CN106279358B - A kind of synthetic peptide for treating cerebral injury - Google Patents

Abstract

The invention belongs to biomedicine technical fields, and in particular to a kind of synthetic peptide and its purposes in preparation treatment cerebral injury drug.The amino acid sequence of the synthetic peptide is X1-X2-L-Ala- (L-Cys-Gly) n-X3(I), in formula, X1 be without or L-Phe；X2 is nothing or L-Cys；X3 is nothing or L-Phe；X1, X3 cannot be nothing simultaneously; n≤8; animal experiments show that such synthetic peptide can pass through intravenous administration approach under extremely low dosage; through blood-brain barrier; reach brain; hydrolysis generates free glycine and cysteine and plays neuroprotection under the action of intracerebral proteolytic enzyme, reduces the ischemic perfusion injury of brain, plays the purpose for the treatment of cerebral apoplexy.

Description

A kind of synthetic peptide for treating cerebral injury

Technical field

The invention belongs to biomedicine technical fields, and in particular to synthetic peptide and its preparation treatment cerebral injury drug in Purposes.

Background technique

Glycine is the simplest amino acid of chemical structure, but has the function of complexity.Glycine is in central nervous system In be a kind of important inhibitory neurotransmitter, play a significant role in terms of controlling neuronal excitability.Zoopery discovery Glycine has apparent neuroprotection, and it has also been found that glycine can obviously improve cognition function in some clinical tests Energy obstacle and dementia especially can be used as the neuroplegic treatment schizophrenia an of routine.In addition to this, glycine Heart injury and cerebral apoplexy can also be treated.

L-cysteine is a kind of neuromodulator and a kind of antioxidant with neuroprotection.Meanwhile It is also the important synthesis unit of intracerebral glutathione, and the glutathione of synthesis is for cerebral ischemia re-pouring injured and Parkinson's disease There is apparent therapeutic effect.

L-phenylalanine is one of essential amino acid, belongs to aromatic amino acid.Existing research shows L-phenylalanine Drug molecule is introduced directly into carcinoma area by the carrier that can be used as anticancer drug, and effect is 3~5 times of other amino acid.

Although glycine and cysteine have an apparent cerebral protection, periphery to glycine (800mg/kg) and Cysteine (500mg/kg) dosage is excessive, causes side reaction serious, limits their clinical application.

Summary of the invention

In view of the problems of the existing technology the present invention, provides a new class of synthetic peptide, it is the half Guang ammonia by glycine Acid, four kinds of natural amino acids of alanine and phenylalanine be raw material dehydration be condensed, be biologically it is active, examining Disconnected learn is useful and has the function for the treatment of cerebral injury.These peptides are particularly stable and easily penetrate blood-brain barrier, therefore logical It is still effective for crossing intravenous route administration.

The first aspect of the present invention, provides a kind of synthetic peptide, structure feature, from N-terminal to C-terminal such as following formula I:

X1-X2-L-Ala- (L-Cys-Gly) n-X3 (I), in formula,

X1 is nothing or L-Phe

X2 is nothing or L-Cys

X3 is nothing or L-Phe

X1, X3 cannot be nothing, the number of repetition of the arrangement that n≤8, n are L-Cys-Gly in the sequence, such synthesis simultaneously There are three kinds of proteolysis cleavage sites in peptide, one be L-Cys and Gly tie point, one is L-Cys and L-Ala Tie point, one be other arbitrary amino acids in peptide both ends L-Phe and Formulas I tie point, generate trip after proteolytic enzyme cutting From Gly and L-Cys as drug or pharmacological activity group, L-Ala is hydrophobic amino acid, promotes synthetic peptide penetrating cell film, Carrier of the L-Phe as penetrating cell film can carry synthesis peptide molecule and pass through blood-brain barrier, into brain.

Amino acid abbreviations are referred to as follows in the present invention:

Glycine: Gly, G；L-cysteine: L-Cys, C；L-phenylalanine: L-Phe, F；L-Alanine: L-Ala, A.

Cys, Phe, Ala mentioned in the present invention are L-types.

In another preferred example, synthetic peptide can selected from it is following any one:

(a)NH₂-L-Phe-L-Ala-L-Cys-Gly-COOH；

(b)NH₂-L-Phe-L-Cys-L-Ala-L-Cys-Gly-COOH；

(c)NH₂-L-Cys-L-Ala-L-Cys-Gly-L-Phe-COOH；

(d)NH₂-L-Phe-L-Ala-(L-Cys-Gly)₄-COOH；

(e)NH₂-L-Phe-L-Ala-(L-Cys-Gly)₈-COOH；

(f)NH₂-L-Ala-(L-Cys-Gly)₄-Phe-COOH；

(g)NH₂-L-Phe-L-Cys-L-Ala-L-Cys-Gly-L-Phe-COOH；

Another aspect of the present invention is to provide application of such synthetic peptide in preparation treatment nervous system disease agent.

Preferably, such synthetic peptide is provided and treats cerebral arterial thrombosis, hemorrhagic apoplexy, cerebral trauma, A Er in preparation Ci Haimo is sick, the application in Parkinson's disease and other neurodegenerative disease drugs.

The drug is injection.

Preferably, such synthetic peptide when treating the nervous system disease by the way of intravenous injection for being administered.

The present invention provides the method by all kinds of synthetic peptides of the synthesis in solid state preparation present invention in a particular embodiment.Specifically with The embodiment of L-Phe-L-Ala-L-Cys-Gly is described.

In order to determine whether the other synthetic peptides of this patent also have cerebral protection, it is sugared in vitro that we establish cortical neuron Oxygen deprives (OGD) model, and neuron handles neuron under OGD degree of impairment, with 500 μM of each synthetic peptides, goes to comment with cck8 The vigor of valence cell, to judge each synthetic peptide, to neuron, whether there is or not protective effects.From Figure 2 it can be seen that each synthetic peptide has protection Effect, wherein FACG, FCACG, CACGF, FCACGF synthetic peptide have obvious protective function to injured neuron.

The present invention uses SD rat as experimental subjects, prepares rats with cerebral ischemia mould using brain middle cerebral artery occlusion method (MCAo) Type, intravenous (IV) drug after ischemic 3 hours take brain, brain tissue frozen section, in fluorescence microscopy microscopic observation brain piece after 1 hour Middle fluorescent material intensity.

Using intravenous injection FITC, (fluorescein isothiocynate, burst of ultraviolel can fluoresced greens；8 μm of ol/kg) label is greatly Mouse cranial nerve cell has no the nerve cell of fluorescent marker in as a result visible non-cerebral ischemic rats brain tissue；Cerebral ischemic rats brain tissue The green fluorescence nerve cell of visible minute quantity FITC label under middle mirror；FITC-CG is injected after cerebral ischemia, is also only observed under mirror Minute quantity fluorecyte；FITC-FCG and FITC-FACG (8 μm of ol/kg), visible a large amount of fluorescent markers under mirror are injected after cerebral ischemia Nerve cell, and the latter is more more than the fluorecyte that the former arrives under the microscope, and fluorescence signal is stronger (see attached drawing 3), Show carrier of the Phe as penetrating cell film, CG can be carried and penetrate blood-brain barrier, into brain, and Ala is as hydrophobicity ammonia Base acid by way of intravenous injection, further increases the ability that FCG penetrates blood-brain barrier, makes FACG in rat ischemia More efficient enters brain.

After entering brain through blood-brain barrier by intravenous injection mode in order to illustrate FACG, it is by proteolytic enzymes hydrolize Gly and L-Cys, increase the horizontal of the Gly and L-Cys of intracerebral, we establish rat cerebral ischemia model, after be divided into two immediately Group, one group is control group, is injected intravenously physiological saline after ischemic 5h；Another group is experimental group, is injected intravenously 12mg/kg FACG, Brain is taken afterwards for 24 hours, with the content of HPLC measurement rat cerebral ischemia damage zone Gly and L-Cys.The result shows that after rat cerebral ischemia, it is quiet Arteries and veins gives FACG group, and the content of rat cerebral ischemia area Gly and Cys are above after ischemic to physiological saline group, illustrates vein to ischemic After rat FACG, FACG penetrates blood-brain barrier, and into brain, hydrolysis generates Gly and L-Cys.(see attached drawing 4)

The present invention uses SD rat as experimental subjects, and FACG (12mg/kg), Edaravone (clinical brain are given in intravenous injection Treatment of Stroke drug；6mg/kg), using MCAo Rat Model with Cerebral Ischemia, 5h after ischemic, vein disposably injects said medicine. After for 24 hours, animal is put to death, takes brain, rear TTC dyeing, Infarction volume is with ([(VC-VL)/V C] indicates that VC is control hemisphere Product, VL is the non-Infarction volume of lesioned hemisphere).

As a result as it can be seen that the rats with cerebral ischemia of injecting normal saline, Infarction volume 0.359 ± 0.041；Give 12mg/kg FACG, Infarction volume 0.021 ± 0.008；6mg/kg Edaravone is injected, Infarction volume is 0.141 ± 0.015；Relative to quiet Arteries and veins has a lower Infarction volume to physiological saline and Edaravone group, to GCAF tetrapeptide medicine group, and statistically significant (p < 0.05) (2 are shown in Table, attached drawing 5).

In short, the present invention has the following advantages:

(1) synthetic peptide provided by the invention can be by intravenous injection, using L-Phe as the carrier of penetrating cell film, L-Ala It is hydrophobic amino acid, promotes synthetic peptide penetrating cell film, through blood-brain barrier, into brain, in intracerebral proteolytic enzyme The lower hydrolysis of effect generates free glycine and cysteine and plays neuroprotection, reduces the ischemic perfusion injury of brain, rises To the purpose for the treatment of cerebral apoplexy.In addition, glycine in addition to it can be used for Treatment of Cerebral Stroke, also can obviously improve cognition dysfunction and Dementia especially can be used as the neuroplegic treatment schizophrenia an of routine.And L-cysteine and with it is The glutathione of basis synthesis can be obviously improved the oxidativestress damage and brain of Parkinsonian as a kind of powerful antioxidant Ischemical reperfusion injury.

(2) Small side effects: the present invention only needs the FACG of 12mg/kg to be injected intravenously dosage, that is, can reach significant treatment Effect.

(3) safety: the amino acid residue of the synthetic peptide is derived from the amino acid of 20 kinds of composition human body proteins, safe and non-toxic.

(4) economical: the synthetic peptide is obtained by synthesis in solid state, and cost is relatively low and is convenient for quality management.

Detailed description of the invention

Fig. 1 is the HPLC map and mass spectrogram of this patent peptide FACG and FCACGF；

Fig. 2 is the influence diagram that synthetic peptide survives to cortical neuron；

Fig. 3 is the synthetic peptide of FITC label in rat cerebral ischemia, through blood-brain barrier, into the signal of brain Figure；

Fig. 4 is HPLC chromatogram

A is the DNFB of blank, and B is Gly and L-Cys standard items, and C is containing for rat cerebral ischemia side damage zone Gly and L-Cys Amount；Peak 1 indicates that the eluting peak of Gly, peak 2 indicate the eluting peak of L-Cys.

Fig. 5 is that cerebral ischemic rats inject schematic diagram after each drug to rat cerebral infarction stereomutation；

Specific embodiment

By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.

[embodiment 1] this patent is by taking L-phenylalanine-l-Alanine-L-cysteine-glycine tetrapeptide (FACG) as an example Illustrate that the preparation process of solid phase synthesis of peptide is as follows:

One, the synthesis of composite I Fmoc-Gly- resin:

The method of this patent tripeptides synthesis in solid state weighs the 2- chlorine trityl chloride resin that degree of substitution is 0.4mmol/g (2-Chlorotrityl Chloride Resin) 10.7g, resin is put into polypeptides reactive pipe, is added methylene chloride (DCM) 160.5ml shakes 30 minutes；DCM solvent is leached out by husky core, the Fmoc-Gly-OH amino acid of 12mmol is added, adds The n,N-diisopropylethylamine (DIEA) of 40mmol is eventually adding a small amount of dimethylformamide (DMF) dissolution, vibrates 1h.Use DMF With DCM alternately cleaning 6 times.Add 160.5ml20% piperidines (piperidines is dissolved in DMF solution), takes out piperidines after five minutes, again 160.5ml20% piperidines is added, piperidine solution is taken out after waiting 15 minutes, Cong Guanzhong negates the resin after answering more than ten, uses ethyl alcohol It washes three times, ninhydrin is added, potassium cyanide solution, each drop of phenol solution, 105 DEG C of -110 DEG C of heating 5min, it is positive for deepening blue Property reaction, illustrate Fmoc-Gly-OH couple with resin successfully, formation composite I Fmoc-Gly- resin.

Two, the synthesis of Complex II Fmoc-Cys (Trt)-Gly- resin:

Polypeptides reactive pipe is washed twice with 160.5ml DMF respectively, and 160.5ml methanol is washed twice, and 160.5ml DMF washes two again It is secondary；Couple second amino acid on Fmoc-Gly- resin complexes afterwards, is added in reaction tube on a small quantity with DMF dissolution 12mmolFmoc-Cys (Trt)-OH and 12mmol benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphate (HBTU), N-methylmorpholine (NMM) 40mmol is added immediately afterwards, reacts 30min, reaction tube is washed once with 160.5ml DMF respectively, 160.5ml methanol is washed twice, and 160.5ml DMF is washed twice again.Add 160.5ml20% piperidines (piperidines is dissolved in DMF solution), Take out piperidines after five minutes, again be added 160.5ml20% piperidines, piperidine solution is taken out after waiting 15 minutes, Cong Guanzhong is negated answer after Resin more than ten, with ethyl alcohol wash three times, be added ninhydrin, potassium cyanide solution, phenol solution it is each one drop, 105 DEG C -110 DEG C plus Hot 5min, deepening blue is positive reaction, illustrates that Fmoc-Cys (Trt)-OH couples successfully with Fmoc-Gly- resin, is formed new Complex II Fmoc-Cys (Trt)-Gly- resin.

Three, the synthesis of Complex II I Fmoc-L-Ala-Cys (Trt)-Gly- resin:

Polypeptides reactive pipe is washed twice with 160.5ml DMF respectively, and 160.5ml methanol is washed twice, and 160.5ml DMF washes two again It is secondary；Couple third amino acid on Fmoc-Cys (Trt)-Gly- resin afterwards, is added in reaction tube on a small quantity with DMF dissolution 12mmolFmoc-L-Ala-OH and 12mmolHBTU, after be added N-methylmorpholine (NMM) 40mmol immediately, react 30min, instead Should pipe washed once with 160.5ml DMF respectively, 160.5ml methanol is washed twice, and 160.5ml DMF is washed twice again.Add 160.5ml20% piperidines (piperidines is dissolved in DMF solution), takes out piperidines after five minutes, and 160.5ml20% piperidines is added again, Deng taking out piperidine solution after 15 minutes, Cong Guanzhong negates the resin after answering more than ten, is washed three times with ethyl alcohol, and ninhydrin, cyanogen is added Change potassium solution, each drop of phenol solution, 105 DEG C of -110 DEG C of heating 5min, deepening blue is positive reaction, illustrates Fmoc-L- Ala-OH couples with Complex II successfully forms new Complex II IFmoc-L-Ala-Cys (Trt)-Gly- resin.

Four, the synthesis of complex IV Fmoc-L-Phe-L-Ala-Cys (Trt)-Gly- resin:

Polypeptides reactive pipe is washed twice with 160.5ml DMF respectively, and 160.5ml methanol is washed twice, and 160.5ml DMF washes two again It is secondary；Couple the 4th amino acid on Fmoc-L-Ala-Cys (Trt)-Gly- resin afterwards, is added in reaction tube and uses DMF on a small quantity The 12mmolFmoc-L-Phe-OH and 12mmolHBTU of dissolution, after N-methylmorpholine (NMM) 40mmol is added immediately, react 30min, reaction tube are washed once with 160.5ml DMF respectively, and 160.5ml methanol is washed twice, and 160.5ml DMF is washed twice again.Add 160.5ml20% piperidines (piperidines is dissolved in DMF solution), takes out piperidines after five minutes, and 160.5ml20% piperidines is added again, Deng taking out piperidine solution after 15 minutes, Cong Guanzhong negates the resin after answering more than ten, is washed three times with ethyl alcohol, and ninhydrin, cyanogen is added Change potassium solution, each drop of phenol solution, 105 DEG C of -110 DEG C of heating 5min, deepening blue is positive reaction, illustrates Fmoc-L- Phe-OH couples with Complex II I Fmoc-L-Ala-Cys (Trt)-Gly- resin successfully forms new complex IV Fmoc- L-Phe-L-Ala-Cys (Trt)-Gly- resin.

Five, crude product peptide is obtained:

Polypeptides reactive pipe is washed twice with 160.5ml DMF respectively, and 160.5ml methanol is washed twice, and 160.5ml DMF washes two again It is secondary, 10min is drained, prepares cutting liquid 107ml, formula is TFA (trifluoroacetic acid) 94.5% respectively；Water 2.5%；EDT (1,2- second Two mercaptan) 2.5%；TIS (tri isopropyl silane) 1%, resin is fitted into flask or centrifuge tube, pours into cutting liquid, constant temperature 2h is shaken, cutting liquid is dried up as far as possible with nitrogen, ether chromatography, then washed 6 times with ether, then room temperature volatilizes, and it is thick to obtain 1.2g Product peptide Phe-Ala-Cys-Gly.

Five, crude product peptide Phe-Ala-Cys-Gly is purified

Instrument: LC3000 type high performance liquid chromatograph

Pillar: Gemini-NX C18 10um 120A 4.6*250mm

Mobile phase A: 0.1%TFA in 100%Acetonitrile

Mobile phase B: 0.1%TFA in 100%Water

According to above-mentioned purification condition, by crude product peptide (1.2g), (each sample introduction 100mg) is entered in HPLC, is separated Main peak fraction is collected in purifying, is put freeze-drying (48h) on freeze dryer, is finally obtained 99% or more sterling 600mg.

The HPLC map of Figure 1A L-phenylalanine-l-Alanine-L-cysteine-glycine tetrapeptide (FACG) tetrapeptide with And mass spectrogram, the visible tetrapeptide purity is 99.5% in HPLC figure；Mass spectrogram result is as it can be seen that [M+H]⁺397.00 therefore the object Matter molecular weight is 396, is consistent with L-phenylalanine-l-Alanine-L-cysteine-glycine tetrapeptide theoretical molecular weight.

Figure 1B L-phenylalanine-L-cysteine-l-Alanine-L-cysteine-glycine-L-phenylalanine hexapeptide (FCACGF) the HPLC map and mass spectrogram of hexapeptide, the visible tetrapeptide purity is 98.72% in HPLC figure；Mass spectrogram result can See, [M+H]⁺647.10, therefore the molecular weight of material is 646.10, with L-phenylalanine-L-cysteine-l-Alanine-L- Cysteine-Glycine-L-phenylalanine hexapeptide theoretical molecular weight is consistent.

Using the method for same synthesis in solid state, this laboratory has synthesized other synthetic peptides in this patent embodiment.

Cell toxicity test of [embodiment 2] this patent synthetic peptide to Primary cortical neurons

The present embodiment uses the Primary cortical neurons of culture 10 days as experimental subjects, and it is sugared in vitro to establish cortical neuron Oxygen deprives (OGD) model, and neuron handles neuron under OGD degree of impairment, with 500 μM of each synthetic peptides, goes to comment with cck8 The vigor of valence cell, to judge each synthetic peptide, to neuron, whether there is or not protective effects.Synthetic peptide used in the present embodiment is as follows:

The synthetic peptide that 1 the present embodiment of table is related to

One, major experimental reagent and instrument

Each synthetic peptide is independently synthesized by this laboratory in experiment；Neurobasal is provided by Gibco company；B27, Gibco company provides；FBS is provided by invitrogen company；Glutamax, invitrogen company provide；Glutamic Acid is provided by sigma company；Poly-D-lysine is provided by sigma company.Incubator cultivates item from SANYO company Part is 37 DEG C, 5%CO2；Microplate reader, Bio-Tek company, the U.S.；CCK8 kit, Japanese colleague's chemistry；Rest part reagent is complete The analytical reagents provided for Sinopharm Chemical Reagent Co., Ltd..

Two, the preparation of main solution

1, HBSS:Hank, S equilibrium liquid: NaCl 8.0g, KCl 0.4g, Glucose 1.0g, KH2PO40.06g, Na2HPO4 0.06g, adds distilled water to dissolve, with NaHCO3 tune PH to 7.3, constant volume to 1000ml.

2, separating liquid: HBSS 500ml, Hepes 1.78g, Sucrose 2.5g, Glucose 10.0g, PH are adjusted to 7.4, It is eventually adding 200ml distilled water, 4 degree of preservations.

3, ECS solution: NaCl 8.01g, CaCl₂ 0.222g,KCl 0.4g,MgCl₂ 0.095g,HEPES2.98g, Glucose 3.27g, adds distilled water to dissolve, and PH7.4 is settled to 1000ml, with the membrane filtration degerming of 0.22um.

Sugar-free ECS solution: NaCl 8.01g, CaCl₂ 0.222g,KCl 0.4g,MgCl₂ 0.095g,HEPES2.98g, Distilled water is added to dissolve, PH7.4 is settled to 1000ml, with the membrane filtration degerming of 0.22um.

4, Glutamic acid stock solution (11.8mM): claiming the glutamic acid of 0.1736g to be dissolved with distilled water, NaOH tune PH To 7.0,100ml is quantitatively arrived, 1060 μ l/tube is dispensed, is stored in 4 degree.

5,0.5%FBS culture medium: B27 takes 1ml, FBS 250ul, glutamax 500ul, glutamic acid106 μ L, it is quantitative to 50ml, 4 degree of preservations with neurobasal.

6, without FBS culture medium: B27 takes 1ml, 500 μ l of glutamax, quantitative to 50ml, 4 degree of preservations with neurobasal.

7,0.15M borate buffer solution: weighing the borax of 28.6g, dissolved with distilled water, 3-5min is stirred, with HCl tune PH To 8.4, constant volume to 500ml.

8, poly-D-lysine: being dissolved into the poly-D-lysine solid of 5mg in the borate buffer solution of 500ml, crosses and filters out Bacterium, -20 DEG C of preservations.

Three, experimental animal

SPF grades of ICR female mices of animal, weight 25-30g.Wuhan University's animal experimental center provides, animal certificate number For No.42000500006525, production licence number: SCXK (Hubei Province) 2014-0004.

Four, experimental principle

The principle of cck8: contain WST-8 (chemical name: 2- (2- methoxyl group -4- nitrobenzophenone) -3- (4- nitro in the reagent Phenyl) -5- (2,4- disulfonic acid benzene) -2H- tetrazolium monosodium salt), it, can be by line grain in the presence of electronics coupled reagent Intracorporal dehydrogenase reduction generates the orange-yellow formazan product (Formazan) of high water soluble.The depth of color and cell It is proliferated directly proportional, is inversely proportional with cytotoxicity.

Five, experimental method

The originally culture of neuron: culture evening before that day is coated with culture dish with poly-D-lysine, overnight；The next morning Coated poly-D-lysine is taken out, is washed 2-3 times before inoculation with PBS.Take the ICR female mice of pregnancy 17-18d, cervical dislocation After execution, tire mouse brain is taken, is placed in separating liquid, taken out full brain after opening cranial cavity and be placed in another glass culture for filling separating liquid In ware, separation removing meninx, takes cerebral cortex to set in another glass capsule for filling separating liquid under anatomical lens；With ophthalmology tweezer Onal layer tissue is at 1mm³Size, suction pipe aspirates tissue are set in the centrifuge tube of a 15ml, stand several seconds, tissue is allowed to precipitate, Separating liquid is discarded, the 0.5%FBS culture medium of 1-2ml is added, is blown and beaten 20-30 times with suction pipe, liquid turns turbid stops, then again 10-20 seconds are stood, big tissue block is made to sink, takes supernatant strainer filtering, filtered liquid first counts cell, with 4*10⁵/ml Cell density cell inoculation into coated ware.The culture medium for abandoning half volume for second day from each ware, changes no FBS into Culture medium change primary full culture medium every three days, what is changed is all the culture medium of no FBS later.

Cell treatment process: the primary neuron of 96 orifice plates of culture 10 days is taken, is simply divided into three groups, is respectively Each synthetic peptide group of Control, OGD, OGD+, synthesis peptide concentration is all 500 μM, 5 multiple holes of every group of repetition, each Kong Xianyong PBS It washes 2-3 times, ECS is added in control group, is put into containing 5%CO₂Incubator in 37 °C continue cultivate 1h, OGD group addition sugar-free ECS is put into 95%N₂/ 5%CO₂Incubator in 37 DEG C of culture 1h, the ECS that rear control group renews continues to cultivate 12h, and It is transferred to after the ECS that OGD group renews containing 5%CO₂Incubator in 37 DEG C continue to cultivate 12h, other OGD+ synthetic peptide groups are added each Synthesis peptide medicine (peptide is dissolved in ECS) is also transferred to containing 5%CO₂Incubator in 37 DEG C continue to cultivate 12h, CCK8 is added in every hole Light absorption value is surveyed after 10 μ l, 2h at 450nm.

Six, experimental result

As a result see Fig. 2, neuron handles neuron under OGD degree of impairment, with 500 μM of each synthetic peptides, finds each conjunction There is protective effect at peptide, wherein FACG, FCACG, CACGF, FCACGF synthetic peptide there is obvious protection to make injured neuron With.

[embodiment 3] establishes rat cerebral ischemia model, is injected intravenously the synthetic peptide of fluorescent marker, on the whole observation synthesis Peptide penetrates the ability of blood-brain barrier

One, main agents and instrument are tested

FITC, the Wuhan biotech inc Ming Hao；FITC-CG, FITC-FCG, FITC-FACG, Wuhan are bright white Biotech inc；OCT glue, Beijing Bioisystech Co., Ltd, Zhong Shan Golden Bridge；Histopathologic slide's machine, Germany Leitz, 1512 types；Fluorescence microscope, Ningbo ShunYu Instruments Co., Ltd；Paraformaldehyde, the limited public affairs of Chinese medicines group chemical reagent Department.

Two, experimental principle

FITC (fluorescein isothiocynate): for yellow or orange-yellow crystalline powder, molecular weight 389.4, absorption maximum light Wavelength is 490~495nm, and bright yellow-green fluorescence is presented in a length of 520~530nm of emission maximum light wave, is current using most Extensive fluorescein.

FITC-FACG: being exactly that FACG carries out fluorescent marker, FITC does not influence FACG tetra- in conjunction with FACG tetrapeptide in conjunction with after The structure and function of peptide, by fluorescence microscopy under the microscope, have strong yellow-green fluorescence, can be used for FACG positioning or Quantitative detection.

Blood-brain barrier (Blood Brain Barrier): refer to the blood plasma that brain capillary wall and Deiter's cells are formed Barrier between barrier between brain cell and the blood plasma and cerebrospinal fluid that are formed by choroid plexus, these barriers can prevent certain Substance (being harmful mostly) enters brain tissue by blood, maintains the normal physiological status of central nervous system.But due to being permitted The drug of more treatment central nervous system diseases is difficult to limit their applications clinically through blood-brain barrier.

Three, experimental result

As a result visible Fig. 3, Control are normal animals iv injecting normal saline group, microscopic observation after slice, unstressed configuration The nerve cell of label；It is injected intravenously 8 μm of ol/kg groups of FITC after ischemic, the mind for having minute quantity fluorescent marker can be observed under mirror Through cell；FITC-CG is injected after cerebral ischemia, also only observes minute quantity fluorecyte under mirror；FITC-FCG is injected after cerebral ischemia With FITC-FACG (8 μm of ol/kg), the nerve cell of visible a large amount of fluorescent markers under mirror, and the latter arrives under the microscope than the former Fluorecyte it is more, fluorescence signal is stronger (see attached drawing 3), shows carrier of the Phe as penetrating cell film, can carry CG Blood-brain barrier is penetrated, into brain, and Ala is as hydrophobic amino acid, in rat ischemia, by intravenous injection side Formula further increases the ability that FCG penetrates blood-brain barrier, makes the entrance brain that FACG is more efficient.

[embodiment 4] establishes rat cerebral ischemia model, is injected intravenously and detects intracerebral glycine and cysteine after FACG It is horizontal

One, laboratory apparatus and reagent

U.S.'s Agilent1100 high performance liquid chromatograph, UV detector and Chromatographic data system；Gly, Cys standard Product (sigma company, the U.S.)；2,4- dinitrofluorobenzene (Chengdu West Asia reagent Co., Ltd)；Acetonitrile, methanol (chromatographic grade, traditional Chinese medicines Chemical reagent Co., Ltd, group)；Ultrapure water；Remaining reagent is that analysis is pure.

Two, experimental method

The preparation precision of amino acid standard stock solution weighs Gly, and each 10mg of Cys standard items is placed in 10ml volumetric flask In, it is dissolved with methanol/water (1:1) and is diluted to scale, make the stock solution for being made into concentration 1mg/ml.

The preparation DNFB of derivatization reagent is made into 0.5% solution with acetonitrile, is kept in dark place.

Sample pretreatment and derivatization establish rat cerebral ischemia model, after be divided into two groups immediately, one group is control group, is lacked Physiological saline is injected intravenously after blood 5h；Another group is experimental group, is injected intravenously 13mg/kgCGPF, takes brain afterwards for 24 hours, divide on ice From ischemic brain damage zone, tissue homogenate is prepared with physiological saline, 15000rpm, 4 degree of centrifugation 20min take supernatant, a part BCA is surveyed, another part adds two volumes methanol, and 15000rpm, 4 degree of centrifugation 10min remove deproteinized, and gained supernatant is For measuring the content of glycine and cysteine.

Chromatographic condition chromatographic column: Dalian Yi Lite Hypersil BDS C18 (4.6 × 200mm, 5 μm)；Mobile phase: A is The sodium-acetate buffer (pH=6.5) of 0.05mol/L, B be acetonitrile-water (1:1, V/V), gradient elution program B by it is initial when 16% increases to 45%, 25min to 85%, 30min to 16% through 10min；Flow velocity: 1mL/min；Detection wavelength: 360nm；Column temperature: 30 DEG C, taking 30 μ L, sample introduction is analyzed.Under the chromatographic condition, glycine and cysteine are efficiently separated in 30min, when reservation Between are as follows: chromatogram obtained by Gly (10.21min), L-Cys (23.71min) is shown in Fig. 4.

Linear relationship precision measures Gly and Cys hybrid standard stock solution, is distinguished stock solution with methanol/water (1:1) dilute It is interpreted into the comparison liquid of 0.1,1,4,10,40,200ug/ml.30ul sample introduction is respectively taken after derivative, is measured with above-mentioned chromatographic condition, Linear regression is carried out with peak area (Y) and standard concentration (X).The result shows that Gly and Cys are within the scope of 0.1~200ug/ml Linear relationship is good, and related coefficient Gly is 0.9991, Cys 0.9997.

Three, experimental result

The assay of sample measurement rat cerebral ischemia damage zone Gly and Cys the results are shown in Table 1.

The content of table 1 rat cerebral ischemia damage zone Gly and Cys

Compared with model group,^**P < 0.01,^#P<0.05

After rat cerebral ischemia, after vein gives FACG group, the content of rat cerebral ischemia damage zone Gly and Cys to be above ischemic To physiological saline group, after illustrating vein to cerebral ischemic rats FACG, FACG penetrates blood-brain barrier, into brain, hydrolysis generate Gly and Cys。

[embodiment 5] establishes rat cerebral ischemia model, determines FACG to the protective effect after cerebral ischemia

One, experimental drug and reagent

FACG is independently synthesized by this experiment；Chloraldurate, Sinopharm Chemical Reagent Co., Ltd.；TTC, Wuhan section are auspicious Bioisystech Co., Ltd provides；Sterile saline, Binghu Shuanghe Pharmaceutical Co., Ltd., Wuhan；Heparin sodium injection, Nanjing Xinbai Pharmaceutical Co provides；Edaravone Injection, Sino Pharm Group Guorui Pharmaceutical Co., Ltd, 30mg/ mono-.

Two, experimental animal

SPF grades of SD male rats of animal, weight 240-260g.Wuhan University's animal experimental center provides, the animal quality certification Number be NO.42000500006247, production licence number: SCXK (Hubei Province) 2014-0004.Mouse feed is purchased from Wuhan University's experiment Animal center.

Three, experimental method

The rat of 240-260g is grouped at random: sham-operation group, ischemia group, ischemic dosing group, and sham-operation group and ischemia group are quiet Arteries and veins all gives the physiological saline of 400 μ l, and the drug concentration of ischemic dosing group is FACG group 12mg/kg, Edaravone group 6mg/ respectively Kg, drug are dissolved into 400 μ l physiological saline, and vein is disposably given after ischemic 5h.

The selection of animal administration mode: there are mainly three types of the peripherally administered modes of animal, be respectively it is oral, intraperitoneal injection and Intravenous injection.Conventional oral drug and intraperitoneal injection of drugs, drug require first by entering blood after intestinal absorption, and drug is being inhaled It needs the regular hour during receiving and has a degree of loss, it is just few into the drug in blood.And vein is infused Drug is penetrated relative to convenient administration mode, drug is to be directly entered blood, blood drug concentration 100%, and is directly played Effect.In the Valid concentration of drug, drug concentration needed for selecting intravenously administrable is lower, and action time is shorter, and pair is made With small, therefore this experimental selection intravenously administrable.

The preparation of arteria cerebri media embolism (MCAO) model: first using 10% chloraldurate according to the weight of animals (0.035ml/kg) carries out intraperitoneal injection of anesthesia, prepares arteria cerebri media ischemia model with the line brush of improvement after anaesthetizing successfully. Selection and the matched line bolt of SD rat body weight, preoperative elder generation's soaking is in the EP pipe for filling heparin.One is cut in external carotid artery distal end After osculum, there is the line bolt of round end from osculum front end, internal carotid is inserted into after adjustment direction, line bolt insertion depth is line bolt head It holds at density bullet, is about 17-19mm.After embolism 90min, then carefully line bolt is transferred to.It sews it up skin, smears chelated iodine, It is postoperative to be placed on electric blanket, isothermal holding is carried out to SD rat.

Sham-operation modeling: carrying out ischemia surgery operation, and line bolt is inserted into after internal carotid to extract rapidly and not stay from external carotid artery Set line bolt.

Testing index carries out clinic nerve function assay using Zea Longa method:

0 point, without any neurological deficit；

1 point, hence it is evident that received in visible SD rat left fore, fore paw is unable to outreach；

2 points, hence it is evident that visible SD rat ischemic side non-to the left at turn-taking with knocking into the back shape；

3 points, hence it is evident that visible SD rat ischemic side non-to the left is at toppling over shape；

4 points, hence it is evident that the disturbance of consciousness of visible SD rat, no autonomous.

For the Testing index for evaluating whether SD rat MCAO ischemia model succeeds, ischemic animal selects 1-2 to be allocated as reality Test animal.

Another index is TTC dyeing: TTC dyestuff participates in the succinic acid dehydrogenation reaction in normal tissue and takes on a red color.Ischemia group Interior albuminous degeneration necrosis is knitted, dehydrogenase activity is remarkably decreased, and succinic acid dehydrogenation reaction can not occur, therefore do not present red and be in White.TTC dyes the ischemic infarction degree that can detecte mammalian tissues.

Staining procedure is as follows:

1. the complete brain tissue of rapidly extracting is put into disposable Tissue Culture Dish on ice.

2. tissue is put into 20min in -20 DEG C of refrigerators.

3. tissue is put the several seconds at room temperature, the hardness of brain tissue is waited slightly to soften

4. the brain piece of 8 equal parts, every 2mm are intercepted in coronal-plane with blade.

5. TTC dyestuff is poured into culture dish, brain piece is made to float on a liquid, brain piece upper and lower surfaces all need to contact TTC liquid.

6. being put into 15min-20min (being protected from light) in 37 DEG C of insulating boxs.

7. toppling over dyestuff after taking out, 4 DEG C of fixations of 4%PFA are changed after being rinsed with physiological saline overnight.

8. taking-up in second day carries out digital image acquisition with scanner and analyzes Infarction volume (infarct using ImageJ software Volume=[(VC-VL)/V C], VC are control hemisphere volumes, VL is the non-Infarction volume of lesioned hemisphere).

Four, experimental result

As a result visible Fig. 5, the cerebral ischemia group of non-administration, Infarction volume are 0.359 ± 0.041；It is injected intravenously FACG The cerebral ischemic rats of 12mg/kg, Infarction volume are 0.021 ± 0.008；It is injected intravenously 6mg/kg Edaravone, Infarction volume is 0.141±0.015；Relative to vein to physiological saline, Edaravone group has lower Infarction volume to FACG tetrapeptide medicine group, And statistically significant (p < 0.05).The result shows that FACG tetrapeptide can improve cerebral apoplexy, and effect is substantially better than clinical application Edaravone (is shown in Table 2, attached drawing 5).

2 synthetic peptide of table is to the protective effect of cerebral ischemic rats and compared with Edaravone

Compared with model group, P < 0.05 * * P < 0.01, *.

[embodiment 5] L-phenylalanine-l-Alanine-four peptide injection of L-cysteine-glycine preparation

L-phenylalanine-l-Alanine-four peptide injection of L-cysteine-glycine, 100g L-phenylalanine-L- third Propylhomoserin-four peptide freeze-dried powder of L-cysteine-glycine is dispensed into 1000 sterilized control freeze-drying bottles, vacuum capping, storage - 20 or -80 DEG C are stored in, every bottle contains L-phenylalanine-l-Alanine-L-cysteine-glycine tetrapeptide 100mg, uses sodium chloride Injection or the dissolution of 5% glucose, vein slowly instil.

Claims (3)

1. a kind of synthetic peptide, which is characterized in that its amino acid sequence is selected from following any:

(a) NH₂-L-Phe-L-Ala-L-Cys-Gly-COOH；

(b) NH₂-L-Phe-L-Cys-L-Ala-L-Cys-Gly-COOH；

(c) NH₂-L-Cys-L-Ala-L-Cys-Gly-L-Phe-COOH；

(d) NH₂-L-Phe-L-Cys-L-Ala-L-Cys-Gly-L-Phe-COOH。

2. application of the synthetic peptide described in claim 1 in preparation treatment nervous system disease agent, which is characterized in that described The nervous system disease be cerebral arterial thrombosis, hemorrhagic apoplexy, cerebral trauma, Alzheimer disease, Parkinson's disease.

3. application according to claim 2, which is characterized in that the therapeutic agent is injection.