CN102327613A - Application of ErbB receptor stimulant to preparation of medicament for treating epilepsy - Google Patents
Application of ErbB receptor stimulant to preparation of medicament for treating epilepsy Download PDFInfo
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- CN102327613A CN102327613A CN201110258064A CN201110258064A CN102327613A CN 102327613 A CN102327613 A CN 102327613A CN 201110258064 A CN201110258064 A CN 201110258064A CN 201110258064 A CN201110258064 A CN 201110258064A CN 102327613 A CN102327613 A CN 102327613A
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Abstract
The invention provides application of an ErbB receptor stimulant to preparation of a medicament for treating epilepsy. The ErbB receptor stimulant has the advantages: the inhibiting effect of the ErbB4 stimulant NRG1 on epilepsy is disclosed, the organism of epilepsy is studied intensively, a research direction for studying a novel effective 'target' and a molecule of an epilepsy inhibiting medicament is provided, and a basis is laid for the screening of a novel medicament for treating brain diseases based on neuronal excitability changes.
Description
(1) technical field
The present invention relates to the application of ErbB receptor stimulating agent in the medicine of preparation treatment epileptics.
(2) background technology
Epilepsy (epilepsy) is the discharge of cerebral neuron paroxysmal abnormality, causes a kind of chronic disease of of short duration cerebral disorder.The epilepsy prevalence is very high, and China has 10,000,000 patients approximately, influences the about 1% of total population, and the epilepsy mortality rate is about 20%.Cause very big burden for family, society.At present, through existing antiepileptic treatment, still have about 30% patient not control.The out of contior epilepsy of medicine is that the mortality rate of intractable epilepsy is higher, can reach 50%, although take operative treatment at present for the out of contior epilepsy of medicine, but the patient who is fit to perform the operation only accounts for sub-fraction more.Therefore, seeking effective and safe medicine is one of biomedical important target.
In brain, it is that GABA can relay cell that 10～20% neuron is arranged approximately, although quantity is few, GABA can relay cell have brought into play important function in the synchronization of the processing of sensory information, neural loop, brain wave rhythm, the activity of inhibition epilepsy.The cell of particularly a kind of expression calbindin parvalbumin (PV) accounts for 50% more than of inhibitory interneuron, because the granting characteristics of its high frequency are known as fast granting relay cell again.From different experiments chamber David Lau, humans such as Ikuo Ogiwara show that in the mode of body eeg recording the low-function of PV relay cell can cause epilepsy to take place.Anatomically, PV relay cell and peripheral nerve unit mainly contain two types of connected modes, and the granting of basivertebral nerve unit is regulated through the cell space and initial the bringing in of aixs cylinder that project basivertebral nerve unit in (1); (2) connect with on every side PV neuron through the slit and extensively be connected, and then influence the synchronization activity of inhibition network.
(Neuregulin NRG) is a member of growth factor family to neuregulin, and is mainly coded by (NRG1～4), and NRG1 is annotated more a kind of.Because different transcriptional start sites and selectivity are sheared, NRG1 can produce 6 kinds of albumen (I-VI) and at least 31 kinds of hypotypes.The receptor ErbB of NRG1 is divided into four receptors of ErbB1～ErbB4 according to hypotype.Wherein ErbB1 and ErbB2 do not have ligand binding domain, but born of the same parents' inner segment possesses hydrolysis and kinase activity; And ErbB3 can combine NRG1 still not possess kinase activity.ErbB4 is a unique receptor that can specificity combines NRG1 and have kinase activity.Can form homodimer or heterodimer between the ErbB receptor.NRG1 is through exciting ErbB tyrosine kinase receptor, and signal transduction pathway in the active cell produces a series of cell effects then, comprises hypertrophy, apoptosis, migration, differentiation and adherent active or inhibition.
2010, people such as Fazzari found that ErbB4 concentrates on and express on the relay cell, particularly on the PV neuron.This results suggest, PV relay cell are likely the main cell target spot of NRG1-ErbB4 signal.
(3) summary of the invention
The present invention has studied the inhibitory action of ErbB4 agonist NRG1 to epilepsy, and for novel effectively " target " and the molecule of exploring epilepsy inhibition medicine provides research direction, the brain of changing into the basis with neuronal excitability for treatment is that disease provides the candidate therapeutic medicine.
The technical scheme that the present invention adopts is:
The application of ErbB receptor stimulating agent in the medicine of preparation treatment epileptics.So-called ErbB4 receptor stimulating agent is meant neuregulin-1 NRG-1 Neu Differentiation Factor NDF glial growth factors GGF (Neuregulin-1).Said Neuregulin-1 is divided into two kinds of endogenous and exogenous agonist.Exogenous Neuregulin-1 is people's recombinant protein N euregulin-1beta 2 (NRG1), the polypeptide chain that it is made up of 61 aminoacid.Endogenous Neuregulin-1 produces used ErbB4 extracellular fragment (aa1-659, the part that ecto-ErbB4) neutralizes here by neuron or glial cell cell.
Preferably, said ErbB receptor stimulating agent is the ErbB4 receptor stimulating agent.
Concrete, said ErbB receptor stimulating agent can be used for preparing the excitatoty medicine that strengthens cortex and hippocampus PV relay cell.
Preferably, said ErbB receptor stimulating agent is NRG1 (people recombinate Neuregulin-1 beta2), is made up of 61 amino acids, and sequence is:
Shlvkcaekektfcvnggecfmvkdlsnpsrylckcpne ftgd rcqnyvmasfykaeelyq; Molecular weight is 7055 dalton.
In a single day ErbB4 combines with NRG1, ErbB4 promptly is activated, and the network propylhomoserin residue generation phosphorylation of its carboxyl terminal is also assembled connection albumen such as Shc and Grb2 etc., and then activate the Ras-Raf-Erk signal path.
The application inventor finds that after deliberation the ErbB4 receptor possibly be the potential target spot of antiepileptic, and ErbB4 agonist NRG1 has certain inhibitory action to epilepsy, and its mechanism is the Kv1.1 potassium channel on the relay cell.
Beneficial effect of the present invention is mainly reflected in: the present invention has disclosed ErbB4 agonist NRG1 and has furtherd investigate to the inhibitory action of epilepsy and to its mechanism; With molecule research direction is provided for exploring novel effectively " target " that epilepsy suppresses medicine, changing into basic brain for treatment with neuronal excitability is that the new medicament screen of disease provides the foundation.
(4) description of drawings
Fig. 1 removes mouse and normal mouse to causing the susceptibility of epilepsy medicine pentylenetetrazole and pilocarpine for the ErbB4 receptor knockout; The result is expressed as meansigma methods ± standard error.
Neuregulin-1 organizes Fig. 2 and the ventricles of the brain are only injected equivalent artificial cerebrospinal fluid group to causing the susceptibility of epilepsy medicine pentylenetetrazole and pilocarpine for the intracerebral ventricle injection people recombinates in advance; The result is expressed as meansigma methods ± standard error.
Fig. 3 is epileptic and normal control group cerebral cortex ErbB4 and ErbB2 expression; The result is expressed as meansigma methods ± standard error.
Fig. 4 be exogenous or endogenous Neuregulin-1 to the regulating action of cerebral cortex or Hippocampus PV relay cell; The result is expressed as meansigma methods ± standard error.
After Fig. 5 knocked out the ErbB4 receptor for the pharmacology blocks ErbB4 receptor or gene specific, the effect of Neuregulin-1 had disappeared; The result is expressed as meansigma methods ± standard error.
Fig. 6 is the adjusting of Neuregulin-1 to Kv1.1 type potassium current on the PV relay cell; The result is expressed as meansigma methods ± standard error.
Fig. 7 can receive the adjusting of NRG1-ErBB4 signal for Kv1.1 tyrosine phosphorylation level on the cell membrane; The result is expressed as meansigma methods ± standard error.
(5) specific embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Specificity knocks out the easier epilepsy of sending out of mouse of ErbB4 receptor on the embodiment 1:PV relay cell
(1) experiment material:
Laboratory animal: male PV-Cre-ErbB4
LoxP/LoxPMice (mice of gene knockout ErbB4 receptor) and brood contrast ErbB4
LoxP/LoxPMice (normal mouse that contains the ErbB4 receptor), each 18, in eight ages in week, body weight 24 ± 2 grams are raised in identical environment, and food and water freely absorb; Give the illumination of 12h every day.Behavioristics's experiment is carried out between 12:00～14:00.
The mice of gene knockout ErbB4 receptor adopts the Cre-LoxP recombinase system of extensive use in the world to obtain.This system contains two kinds of compositions: the 1. DNA sequence of a segment length 34bp, contain the inverted repeat of two 13bp and the core sequence of a 8bp, and its sequence is following: 5 '-ATAACTTCGTATA-ATGTATGC-TATACGAAGTTAT-3 '.This section 34bp sequence is the site of recombinase identification, is called as the loxP site; 2. Cre recombinase (cyclizationrecombination), it is a kind of monomeric protein of being made up of 343 aminoacid, can cause the DNA reorganization in loxP site, Cre recombinase gene coding region sequence total length 1029bp (EMBL database login X03453).The DNA of any sequence, when its between two loxP sites the time, under the effect of Cre recombinase or by disappearance (direction in two loxP sites is identical), or direction is reversed (two loxP sites in the opposite direction).Wherein the PV-Cre mouse is given the cold spring port Dr.ZJ Huang laboratory in the U.S., and promptly No. five exon 3 ' UTR end at the parvalbumin gene inserts the Cre recombinase gene, and its article number in U.S. Jacksonlab company is: 008069.ErbB4
LoxP/LoxPMouse is given the laboratory in the Dr.LMei of georgia ,u.s.a university, and promptly one section loxp sequence places the erbb4 upper reaches, and another section places the erbb4 downstream.Appeal the hybridization of two kinds of mouse, in the filial generation mouse of generation, filter out through gene identification and to have PV-Cre-ErbB4
LoxP/LoxPGenotypic mouse.
Reagent: PTZ (pentylenetetrazole), pilocarpine (pilocarpine) is available from U.S. Sigma company.
Instrument: clear glass cage
(2) experimental technique:
I, according to document and preliminary experiment result, selecting PTZ concentration for use is 40mg/kg, pilocarpine200mg/kg is dissolved in PBS, and and intraperitoneal injection.After the injection PTZ, mice is put separately and gives in the clear glass cage, and observed behavior changes 30min.The epilepsy order of severity is according to the document classification: 0 grade: reactionless; 1 grade: ear is twitched with facial, comprises that nictation, moving palpus, rhythm chew; 2 grades: myoclonus, but do not have upright; 3 grades: myoclonus, the bilateral forelimb lifts; 4 grades: lean to one side to fall down to the ground; 5 grades: the back falls down to the ground, complete tetanus. grand mal: 6 grades: death.
II, two kinds of mouse have been assessed in the incidence rate of bringing out generalized seizure by PTZ.
III, also two kinds of mouse have been assessed in the incidence rate that causes status epilepticus by pilocarpine.
(3) experimental result:
I, Fig. 1 a, b are depicted as PV-Cre-ErbB4
LoxP/LoxPThe higher outbreak rank of epilepsy performance that mice is brought out pentylenetetrazole, and the percentage ratio that more shows effect.
II, Fig. 1 c are depicted as PV-Cre-ErbB4
LoxP/LoxPMice is brought out the higher incidence rate of epilepsy performance to pilocarpine.
(4) conclusion: specificity knocks out the easier epilepsy of sending out of mouse of ErbB4 receptor on the PV relay cell.
Embodiment 2: exogenous Intraventricular is injected NRG1 can suppress induced seizures
(1) experiment material:
Laboratory animal: male ErbB4
LoxP/LoxPMice, totally 20, in eight ages in week, body weight 24+2 gram is raised in identical environment, and food and water freely absorb; Give the illumination of 12h every day.Behavioristics's experiment is carried out between 12:00～14:00.
Reagent: PTZ, pilocarpine is available from U.S. Sigma company; Isoflurane (isoflurane) sends drugmaker available from Hebei nine;
Instrument; Stereotaxic instrument, model 512600 originates in U.S. Stoelting company; Anesthetic machine.
(2) experimental technique:
The result who draws according to the front can make such hypothesis: medicine irritation NRG1-ErbB4 axle possibly have antiepileptic effect.Continue isoflurane (2%) and suck anesthesia, mice is fixed under the stereotaxic instrument, treat anesthesia steadily after, (AP:-0.5mm, L:-1mm inject human recombination protein Neuregulin-1beta 2 (6mmol in 3 μ l) in V:-2.2mm) to right ventricle.After 15 minutes recovery, carry out intraperitoneal injection PTZ concentration be 60mg/kg or, pilocarpine 250mg/kg.Accordingly, matched group takes to inject right ventricle respective volume cerebrospinal fluid, after 15 minutes, intraperitoneal inject PTZ concentration be 60mg/kg or, pilocarpine250mg/kg.The epilepsy grading is the same.
(3) experimental result:
I, all mouse all can show the epilepsy symptom behind lumbar injection 60mg/kg PTZ, yet the rank and the attack times of injecting the mouse outbreak of NRG1 are starkly lower than matched group.
II, mouse are behind lumbar injection 250mg/kg pilocarpine, and be longer when diving in advance lower the and outbreak of the mouse status epilepticus incidence rate of NRG1.
(4) conclusion: exogenous Intraventricular is injected NRG1 can suppress induced seizures.
Embodiment 3:ErbB4 receptor expression in epileptic's brain of people descends
(1) experiment material:
Human brain tissue: 5 routine intractable epileptic's postoperative cerebral tissue meet following standard: I characteristic electroencephalogram, Drug therapy still have epilepsy, application 2～3 kind of a line antiepileptic and have reached blood drug level more than 2 years.II epilepsy symptom meets international epilepsy tissue typing standard in 1981.IIICT or MRI guide down not carrying out property kitchen range point.IV does not have the preceding assessment of other nervous system disease V art to belong to the relevant ED in position except that epilepsy.
The matched group cerebral tissue meets following standard: I does not have neural history of disease.II does not have other brain structures that can cause epilepsy or the damage of changing function except that wound.The age does not have significant difference between these two groups.
Reagent: anti-ErbB4 (ErbB4 antibody) and anti-ErbB2 (ErbB2 antibody) are available from U.S. Santa Cruz Biotechnology company, and anti-caveolin-1 (little recessed albumen 1 antibody) is available from U.S. Cell Signaling Technology company.
Instrument: the basic molecule experimental facilities that Western Blotting is required.
(2) experimental technique:
Protein quantification: cell lysis, extract protein lysate, be used for Western Blotting.Western Blotting one anti-concentration is: anti-ErbB4 (1: 1000), anti-ErbB2 (1: 1000) and anti-caveolin-1 (1: 1000).
(3) experimental result: epileptic's cerebral cortex shown in Figure 3 ground ErbB4 expression has only 60% of normal control group, yet the expression of ErbB2 does not but change.Fig. 3 b is seen in statistical result.
(4) conclusion: ErbB4 receptor expression in epileptic's brain of people descends.
Embodiment 4:NRG1 strengthens the irritability of cortex and hippocampus PV relay cell
(1) experiment material:
Laboratory animal: B13 and G42 mice are given the cold spring port Dr.ZJ Huang laboratory in the U.S..G42, promptly one section GAD67-GFP BAC recombinant is inserted in first section exon of GAD67gene, and its article number in U.S. Jacksonlab company is: 007677.B13, promptly one section Pv-GFPBAC recombinant is inserted in the PV gene.On the PV relay cell of these two kinds of mouse by the specific green fluorescence that indicates.
Reagent: NRG1 is available from U.S. Prospec company, and ecto-ErbB4: transfection ecto-ErbB4 (contain the ErbB4 extracellular fragment: the pErbB4ex/Fc of aa 1-659 and HEK293, cultivate, purification extracts ecto-ErbB4 by warp.The MEM culture medium, hyclone is available from U.S. Gibco company.
Instrument: cell cultivation equipment, culture bottle and incubator.Microtome is available from U.S. Leica company.The infrared interference microscope is available from Japanese Nikon company.The electrophysiological recording system: MultiClamp 700B amplifier, Digidata 1440A digital to analog converter and pClamp 10.2 logging softwares are available from U.S. Axon Instruments/Molecular Devices company.
(2) experimental technique:
Artificial cerebrospinal fluid preparation: 125mM NaCl, 3mM KCl, 1.25mM NaH
2PO
4, 2mMMgSO
4, 2mM CaCl
2, 25mM NaHCO
3, and 10mM glucose, solvent are deionized water;
Liquid preparation in the electrode: 130mM K-gluconate, 20mM KCl, 10mM Hepes, 4mMMg2ATP, 0.3mM NaGTP, 10mM disodium phosphocreatine and 0.2mMEGTA (pH 7.2with KOH, 288mOsm), solvent is a deionized water;
The brain sheet is prepared: after the Animal Anesthesia, get brain as far as possible fast, and keep low temperature.Crownly then cut 300 microns, after 33 ℃ of artificial cerebrospinal fluids are hatched 60 minutes, transfer under 22 ℃ of room temperatures, omnidistance 32 ℃～33 ℃ of electrophysiological recording.At first on relay cell, made full cell record of the green fluorescence labelling.The omnidistance inflation of used liquid 95%O
2/ 5%CO
2
Electrophysiological recording: brain sheet 2 or 3 layers, through 40 times of hydroscopes, fluorescence excitation guides down and finds the neuron that has green fluorescence, does full cell record, injects 500 milliseconds of electric currents under the current clamp pattern.
(3) experimental result:
The NRG1 of exogenous perfusion 10 nanomoles shown in I, Fig. 4 a has accelerated the granting frequency of cortex PV relay cell action potential.Perfusion again behind the heat inactivation NRG1 simultaneously is to not influence of frequency.Fig. 4 c is seen in statistical result.
Shown in II, Fig. 4 b, with the endogenic NRG1 of ecto-ErbB4 antagonism, the frequency of cortex PV relay cell action potential obviously descends.Perfusion again behind the heat inactivation ecto-ErbB4 simultaneously is to not influence of frequency.Fig. 4 c is seen in statistical result.
Shown in III Fig. 4 d, repeat as above to test, obtain similar effects in the hippocampus.
(4) conclusion: NRG1 strengthens the irritability of cortex and hippocampus PV relay cell
Embodiment 5:ErbB4 receptor is necessary for the irritability that NRG1 regulates relay cell
(1) experiment material:
Laboratory animal: G42-ErbB4
LoxP/LoxPMice is by G42 and ErbB4
LoxP/LoxPHybridization obtains; G42-PV-Cre-ErbB4
LoxP/LoxPMice is by G42-ErbB4
LoxP/LoxPWith PV-Cre-ErbB4
LoxP/LoxPHybridization obtains.
Reagent: NRG1 is available from U.S. Prospec company, and ecto-ErbB4: transfection contains pErbB4ex/Fc and the HEK293 of ecto-ErbB4, extracts ecro-ErbB4 through cultivation, purification.The MEM culture medium, hyclone is available from U.S. Gibco company.AG1478 is available from U.S. Sigma company.
Instrument: cell cultivation equipment, culture bottle and incubator.Microtome is available from U.S. Leica company.The infrared interference microscope is available from Japanese Nikon company.The electrophysiological recording system: MultiClamp 700B amplifier, Digidata 1440A digital to analog converter and pClamp 10.2 logging softwares are available from U.S. Axon Instruments/Molecular Devices company.
(2) experimental technique:
Artificial cerebrospinal fluid preparation: 125mM NaCl, 3mM KCl, 1.25mM NaH
2PO4,2mMMgSO
4, 2mM CaCl
2, 25mM NaHCO
3, and 10mM glucose, solvent are deionized water;
Liquid preparation in the electrode: 130mM K-gluconate, 20mM KCl, 10mM Hepes, 4mMMg2ATP, 0.3mM NaGTP, 10mM disodium phosphocreatine and 0.2mMEGTA (pH 7.2with KOH, 288mOsm), solvent is a deionized water;
The brain sheet is prepared: after the Animal Anesthesia, get brain as far as possible fast, and keep low temperature.Crownly then cut 300 microns, after 33 ℃ of artificial cerebrospinal fluids are hatched 60 minutes, transfer under 22 ℃ of room temperatures, omnidistance 32 ℃～33 ℃ of electrophysiological recording.At first on relay cell, made full cell record of the green fluorescence labelling.The omnidistance inflation of used liquid 95%O
2/ 5%CO
2
Electrophysiological recording: brain sheet 2 or 3 layers, through 40 times of hydroscopes, fluorescence excitation guides down and finds the neuron that has green fluorescence, does full cell record, injects 500 milliseconds of electric currents under the current clamp pattern.
(3) experimental result:
Shown in I, Fig. 5 a, used pharmacological method earlier, specific inhibition agent-AG1478 of ErbB4 can hinder the adjusting of NRG1 to middle neuronal excitability.And just can significantly the slow down granting frequency (figure) of PV relay cell of AG1478 itself, this further provides evidence for the effect of endogenous NRG1 again.
II shown in Fig. 5 b, knocks out the ErbB4 receptor on the PV relay cell with the transgenic technology method specificity of Cre-LoxP.Find that NRG1 no longer can accelerate the irritability of PV relay cell.This and pharmacological result are consistent, further produce evidence for the necessity of ErbB4.
(4) conclusion: the ErbB4 receptor is necessary for the irritability that NRG1 regulates relay cell.
Embodiment 6:
(1) experiment material:
Laboratory animal: B13 and G42 mice are given the cold spring port Dr.ZJ Huang laboratory in the U.S..G42, promptly one section GAD67-GFP BAC recombinant is inserted in first section exon of GAD67gene, and its article number in U.S. Jacksonlab company is: 007677.B13, promptly one section Pv-GFPBAC recombinant is inserted in the PV gene.On the PV relay cell of these two kinds of mouse by the specific green fluorescence that indicates;
Reagent: NRG1 is available from U.S. Prospec company, and ecto-ErbB4: transfection contains pErbB4ex/Fc and the HEK293 of ecto-ErbB4, extracts ecro-ErbB4 through cultivation, purification.The MEM culture medium, hyclone is available from U.S. Gibco company.DTx-K is available from U.S. Alomone Labs. company.
Instrument: cell cultivation equipment, culture bottle and incubator.Microtome is available from U.S. Leica company.The infrared interference microscope is available from Japanese Nikon company.The electrophysiological recording system: MultiClamp 700B amplifier, Digidata 1440A digital to analog converter and pClamp 10.2 logging softwares are available from U.S. Axon Instruments/Molecular Devices company.
(2) experimental technique:
Artificial cerebrospinal fluid preparation: 125mM NaCl, 3mM KCl, 1.25mM NaH
2PO
4, 2mMMgSO
4, 2mM CaCl
2, 25mM NaHCO
3, and 10mM glucose, solvent are deionized water;
Liquid preparation in the electrode: 130mM K-gluconate, 20mM KCl, 10mM Hepes, 4mMMg
2ATP, 0.3mM NaGTP, 10mM disodium phosphocreatine and 0.2mMEGTA (pH 7.2with KOH, 288mOsm), solvent is a deionized water;
The brain sheet is prepared: after the Animal Anesthesia, get brain as far as possible fast, and keep low temperature.Crownly then cut 300 microns, after 33 ℃ of artificial cerebrospinal fluids are hatched 60 minutes, transfer under 22 ℃ of room temperatures, omnidistance 32 ℃～33 ℃ of electrophysiological recording.At first on relay cell, made full cell record of the green fluorescence labelling.The omnidistance inflation of used liquid 95%O
2/ 5%CO
2
Electrophysiological recording: brain sheet 2 or 3 layers, through 40 times of hydroscopes, fluorescence excitation guides down and finds the neuron that has green fluorescence, does full cell record, the voltage clamp pattern.Used the analytical method of current subtraction to isolate the responsive potassium current Kv1.1 of DTx-K and receive the electric current that ecto-ErbB4 regulates.Obtain receiving the outward current that ecto-ErbB4 regulates to the current subtraction before and after the perfusion ecto-ErbB4.Or obtain the responsive potassium current Kv1.1 of DTX-k to current subtraction before and after the perfusion DTx-K.
(3) experimental result:
Shown in Figure 6, ecto-ErbB4 can increase whole export-oriented potassium current.The part of this increase is fallen (figure) by DTx-K complete inhibition afterwards.When hatching with DTx-k under the situation of brain sheet in advance, ecto-ErbB4 no longer can increase potassium current (figure).
(4) conclusion: this result shows that the part electric current that ecto-ErbB4 regulates is the responsive potassium current of DTx-K, i.e. Kv1.1 electric current.
Embodiment 7: Kv1.1 tyrosine phosphorylation level can receive the adjusting of NRG1-ErBB4 signal on the cell membrane.
(1) experiment material:
Laboratory animal: PV-Cre-ErbB4
LoxP/LoxPMice and brood control mice ErbB4
LoxP/LoxP
Reagent: anti-Kv1.1 (Kv1.1 antibody) is available from U.S. NeuroMab company, and anti-caveolin-1 (little recessed albumen-1 antibody) and anti-phosphotyrosine (tyrosine phosphorylation antibody) are available from U.S. Cell Signaling Technology company.
Instrument:
(2) experimental technique:
Immunoprecipitation: the cracking tissue, extract the memebrane protein lysate, the Kv1.1 albumen on the enrichment film is used for Western Blotting.
Protein quantification: protein quantification: the cracking tissue, extract protein lysate, be used for Western Blotting.Western Blotting one anti-concentration is: anti-Kv1.1 (1: 1000), anti-phospho-tyrosine and anti-caveolin-1 (1: 1000)
(3) experimental result:
Shown in I, Fig. 7 a, the Kv1.1 total protein is handled at NRG1 all not to be had in back 20 minutes obviously to change, yet the Kv1.1 passage tyrosine level on the cell membrane raise in 20 minutes gradually.
Shown in II, Fig. 7 b, PV-Cre-ErbB4
LoxP/LoxPMice Kv1.1 phosphorylation level is lower than ErbB4
LoxP/LoxPMice Kv1.1 level.
(4) conclusion: Kv1.1 tyrosine phosphorylation level can receive the adjusting of NRG1-ErBB4 signal on the cell membrane.
SEQUENCE LISTING
< 110>Zhejiang University
< 120>application of ErbB receptor stimulating agent in the medicine of preparation treatment epileptics
<130>
<160> 1
<170> PatentIn version 3.4
<210> 1
<211> 61
<212> PRT
<213> Unknown
<220>
< 223>artificial sequence
<400> 1
Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn
1 5 10 15
Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
20 25 30
Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr
35 40 45
Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln
50 55 60
Claims (4)
1.ErbB the application of receptor stimulating agent in the medicine of preparation treatment epileptics.
2. application as claimed in claim 1 is characterized in that said ErbB receptor stimulating agent is the ErbB4 receptor stimulating agent.
3. application as claimed in claim 2 is characterized in that said ErbB receptor stimulating agent is used to prepare the excitatoty medicine that strengthens cortex and hippocampus PV relay cell.
4. application as claimed in claim 3; It is characterized in that said ErbB receptor stimulating agent behaviour recombinant protein N euregulin-1 beta 2, its aminoacid sequence is following: shlvkcaekektfcvnggecfmvkdlsnpsrylckcpneftgdrcqnyvmasfyka eelyq.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108168827A (en) * | 2018-01-19 | 2018-06-15 | 清华大学 | A kind of physics head model and test system |
| CN108277272A (en) * | 2018-04-09 | 2018-07-13 | 重庆医科大学附属第医院 | A kind of marker and its detection kit of auxiliary Diagnosis of Epilepsy |
| CN110721307A (en) * | 2019-12-03 | 2020-01-24 | 广州中医药大学(广州中医药研究院) | Application of neuregulin 1 in preparing product for enhancing TRPC6 channel activity |
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| CN101243178A (en) * | 2005-06-16 | 2008-08-13 | 特拉维夫大学拉莫特有限公司 | Isolated cells and populations comprising same for the treatment of CNS diseases |
| CN101310779A (en) * | 2007-05-25 | 2008-11-26 | 上海泽生科技开发有限公司 | Device and medicinal preparation containing neuroregulation protein |
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2011
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| CN101243178A (en) * | 2005-06-16 | 2008-08-13 | 特拉维夫大学拉莫特有限公司 | Isolated cells and populations comprising same for the treatment of CNS diseases |
| CN101310779A (en) * | 2007-05-25 | 2008-11-26 | 上海泽生科技开发有限公司 | Device and medicinal preparation containing neuroregulation protein |
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| CN108168827A (en) * | 2018-01-19 | 2018-06-15 | 清华大学 | A kind of physics head model and test system |
| CN108168827B (en) * | 2018-01-19 | 2019-08-02 | 清华大学 | A kind of physics head model and test macro |
| CN108277272A (en) * | 2018-04-09 | 2018-07-13 | 重庆医科大学附属第医院 | A kind of marker and its detection kit of auxiliary Diagnosis of Epilepsy |
| CN110721307A (en) * | 2019-12-03 | 2020-01-24 | 广州中医药大学(广州中医药研究院) | Application of neuregulin 1 in preparing product for enhancing TRPC6 channel activity |
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