious toxic-side effects.The transfer efficiency of TAT is high, can lead to Blood transportation is crossed, and neuron and spongiocyte can be entered through blood-brain barrier, and entrained protein or polypeptide can Its original bioactivity is kept, its corresponding biological action is played.
CTM is autophagocytosis element (the Chaperone-mediated autophagy Targeted that molecular chaperones mediate Motif), core element is Lys-Phe-Glu-Arg-Gln (KFERQ), and albumen in combination can be mediated to enter lyase It is degraded in vivo.
Recent research and TAT technology based on applicant, applicant have synthesized one section of amino acid sequence by NR2B (Arg-Arg-Pro-Pr o-Arg-Ser-Pro-Asp-His-Lys-Arg-Tyr-Phe-Arg-Asp-Lys-Glu, ) and CTM sequence (L ys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln-Arg-Phe- RRPPRSPDHKRYFRDKE Phe-Glu, KFERQKILDQRFFE) and TAT cell-penetrating peptide (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg- Arg, YGRKKRRQRRR) composition film penetrating small-molecular peptide T AT-CDK5-C TM, applied in vitro and carrier ischemic Property cerebral apoplexy model in, effectively play its block N- methylaspartic acid receptor (NMDAR) N R2B subunit and cyclin according to Rely property protein kinase 5 (CDK5) to combine and CDK5 is inducted into the effect of lysosomal degradation, inhibits the downstream CDK5 to reach The Neuron Apoptosis and necrosis of initiation, the cerebral injury after reducing cerebral arterial thrombosis, for further exploitation clinical treatment ischemic The drug of cerebral apoplexy provides molecular target.
Summary of the invention
The task of that invention is to provide a kind of polypeptides, and provide the polypeptide in preparation prevention or treatment cerebral arterial thrombosis drug In application.
Realize the technical scheme is that
Polypeptide provided by the invention is micromolecule polypeptide TAT-CDK5-CTM, sequence 1 in amino acid sequence such as sequence table Shown in (SEQ ID NO.1).
Micromolecule polypeptide TAT-CDK5-CTM provided by the invention can be used to prepare prevention or treat cerebral arterial thrombosis Drug, and be used to prepare reduce ischemic after neuronal necrosis drug and be used to prepare reduce ischemic after Neuron Apoptosis Drug.
The present invention by TAT cell-penetrating peptide (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, YGRKKRRQRRR) with one section of amino acid sequence (Arg-Arg-Pro-Pro-Arg-Ser-Pro-Asp-His-Lys- of NR2 B Arg-Tyr-Phe-Arg-Asp-Lys-Glu, RRPP RSPDHKRYFRDKE) and CTM sequence (Lys-Phe-Glu-Arg-Gln- Lys-Ile-Leu-Asp-Gln-Arg-Phe-Phe-Glu, KF ERQKILDQRFFE) connection, obtain that there is biological activity TAT-CDK5-CTM micromolecule polypeptide.CDK5 polypeptide is carried using TAT, penetrates blood-brain barrier through blood transportation, and by brain mind Through member intake to play the biological function that it induces CDK5 degradation.
The present invention provides micromolecule polypeptide TAT-CDK5-CTM in preparation prevention or treatment cerebral arterial thrombosis drug Application, it is incubated for neuronal cell or mouse tail vein injection altogether, it is found that it can effectively reduce ischemic cerebral apoplexy Middle cerebral infarction area inhibits apoptosis or the necrosis of neuron, improves neurological symptom and corresponding behaviouristics phenotype.
Present inventor's discovery, after cerebral arterial thrombosis, the NR2B subunit of N- methylaspartic acid receptor (NMDAR) with Cyclin dependent kinase 5 (CDK5) interaction, and mediate downstream neuronal death (necrosis and apoptosis).It blocks The phase interaction of the NR2B subunit and Cyclin dependent kinase 5 (CDK5) of N- methylaspartic acid receptor (N MDAR) With neuronal death after cerebral arterial thrombosis can be effectively reduced.For this discovery, applicant is by TAT cell-penetrating peptide (Tyr- Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, YG RKKRRQRRR) with one section of amino acid sequence of NR2B (Arg-Arg-Pro-Pro-Arg-Ser-Pro-Asp-His-Lys-Arg-Tyr-Phe-A rg-Asp-Lys-Glu, ) and CTM sequence (Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu-Asp-Gln- Arg-Phe- RRPPRSPDHKRYFRDKE Phe-Glu, KFERQKILDQRFFE) connection, obtain the TAT-CDK5-CTM with biological activity.By TAT-CD K5-CTM It is incubated for jointly with the neuron of in vitro culture, TAT-CDK5-CTM polypeptide can be directly by neuronal uptake.Pass through mouse tail vein Injection can make TAT-CDK5-CTM polypeptide enter blood and be absorbed across blood-brain barrier by cerebral neuron, to play its life Object effect.
Micromolecule polypeptide TAT-CDK5-CTM sequence is as follows:
Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-Arg-Pro-Pro-Arg-Ser- Pro-Asp-His-Lys-Arg-Tyr- Phe-Arg-Asp-Lys-Glu-Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu- Asp-Gln-Arg-Phe-Phe-Glu(YGRKKRRQRRR- RRPPRSPDHKRYFRDKE-KFERQKILDQRFFE)
The control of TAT-CDK5-CTM is TAT-scramble-CDK5 (TAT-s-CDK5), and sequence is as follows:
Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-His-Pro-Arg-Ser-Arg- Pro-Arg-Lys-Glu-Asp-Asp- Lys-Arg-Tyr-Phe-Arg-Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu- Asp-Gln-Arg-Phe-Phe-Glu(YGRKKRRQRRR-P HPRSRPRKEDDKRYFR-KFERQKILDQRFFE)
TAT-CDK5-CTM and its control TAT-s-CDK5 is the synthesis of commercialization company.
Application of the micromolecule polypeptide TAT-CDK5-CTM in preparation prevention or treatment cerebral arterial thrombosis drug, application Process is as follows:
(1) application of the TAT-CDK5-CTM in cerebral arterial thrombosis cell model
Oxygen sugar deprives the In vitro cell model that (Oxygen Glucose Deprivation, OGD) is cerebral arterial thrombosis. Experiment carries out OGD to 10 days neurons of primary culture in vitro and handles 60 minutes and 90 minutes, handles 60 minutes feelings in OGD Under condition, PI marks 44% meronecrosis, and TUNNEL marks 20% Apoptosis.In the case where processing in OGD90 minutes, PI marks 86% meronecrosis, and TUNNEL marks 18% Apoptosis.It can be seen that most neurons are dead under OGD processing It dies.Incubation processing is carried out with primary neuron of the TAT-CDK5-CTM of 5um in vitro culture, the primary nerve handled through OGD The cell quantity of member, PI and the TUNNEL positive substantially reduces, and illustrates that TAT-CDK5-CTM processing can be reduced in cellular level and lacks Neuronal death after blood.
(2) application of the TAT-CDK5-CTM in cerebral arterial thrombosis animal model
Arteria cerebri media embolism (middle cerebral artery occlusion, MCAO) model is that generally acknowledged brain lacks Blood animal model.It is inserted into line bolt by mouse arteria carotis, blocks arteria cerebri media, the skin that arteria cerebri media can be caused to dominate Lamellar body ischemic.Blood flow Reperfu- sion after Outlet bolt is pulled out, ischemical reperfusion injury can be caused.Clinical cerebral embolism is simulated with this Or the symptom of cerebral infarction.After model foundation, detection ischemic infarction volume, FJ and TUNNEL dyeing detection mind are dyed by TTC Through first dead, the balance ability of tired turn-club test Rotarod reflection mouse, scores of nervous system (Neurological Score, N.S.) detect nervous function after ischemic.Respectively after model mice ischemia-reperfusion at 3 hours and 6 hours, Give the TAT-CDK5-CTM solution of tail vein injection 1mg/ml, the progress TTC dyeing in the 3rd day of animal ischemia-reperfusion and nuclear-magnetism Resonance detects, and the 7th day progress FJ is dyed and TUNNEL dyeing, and interior progress Animal Behavior Science experiment is interrupted when corresponding.TTC Coloration result show the focal cerebral ischemia Infarction volume for giving TAT-CDK5-CTM be substantially less than control group TAT-s-CDK5 or Physiological saline Vehicle group.FJ and TUNNEL coloration result is shown, gives neuron after the mouse ischemic of TAT-CDK5-CTM Dead quantity is substantially less than control group TAT-s-CDK5 or physiological saline Vehicle group.In addition, tired transfer rod and water fan Palace the experimental results showed that, the balance ability and ability of learning and memory for injecting the mouse of TAT-CDK5-CTM are compared to right It is significantly improved according to group TAT-s-CDK5 or physiological saline Vehicle group.Result above proves, TAT-CDK5-CTM pairs Cerebral arterial thrombosis has exact therapeutic effect.
Compared with prior art, the invention has the characteristics that: the present invention designed by micromolecule polypeptide TAT-CDK5- CTM synthesis purity is high, it is soluble good, it is suitable for intravenous injection, has no toxic side effect, has in conversion production and clinical application It is advantageous.
The present invention is with artificial synthesized TAT protein transduction structural domain and the fusion protein polypeptide for combining CDK5, it is intended that uses The mode of intravenous injection treats cerebral arterial thrombosis, reduces nerve cell death and cerebral injury caused by cerebral arterial thrombosis, changes Neurological symptom after kind cerebral arterial thrombosis.In TAT-CDK5-CTM recombinant protein polypeptide disclosed by the invention, TAT conduct A kind of efficient transducin can carry polypeptide through blood transportation through blood-brain barrier and by neuronal uptake, can convert and answer For in the medium the nervous system disease of ischemic cerebral apoplexy, to there is the feasibility of practical operation.
Specific embodiment
This method is described further below in conjunction with attached drawing and specific implementation method.What is be related in embodiments is all All operating methods of cell culture, apoptosis and necrosis dyeing, animal ischemia model, tail vein injection and behaviouristics detection, It is known to the researcher of this field.Do not elaborated in the present invention refer to document (Tu W, Xu X, Peng L, Zhong X,Zhang W,Soundarapandian MM,Balel C, Wang W,Jia N,Zhang W,Lew F,Chan SL,Chen Y,Lu Y(2010)DAPK1interaction with NMDA receptor NR2B subunits Mediates brain damage in stroke.Cell 140:222-234) materials and methods part.
Embodiment 1:
TAT-CDK5-CTM's is artificial synthesized
The sequence of TAT-CDK5-CTM is as shown in SEQ ID NO.1, and by Jiangsu, Qiang Yao Biotechnology Co., Ltd is manually closed At, synthesis be reported as follows shown in, chromatography is as shown in Figure 1.
The artificial synthesized HPLC report of TAT-CDK5-CTM
Table 1
Wavelength Time Peak area Peak area percent (%)
1 220nm 7.310 17438 0.2265
2 220nm 9.067 55117 0.716
3 220nm 9.360 7404161 96.18
4 220nm 9.933 118775 1.543
5 220nm 11.207 38910 0.5055
6 220nm 12.239 63670 0.8271
The purity of the TAT-CDK5-CTM polypeptide of synthesis is 96.18%, every 1mg, is white powder, is completely soluble in Water, sealing are kept in dark place in -20 DEG C.Before use, configuration is diluted by prescribed concentration with injection physiological saline, now with existing With.
The control of TAT-CDK5-CTM is TAT-scramble-CDK5 (TAT-s-CDK5), and sequence is as follows:
Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-His-Pro-Arg-Ser-Arg- Pro-Arg-Lys-Glu-Asp-Asp-Lys- Arg-Tyr-Phe-Arg-Lys-Phe-Glu-Arg-Gln-Lys-Ile-Leu- Asp-Gln-Arg-Phe-Phe-Glu (YGRKKRRQRRR-PHP RSRPRKEDDKRYFR-KFERQKILDQRFFE), is also this Company's synthesis.
Embodiment 2:
Neuron NR2B receptor inhibits the downstream CDK5 to draw in conjunction with CDK5 under the conditions of TAT-CDK5-CTM blocks hypoxic-ischemic The Neuron Apoptosis and necrosis risen.
The 10th day primary neuron of in vitro culture carries out glycosyloxy and deprives (OGD) processing, the cell of simulated ischemia stroke Model.OGD is normally cultivated 2 hours after handling 90 minutes, gives the TAT-CDK5-CTM polypeptide or control TAT-s-CDK5 of 5uM Polypeptide or vehicle are incubated for.Cell protein is extracted after 2 hours.First with anti-NR2B antibody sedimentation cell albumen, then with anti-CDK5 The albumen that antibody test precipitates detects that the black trace on NC film shows the interaction of NR2B and CDK5.As a result it shows Show: after giving TAT-CDK5-CTM, the antibody of anti-CDK5 can not almost detect the CDK5 albumen on NC film, prompt TAT- CDK5-CTM has blocked the interaction of NR2B and CDK5.And the control group for giving TAT-s-CDK5 then obviously can be detected CDK5 albumen then illustrates that TAT-s-CDK5 can not block the interaction of NR2B and CDK5.
Embodiment 3:
Application of the TAT-CDK5-CTM in cerebral arterial thrombosis cell model
(1) primary neuronal culture and glycosyloxy deprive the building of simulated ischemia stroke cell model
In dissection liquid (Hank ' s Balance after the tire mouse of pregnant mouse uterus taking-up embryonic period, embryonic phase the 18.5th day, broken end Solution Prefrontal Cortex cortex, 0.125% pancreatin digestion separation cell are taken out in).Cell seeding is in the more of 20ng/ul On polylysine and the coated coverslip of Laminin ELISA, coverslip is placed in 12 orifice plates, and cell seeding density is 100-150 Every square millimeter of cell, culture medium was serum-free Neurobasal culture medium (21103, gibco) plus 2%B27, every three days Liquid is partly changed with identical culture medium, i.e. the old culture medium of discarding half adds the fresh culture of half.Use neuron Marker β-tubulin III (Tuj1) carries out immunofluorescence label to the cell of culture, it was demonstrated that 90% or more the cell cultivated For neuron.
For the cell model for establishing Ischemic Stroke, the primary neuron cultivated in 12 orifice plates gave glycosyloxy at the 10th day (OGD) processing is deprived, that is, discards original cell culture medium, after bicarbonate buffer rinse 3 times of deoxygenation sugar-free, adds Enter the 500ul buffer, and cell is placed in 37 degree of anaerobic incubators and cultivates 60 minutes and 90 minutes, simulated ischemia respectively Property stroke.The bicarbonate buffer of normal incubation medium displacement anaerobic sugar-free with oxygen-containing containing sugar later, in 37 DEG C/5%CO2/ 10%H2/ 85%O2Humidified incubator in maintain culture 72 hours, simulate reperfusion injury.So far, cerebral arterial thrombosis Cell model, which is established, to be completed, and can accordingly be detected to neuron.
(2) application of the TAT-CDK5-CTM in Ischemic Stroke cell model
In order to probe into the best use concentration of TAT-CDK5-CTM, the neuron of above-mentioned originally culture is handled by OGD After 90 minutes, be restored to regular culture conditions it is 2 hours lower when, give 10ul respectively in the every hole 500ul culture medium of 12 orifice plates The TAT-CDK5-CTM of 1uM/3uM/5uM is incubated for, its effect for inhibiting NR2B to interact in CDK5 is observed.Inhibit to make It is used in subsequent experiment with strongest one group of optium concentration for being selected as TAT-CDK5-CTM.
The best applications concentration of TAT-CDK5-CTM: after being incubated for OGD processing with the TAT-CDK5-CTM of various concentration Neuronal cell, and extract corresponding cell protein, carry out co-immunoprecipitation experiment.It is first precipitated with the antibody of anti-NR2B thin Born of the same parents' albumen, then the albumen to be precipitated with the antibody test of anti-CDK5 detect that the black trace on NC film is more shallow, explanation The interaction of NR2B and CDK5 is weaker.When the TAT-CDK5-CTM concentration given is 5uM, the black stripe on NC film is several It disappears, illustrates that TAT-CDK5-CTM polypeptide can completely inhibit the interaction of NR2B and CDK5 when the concentration, also illustrate 5uM TAT-CDK5-CTM be best administration concentration.
The neuron of originally culture carries out OGD processing when cultivating the tenth day, and in the case where restoring regular culture conditions 2 hours Afterwards, in the every hole 500ul culture medium of 12 orifice plates be added 10ul 5uM TAT-CDK5-CTM polypeptide be incubated for, continue 37 DEG C/ 5%CO2/ 10%H2/ 85%O2Humidified incubator in maintain culture 70 hours, PI and TUNNEL are carried out to cell later and contaminated Color, the quantity of Tongji University's meronecrosis and apoptosis.(see Fig. 3).
(3) TAT-CDK5-CTM application effect is assessed
Propidium iodide (propidine iodide, PI) is a kind of nucleic acid dye.It cannot penetrate the cell of normal cell Film, but the cell membrane of non-viable non-apoptotic cell can be penetrated, and by the nuclei dyeing of non-viable non-apoptotic cell at red.Therefore PI dyeing can be anti- Reflect the quantity of non-viable non-apoptotic cell.DAPI is also a kind of nucleic acid dye, can both mark living cells or mark dead cell, therefore DAPI dyeing can be with the total quantity of reacting cells.Specific experiment step are as follows: configure the PI solution of 10uM with PBS, and say the solution It is added in cell culture medium (50ul/500ul) by 1/10 volume.After being incubated for 15 minutes under the conditions of 37 degree, buffered with PBS Liquid is washed twice.500ul nucleus dyestuff DAPI (1/1000 PBS dilution) is added in the every hole of 12 orifice plates again, is incubated for after five minutes It is washed twice with PBS buffer solution.Then it directly takes pictures under the microscope in fluorescence microscopy, red-label cell is non-viable non-apoptotic cell, blue The cell of label is total cell.The percentage of non-viable non-apoptotic cell is calculated with this.
The end in situ disconnected transferase labelling method (TUNNEL) of apoptotic cell can be used for marking apoptotic cell.When Apoptosis, Endogenous cellular endonuclease can be activated, and lead to DNA double chain break.The DNA fragmentation of fracture can expose 3 '-OH, Terminal deoxynucleotidyl transferase (Terminal Deoxynucleotidyl Transferase, TdT)) and DNA polymerase Catalysis under, with fluorescein (TRITC) label nucleotide in conjunction with, make the cell of apoptosis by specific marker, therefore can use In the apoptosis degree of reacting cells.Specific experiment step are as follows: attached cell is washed 2 times with PBS buffer solution, with 4% paraformaldehyde It fixes after ten minutes, is washed twice with PBS buffer solution on ice.It is slow with the sodium citrate containing 0.1%Trition-X-100 afterwards Fliud flushing is incubated for 2 minutes on ice.The TUNNEL reaction solution (enzyme solutions: label solution, 1:9 mixing) of the fresh configuration of 30ul is added Enter on Cell sheet glass, 37 degree be protected from light incubation one hour after, then with DAPI (1/1000PBS dilution) incubation 5 minutes, after with PBS delay Fliud flushing is washed twice.It is taken pictures directly under fluorescence microscope with being observed under the irradiation of 562nm exciting light, is labeled as red cell Apoptotic cell irradiates lower observation with 660nm exciting light and takes pictures, and the cell labeled as blue is total cell.It is thin that apoptosis is calculated with this The percentage of born of the same parents.
We carry out OGD processing in vitro culture 10 days Yuan Dynasty's nerves, handle 60 minutes and 90 minutes respectively.In OGD In the case where processing 60 minutes, PI marks 44% meronecrosis, and TUNNEL marks 20% Apoptosis.At OGD90 minutes In the case where processing, PI marks 86% meronecrosis, and TUNNEL marks 18% Apoptosis.It can be seen that most neurons It is dead under OGD processing.Incubation processing is carried out with primary neuron of the TAT-CDK5-CTM of 5um in vitro culture, through OGD 90 minutes primary neurons of processing, are substantially reduced (Fig. 4 B) by the cell quantity of PI and TUNNEL positive, Apoptosis with Necrosis rate is 3% and 11% respectively, illustrate TAT-CDK5-CTM processing can after cellular level reduces ischemic neuron it is dead It dies, shows that TAT-CDK5-CTM has the function for the treatment of Ischemic Stroke.
Embodiment 4:
Application of the TAT-CDK5-CTM in cerebral arterial thrombosis mouse model
(1) foundation of cerebral arterial thrombosis mouse model
Arteria cerebri media embolism (middle cerebral artery occlusion, MCAO) model is that generally acknowledged brain lacks Blood animal model, concrete operations are as follows: carry out preoperative anesthesia (0.1ml/10g weight, intraperitoneal injection) with 6% chloraldurate, lie on the back It fixes, neck preserved skin, neck does 1-2 centimetres of median incision after iodophor disinfection, separates right carotid, uses in its proximal part Slip-knot ligation, exposure external carotid artery and internal carotid bifurcated, in the 1.5 centimeters ligation of external carotid artery distal end, from external carotid artery to Internal carotid is inserted into the coated nylon wire bolt of diameter about 0.22mm silica gel, and insertion depth is about 0.9-1.0 centimetres, until in brain Artery section start.It moves back except line bolt, and unclamps common carotid artery occlusion within 1 hour, restore its perfusion, sew up a wound.Sham-operation group (sham) for animal in addition to being not inserted into line bolt, other steps are identical as MCAO group model mouse.In entire surgical procedure, as far as possible Bleeding is reduced, implements sterile working, and maintain animal heat with heating cushion.
(2) TAT-CDK5-CTM is injected intravenously
After mouse ischemia-reperfusion 3 hours and 6 hours, 1mg/kg peptide T AT- is injected through mouse femoral vein word CDK5-CTM and control peptide T AT-s-CDK5 or injection physiological saline (vehicle).Peptide concentration is 1mg/ml, with note It penetrates and uses physiological saline solution.After intravenous injection, Mice brain tissues is taken to carry out TTC dyeing and nuclear magnetic resonance after surgical procedure 3 days Infarction volume is detected, TUNNEL and FJ dyeing observation Apoptosis and necrosis situation is carried out after 7 days, is carried out within the 7th day to the 14th day Water maze laboratory detects the ability of learning and memory of mouse, and the corresponding time carries out tired turn-club test in 28 days of ischemia-reperfusion The balance ability of mouse is detected, and in the 28th day progress scores of nervous system (N.S.), evaluates the nervous system function of mouse Can, the therapeutic effect with above-mentioned the results show TAT-CDK5-CTM to Ischemic Stroke.
(3) assessment of TAT-CDK5-CTM therapeutic effect
TTC dyeing: TTC is dyed for detecting ischemic infarction volume.TTC (2,3,5-triphenyltetrazolium chloride) is one The fat-soluble photosensitive compounds of kind.It can be reacted with the succinate dehydrogenase in living cells mitochondria, generate red formazan.And it lacks The activity of succinate dehydrogenase declines in cell mitochondrial in haemal tissue, cannot react, therefore organizing is in pale asphyxia.Specifically Operating procedure is as follows: MCAO model mice anaesthetizes execution after ischemia-reperfusion 3 days, takes out complete brain tissue rapidly, and -20 DEG C It is rapidly frozen 20 minutes, configures 1%TTC-PBS solution, be kept in dark place in 37 degree of insulating boxs.After -20 DEG C of taking-up brain tissues, set In on ice, makees about 1 millimeter of thickness of continuous coronal slice from front to back rapidly, slice is soaked in 1%TTC-PBS solution, 37 DEG C are protected from light incubation 15 minutes, and overturning slice is incubated for 15 minutes again.Coloration result as it can be seen that normal cerebral tissue region be red, Ischemic area is pale asphyxia.After dyeing, slice, which is placed in 4% paraformaldehyde PBS solution, fixes 15 minutes, finally to fixed Brain piece carries out photographic analysis.Continuous 6 slices for choosing each 0.3 cm section in bregma front and back, with image analysis software Image J calculates analysis ischemic volume (Infarction, mm3).
Nuclear magnetic resonance: nuclear magnetic resonance (Magnetic Resonance Imaging, MRI) is for detecting ischemic areas.MRI It is one kind of tomographic imaging, is a kind of biological magnetic spin imaging technique.It utilizes the characteristic of nuclear spin campaign, is adding outside Under magnetic field condition, signal is generated after RF pulse-to-pulse impulse, inputs computer after detector detects, handles and converts through computer After show image.MRI can detect the ischemic areas of mouse under the premise of not putting to death mouse, and result is more accurate.Specifically Steps are as follows: after mouse weighing, preoperative anesthesia (0.1 ml/10g weight, intraperitoneal injection) is carried out with 6% chloraldurate, after anesthesia Careful removal metal ear tag, mouse is fixed in Nuclear Magnetic Resonance, and adjustment mouse form guarantees to scan its head, setting ginseng Number starts to scan.
Fluoro-Jade C (FJ-C) and TUNNEL dyeing: MCAO model mice is anaesthetized after ischemia-reperfusion 7 days, warp Heart aorta perfusion.Blood in Mice Body is rinsed with 0.9% physiological saline quick filling of pre-cooling, until atrium dextrum is flowed out Liquid becomes colorless, thereafter with 4 DEG C of 4% paraformaldehyde solution perfusion 10 minutes be pre-chilled.It is complete to take out brain tissue, it is soaked in phase Behind in paraformaldehyde solution 12-24 hours, it is placed in 30% sucrose solution and is dehydrated, brain tissue carries out frozen section after sinking to the bottom, 30 microns of slice thickness, slice is soaked in PBS buffer solution, 4 degree of preservations.1. FJ-C is dyed: FJ-C dye liquor is a kind of band fluorescence Anion ligand group dyestuff, can be issued under microscope 488nm excitation green in conjunction with the neuron of denaturation Color fluorescence, and normal neurons will not be in conjunction with FJ-C dye liquor, so not fluorescing.Concrete operations are as follows: slice is affixed on gelatin It is dried after coated glass slide, is respectively placed in 100% ethyl alcohol, 1 minute in 70% ethyl alcohol and distilled water.Then containing 0.01% Room temperature is protected from light diformazan after incubation 30 minutes, distilled water rinse 3 times in the mixed liquor of Fluoro-Jade and 0.1% acetic acid (1:10) Benzene fast transparent can be observed directly under the microscope after DPX mountant mounting, and 488nm exciting light observes the cell of Green Marker As it is denaturalized dead cell.2. TUNNEL is dyed: TUNNEL dyeing theory is for example above-mentioned.Concrete operations are as follows: slice is affixed on bright Dried after the coated glass slide of glue, first use PBS rinse, after with the sodium citrate buffer solution containing 0.1%Trition-X-100 It is incubated for 2 minutes on ice.The TUNNEL reaction solution (enzyme solutions: label solution, 1:9 mixing) of the fresh configuration of 50ul is added every Brain on piece is opened, after 37 degree are protected from light incubation one hour, is taken pictures directly under fluorescence microscope with being observed under the irradiation of 562nm exciting light, It is apoptotic cell labeled as red cell.
The scoring of longa behavior disorder and tired turn-club test: 1. Longa the five-grade marking system method scores: 0 point, impassivity damages disease Shape；1 point, when lifting tail, buckling is received in damage opposite side forelimb, is unable to full extension；It 2 points, turn-takes when creeping to opposite side；3 Point, when standing, topples over to opposite side；It 4 points, cannot spontaneous walking or the loss of consciousness.Score value is higher, illustrates that animal behavior obstacle is tighter Weight.2. tired turn-club test: respectively in mouse operation consent (the 0th day), the 7th day after operation, 14 days, 21 days, the fatigue of progress in 28 days The sports coordination ability of turn-club test test animal.Concrete operations are as follows: under conditions of 4 revs/min, from place mouse to The time interval that mouse falls is denoted as retention time, reacts the limb force and sports coordination ability of animal.Operation consent is by mouse Training two days, 3 times a day, is denoted as reference baseline for their mean residence time.After formal experiment, carry out 3 times daily, Every detection 28 days.The mouse residence time is longer, illustrates that its locomitivity is stronger.
Morris water maze laboratory: Morris water maze laboratory was established in 1981 by Morris, was test experience animal The Classic Experiments of Spatial memory, concrete operations are as follows: 1. orientation navigation experiment detects the Spatial learning ability of mouse: One round pool (diameter 120cm, high 60cm) is filled into water, one diameter of specified quadrant position placement is at pond center The circular platform of 15cm is in water surface 1cm or less.Experimenter holds up mouse towards pool wall gently into the water, if mouse was at 60 seconds Interior to find platform, then the time for finding platform is the incubation period of the mouse secondary experiment.If being found not successfully in 90s flat Platform is then guided it on platform and is taken out after 30s.So training 7 days, daily training four times are put by different item limit every time Enter.The incubation period of daily each group mouse is calculated, distance of swimming, incubation period, smaller learning ability was stronger.2. space exploration experiment inspection Survey the spatial memory capacity of mouse: training is taken a day off after a week removes platform, and mouse is vertically put into from platform opposite side quadrant It in water, swims 60 seconds, statistics mouse reacts the spatial memory capacity of mouse in the run duration percentage of target quadrant.
At mouse ischemia-reperfusion 3 hours and 6 hours, through mouse tail vein injection 1mg/kg TAT-CDK5-CTM solution Or control group TAT-s- CDK5 solution or physiological saline vehicle group.After TTC is the results show that give TAT-CDK5-CTM Mouse ischemic infarction volume is 10.1 ± 2.2mm3, substantially less than 19.1 ± 2.3mm of TAT-s-CDK5 control group3And physiology salt 19.6 ± 2.0mm of water vehicle group3(Fig. 7 A).It can be reduced Ischemic Stroke mouse brain after illustrating TAT-CDK5-CTM injection Ischemic infarction volume.As the result is shown, mouse ischemic infarction area is 0.18 ± 0.1cm to MRI after giving TAT- CDK5-CTM2, show It writes and is lower than 0.75 ± 0.3cm of TAT-s-CDK5 control group2And 0.81 ± 0.3cm of physiological saline vehicle group2(Fig. 7 B), says It can be reduced Ischemic Stroke murine cerebral ischemia infarct size of tumor after bright TAT-CDK5-CTM injection.FJ-C and TUNNEL contaminates Color shows that the quantity for giving TAT-CDK5-CTM group Mouse Neuron necrosis and apoptosis is respectively 30 ± 2 and 6 ± 1, significantly Lower than 139 ± 27 and 38 ± 12 of TAT-s-CDK5 control group TAT-s-CDK5 or physiological saline vehicle group 147 ± 25 and 49 ± 12, and it is horizontal (16 ± 2 and 6 ± 1) (Fig. 7 A/ Fig. 7 B) close to sham group.Illustrate that TAT-CDK5-CTM is injected The necrosis of neuron and apoptosis after mouse Ischemic Stroke can be improved.And mouse fatigue turn-club test is the results show that after ischemic Seven days, the residence time of TAT- CDK5-CTM group mouse was 72 ± 5 seconds, was significantly higher than 42 ± 3 seconds of TAT-s-CDK5 control group With 39 ± 4 seconds of physiological saline vehicle group, and this trend is continued until the 28th day (Fig. 8 A).Illustrate to inject TAT- CDK5-CTM can improve the sports coordination ability of mouse after cerebral arterial thrombosis.28th day Neuroscore after ischemic (N.S.) the results show that the scoring of TAT-CDK5-CTM group mouse is 1.3 ± 0.2 points, substantially less than TAT-s-CDK5 control 3.3 ± 0.5 points and 3.5 ± 0.5 points of physiological saline vehicle group of group.(Fig. 8 B).Illustrate to inject TAT-CDK5-CTM energy The nervous function of mouse after improvement cerebral arterial thrombosis.Water maze Behaviors survey is the results show that TAT- CDK5-CTM group The incubation period that mouse found platform at the 7th day is 17 ± 5 seconds, substantially less than the 33 ± 5 of TAT-s-CDK5 control group second and physiology 35 ± 6 seconds of salt water vehicle group, and close to 15 ± 5 seconds of sham group.(Fig. 9 A).TAT-CDK5-CTM group mouse is found flat Time needed for platform is shorter, illustrates that TAT-CDK5-CTM can improve the learning ability of Ischemic Stroke mouse.In the 9th day sky Between in explorative experiment, TAT- CDK5-CTM group mouse is 62 ± 5% in target item limit residence time percentage, is significantly higher than The 31 ± 3% of 29 ± 2% and physiological saline vehicle group of TAT-s-CDK5 control group, and close to the 67 ± 5% of sham group. (Fig. 9 B).The result shows that the memory capability of Ischemic Stroke mouse can be significantly improved after TAT-CDK5-CTM injection.
Above-mentioned histology is shown with ethological experimental result, after injecting TAT-CDK5-CTM polypeptide, can be substantially reduced The Infarction volume of cerebral arterial thrombosis mouse reduces necrosis and the apoptosis of neuron, improves the moving equilibrium ability and sky of mouse Between ability of learning and memory, alleviate ischemic after neurological symptom, be develop cerebral arterial thrombosis clinical treatment drug it is potential Target spot.
Detailed description of the invention
Fig. 1 is the artificial synthesized chromatogram of micromolecule polypeptide TAT-CDK5-CTM a kind of.
Fig. 2 is that TAT-CDK5-CTM interferes result schematic diagram of the neuron NR2B receptor in conjunction with CDK5.Figure A exempts from for albumen Epidemic disease Blot results figure, figure B are result statistical chart.
Fig. 3 is the flow chart that TAT-CDK5-CTM treats cerebral arterial thrombosis on primary neuron.
Fig. 4 is best polypeptide concentration for the treatment of the selection result figure.Figure A is protein immunoblot result figure, and figure B is result statistical chart.
Fig. 5 is that TAT-CDK5-CTM reduces Neuron Apoptosis and the result of necrosis statistics after OGD processing on primary neuron Figure.Figure A is TUNNEL coloration result statistical chart, and figure B is PI coloration result statistical chart.
Fig. 6 is the flow chart that TAT-CDK5-CTM treats cerebral arterial thrombosis in animal model.
Fig. 7 is the MRI detection knot that TAT-CDK5-CTM reduces ischemic infarction area after cerebral arterial thrombosis in animal model Fruit and statistical chart.Figure A is control group, and figure B is injecting normal saline group.Scheming C is to inject out-of-order polypeptide group (TAT-s-CTM), figure D is to inject normal polypeptide group (TAT-CDK5-CTM).Figure E is result statistical chart.
Fig. 8 is that TAT-CDK5-CTM reduces ischemic infarction volume (A) after cerebral arterial thrombosis in animal model, and neuron is bad The extremely result statistical chart of (B) and apoptosis (C).
Fig. 9 is that TAT-CDK5-CTM improves motor function (A) and nervous system disease after cerebral arterial thrombosis in animal model The result statistical chart of shape (B).
Figure 10 is the result statistics that TAT-CDK5-CTM improves learning and memory function after cerebral arterial thrombosis in animal model Figure.Figure A is incubation period statistical chart, and figure B is that target item limits residence time percentage statistical chart.
The following are the amino acid sequence tables of polypeptide involved in present patent application, and wherein sequence 1 is micromolecule polypeptide TAT-CDK5- CTM；Sequence 2 is TAT-scramble-CDK5(TAT-s-CDK5).
Sequence table
SEQUENCE LISTING
<110>Central China University of Science and Technology
<120>a kind of micromolecule polypeptide and its application in preparation prevention and treatment cerebral arterial thrombosis drug
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 42
<212> PRT
<213>artificial sequence
<400> 1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg Arg Pro Pro Arg
1 5 10 15
Ser Pro Asp His Lys Arg Tyr Phe Arg Asp Lys Glu Lys Phe Glu Arg
20 25 30
Gln Lys Ile Leu Asp Gln Arg Phe Phe Glu
35 40
<210> 2
<211> 42
<212> PRT
<213>artificial sequence
<400> 2
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro His Pro Arg Ser
1 5 10 15
Arg Pro Arg Lys Glu Asp Asp Lys Arg Tyr Phe Arg Lys Phe Glu Arg
20 25 30
Gln Lys Ile Leu Asp Gln Arg Phe Phe Glu
35 40

Claims (4)

1. a kind of artificial synthesized micromolecule polypeptide, amino acid sequence is shown in SEQ ID NO.1.
2. application of the polypeptide described in claim 1 in preparation prevention or treatment cerebral arterial thrombosis drug.
3. polypeptide described in claim 1 reduces the application after ischemic in neuronal necrosis drug in preparation.
4. polypeptide described in claim 1 reduces the application after ischemic in Neuron Apoptosis drug in preparation.
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CN110787284A (en) * 2019-11-23 2020-02-14 胡书群 Mixed small peptide TAT-SHC for treating ischemic brain injury and application thereof
CN112679595A (en) * 2020-04-10 2021-04-20 南京市儿童医院 Application of brain-derived peptide or fusion peptide in medicine for treating neonatal hypoxia ischemic brain injury
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CN112142856A (en) * 2020-09-30 2020-12-29 中国人民解放军空军军医大学 Specific degradation NDRG2 targeting peptide for improving cerebral stroke excitotoxic injury and application thereof
CN112142856B (en) * 2020-09-30 2022-05-03 中国人民解放军空军军医大学 Specific degradation NDRG2 targeting peptide for improving cerebral stroke excitotoxic injury and application thereof
CN114344446A (en) * 2021-11-17 2022-04-15 湖南大学 Polypeptide capable of relieving neuron hypoxia and glucose-deficiency injury
CN114344446B (en) * 2021-11-17 2024-03-19 湖南大学 Polypeptide capable of relieving neuronal hypoxia and glucose-deficient injury
CN114053413A (en) * 2021-12-29 2022-02-18 暨南大学附属第一医院（广州华侨医院） Application of COL4A4 gene as acute ischemic stroke treatment target

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