CN107047538A - Application and blood platelet store method of the protein kinase A activator in blood platelet preservation - Google Patents
Application and blood platelet store method of the protein kinase A activator in blood platelet preservation Download PDFInfo
- Publication number
- CN107047538A CN107047538A CN201710158221.5A CN201710158221A CN107047538A CN 107047538 A CN107047538 A CN 107047538A CN 201710158221 A CN201710158221 A CN 201710158221A CN 107047538 A CN107047538 A CN 107047538A
- Authority
- CN
- China
- Prior art keywords
- blood platelet
- platelet
- activator
- blood
- protein kinase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
Abstract
The invention discloses protein kinase A activator blood platelet preservation in application and blood platelet store method.From the embodiment of the present invention it can be seen that; the present invention is making public for the first time application of the protein kinase A activator in suppressing blood platelet apoptosis or keeping biologically active pdgf in the case of the induction of blood platelet apoptosis and regulation mechanism are knowable not yet; protein kinase A activator, which can suppress the function caused by blood platelet apoptosis, to be lost; influence of the blood platelet from apoptosis under storage and pathologic stressed condition can be protected, so as to extend platelet survival time.It is a kind of new strategy for improving and storing blood platelet life cycle, as a result show that the present invention determines hematoblastic life-span and existence by modulating apoptosis, to extension platelet storage times, improve hematoblastic clinical practice efficiency, avoid because platelet function forfeiture causes stock's blood platelet to inactivate and be scrapped, with far reaching significance.
Description
Technical field
The invention belongs to blood platelet Techniques of preserving field, and in particular to protein kinase A activator is in blood platelet preservation
Using and blood platelet store method.
Background technology
Blood platelet is the key factor of regulation thrombosis and Pathology bleeding in the circulatory system, while in the immune of body
Also played an important role in the pathophysiological processes such as reaction, infection, atherogenesis and metastases.However,
Blood platelet life cycle is shorter, can only be survived 8-9 days in peripheral blood.Fatefulue bleeding is often felt caused by decrease of platelet
In many common diseases such as dye, immune thrombocytopenia, diabetes and some drug therapies, and these diseases are drawn
The mechanism that the platelet life span risen shortens is not yet completely clearly.In addition, short-life blood platelet, especially stores lesion, limit
The term of validity of blood platelet clinical practice is made.Therefore, the mechanism for inquiring into regulation and control platelet life span and existence has important pathology
Physiological significance.
The damage that storage blood platelet is caused seriously limits clinical practice of the blood platelet in thrombopenia, and this is one
Generally acknowledged global problem, apoptosis seemingly cause storage damage main cause, be attempted to by suppress Caspase and
Related enzyme mitigates hematoblastic storage lesion, but does not up to the present obtain substantial progress.Gush in recent years
The Related Experimental Study of existing a large amount of blood platelet apoptosis discloses the secret of blood platelet life cycle, and research shows apoptosis
Albumen regulation platelet life span and blood platelet existence in play an important roll, and under physiology or pathological conditions blood platelet apoptosis
Startup and suppression mechanism it is still not very clear at present.
Blood platelet is one of blood main component, participates in body coagulation process, the hemostatic function brought into normal play, and prevents damage
Blood loss afterwards, therefore platelet transfusion turns into a kind of common method in clinical treatment.After platelets gathering, if can not be very fast
For clinic, it is necessary to preserve as early as possible.So blood platelet there must be suitable Techniques of preserving, blood resource could be both saved, again may be used
Storage.But because platelet life span is short, 26S Proteasome Structure and Function is easily influenceed by many factors, rationally need to effectively it be protected applied to preclinical
Deposit.The Inefficacy of Platelets Transfusion treatment that washing blood platelet can not only solve the patients such as blood plasma allergy, IgA shortages, isoimmunization is asked
Topic, and to preventing nonhemolytic febrific reaction, acute intravascular hemolysis reaction to have certain effect in platelet transfusion, simultaneously
The leucocyte remained in Platelet Concentrate product competes the nutrition in blood plasma with blood platelet, platelet storage can be accelerated to damage, and
And adverse effect can be directly produced, cause isoimmunization and ebv infection.Current blood platelet room temperature preservation temperature is 22 ± 2
DEG C, but the holding time is 3 days or so.
The content of the invention
It is an object of the invention to provide application and blood platelet preservation of a kind of protein kinase A activator in blood platelet preservation
Method, the life-span is preserved to extend blood platelet.
The present invention adopts the following technical scheme that protein kinase A activator is suppressing blood platelet apoptosis or keeping blood platelet
Application in activity.
The invention also discloses application of the protein kinase A activator in blood platelet preservation.
The invention also discloses application of the protein kinase A activator in extension platelet survival time.
The application during blood platelet preserves addition reagent is being prepared the invention also discloses protein kinase A activator.
In above-mentioned technical proposal, the blood platelet is stored at 4~15 DEG C, preferably 10 DEG C.10 DEG C of environment and protein kinase A
Activator synergy can further suppress apoptotic proteins caspase-3 activation and up-regulation anti-apoptotic proteins Bcl-xl expression,
Extend the blood platelet holding time.
In above-mentioned technical proposal, the protein kinase A activator includes Pimobendane, adenyl cyclase and swashed
Agent living, adenosine cyclophosphate, adenosine cyclophosphate derivative；The Pimobendane include PDE inorganic matter inhibitor,
PDE organic matter inhibitor；The adenyl cyclase activator includes adenyl cyclase inorganic matter activator, gland
Thuja acid cyclase organic matter activator；The adenosine cyclophosphate derivative includes the trim using adenosine cyclophosphate as nucleus.This
Inventive embodiments can be seen that protein kinase A activator can reduce PS and turn up, raise anti-apoptotic proteins Bcl-xl expression, illustrate it
Inhibitory action is played for hematoblastic apoptosis.
Such as protein kinase A activator of the invention is Amrinone, milrinone, Enoximone, aminophylline, promise prostatitis
In ketone, iloprost, Cilostazol, cilostamide, Dipyridamole, ginkgo biloba p.e, Quercetin, meglumine adenosine cycle phosphate,
Adenosine cyclophosphate, Forskolin, 8- bromines adenosine -3 ', 5 '-ring monophosphate, 8- bromos-adenosine cyclophosphate, 8- piperidyls adenosine-ring phosphorus gland
Glycosides, 8- chloros-adenosine cyclophosphate, adenylate 3,5- rings monophosphate, N6- benzoyls-adenosine cyclophosphate, (S)-adenylate, ring
3', 5'- (hydrogen thiophosphate) triethyl group, 3-isobutyl-1-methylxanthine, 8- chlorobenzenes-adenosine cyclophosphate, adenylate 3,
5- rings monophosphate, adenylate 3,5- rings single thiophosphate ester, 8- bromos-adenosine cyclophosphate, specificity 5,6- 4,5- dicyano miaows
Azoles-ring phosphorus bismuth glycosides, specificity 8- chlorobenzenes-cyclic guanylic acid sodium, specific adenylate 3', 5'- ring single thiophosphate ester triethyl group
Salt, specific adenosine cyclophosphate, dibutyryl-adenosine cyclophosphate, mono- acyl adenosine 3', the 5'- ring monophosphates of N6-, 8- bromo adenylates
3', 5'- ring monophosphate thioesters, 8- bromo adenylate 3', 5'- rings monophosphate, N6- benzoyls-adenosine cyclophosphate, red -9- ammonia
One or more in base-β-hexyl-Alpha-Methyl -9H- purine -9- acidic alcohol -9- adenine hydrochloric acid；Wherein Forskolin
（Forsklin）, i.e. forskolin is a kind of natural diterpene product for being isolated from Indian plant Coleus forskohlii, is true
Nucleus adenyl cyclase（AC）Activator.
The invention also discloses a kind of blood platelet store method, comprise the following steps, protein kinase A activator is added and washed
In blood platelet after washing, then preserve.
In above-mentioned technical proposal, blood platelet is in 4~15 DEG C of preservations, preferably 10 DEG C.
The present invention further discloses a kind of preparation method for the reagent for extending the blood platelet holding time, by protein kinase A
Activator is prepared.Simultaneously the invention discloses a kind of addition reagent preserved for blood platelet, including protein kinase A activation
Agent, can also include decentralized medium, such as buffer medium.
The present invention makes public for the first time protein kinase A in the case of the induction of blood platelet apoptosis and regulation mechanism are knowable not yet
Application of the activator in suppressing blood platelet apoptosis or keeping biologically active pdgf, can suppress phosphatidylserine and turn up, raise
Anti-apoptotic proteins Bcl-xl is expressed, and enough protects influence of the blood platelet from apoptosis under storage and pathologic stressed condition, so that
Extend platelet survival time.
Brief description of the drawings
Fig. 1 is that Flow cytometry is washed hematoblastic PS after Forskolin and DMSO processing and turned up result figure；
Fig. 2 is the expression figure that Western Blot methods detect blood platelet anti-apoptotic proteins Bcl-xl after distinct methods processing；
Fig. 3 is washing blood platelet anti-apoptotic proteins Bcl-xl expression figures after the detection two methods processing of Western blot methods；
Fig. 4 is washing blood platelet △ Ψ m depolarising result figures；
Fig. 5 turns up result figure for washing blood platelet PS；
Fig. 6 is flow cytometry analysis result figure；
Fig. 7 is the platelet electrophoresis figure after H89, forskin and DMSO are handled；
Fig. 8 is the electrophoretogram after the platelet lysates of pretreatment；
Fig. 9 is combined for BAD with Bcl-xL in mitochondria coexists figure；
Figure 10 is expression figures of the BAD on mitochondria；
Figure 11 is the platelet aggregation figure that ristomycin is induced；
Figure 12 is collagen-induced platelet aggregation figure；
Figure 13 is the situation map of thrombosis and fluorescence labeling blood platelet in thrombus.
Embodiment
The reagent and material of the present embodiment：
JC-1, Forskolin, calcium ion carrier A 23187, anti-caspase-3 monoclonal antibodies are green skies Products, molten
Agent DMSO（Dimethyl sulfoxide, DMSO）For Sigma Co., USA's product.Annexin V-FITC apoptosis is detected
Kit is Jia Mei biotech firms product.Phenylmethylsulfonyl fluoride（PMSF）For U.S.'s Amresco Products, polyvinylidene fluoride
Film（Pvdf membrane）For U.S.'s Bio-Rad Products, rabbit-anti people Bcl-xl, β-actin are U.S.'s CST Products, horseradish
The goat anti-rabbit antibodies of peroxidase labelling are U.S.'s Santa Cruz Products, and ECL produces for Advansta companies of the U.S.
Product.Vulgar centrifuge 1-16 types（Sigma, USA）；The types of flow cytometer FC 500（Beckman coulter, USA）;Vertical electricity
Swimming equipment（Bio-Rad, USA）.
Blood platelet is washed, the venous blood of the healthy blood donation personnel without hemorrhagic disease is extracted, by 7:1 volume ratio adds
Enter anti-coagulants glucose sodium citrate（The trisodium citrates of ACD 2.5%, 2.0% glucose, 1.5% citric acid）.It is complete after anti-freezing
Blood is in 100g（1100 r.p.m）Lower centrifugation 11 minutes, lower floor is red blood cell after centrifugation, and upper strata is rich in hematoblastic blood
Slurry.Supernatant liquid is transferred to new centrifuge tube.Rich in hematoblastic blood plasma in 825 g（3000 r.p.m）Lower 2 points of centrifugation
Clock, is precipitated as blood platelet, and supernatant liquid is platelet-poor plasma.Abandon after supernatant, blood platelet is resuspended in being rich in blood platelet blood
The isometric CGS buffer solutions of slurry（0.123 mol/L sodium chloride, 0.033 mol/L glucose, 0.013 mol/L lemons
Sour sodium, PH 6.5）In, in 270 g（2000 r.p.m）It is lower to centrifuge 2 minutes to wash away plasma protein.The blood platelet of precipitation is most
The improvement Tyrode buffer solutions of certain volume are resuspended in eventually（2.5 mmol/L Hepes, 150 mmol/L sodium chloride, 2.5
Mmol/L potassium chloride, 12 mmol/L sodium acid carbonates, 5.5 mmol/L glucose, 1 mmol/L calcium chloride, 1 mmol/L
Magnesium chloride, PH 7.4）, concentration is 3 × 10 8/ml.Washing blood platelet after resuspension places 1 hour so that its is extensive at room temperature
Answering is used for subsequent experimental to physiological status.
Flow cytometry △ Ψ m are depolarized, under aseptic condition by untreated washing blood platelet respectively 0 DEG C, 4 DEG C,
Preserved 5 days in 5 temperature such as 10 DEG C, 22 DEG C and 37 DEG C.Take what each different temperatures was preserved in 1d, 2d, 3d, 4d and 5d respectively
Wash blood platelet（50μL）And the washing blood platelet of A23187 processing is added under normal temperature（50μL）, add JC-1 (2 μ g/
Ml 10 min), are incubated at room temperature in DMSO and MTB mixed liquors, flow cytometry analysis is finally carried out.
Flow cytometer determines PS and turned up, and blood platelet is washed under aseptic condition and is divided into 3 groups：（Ⅰ）Untreated fish group,（Ⅱ）Add
Forskolin,（Ⅲ）Add DMSO（0.5%）, by three kinds of blood platelets in 5 temperature such as 0 DEG C, 4 DEG C, 10 DEG C, 22 DEG C and 37 DEG C
It is middle to preserve 5 days, respectively take three kinds of blood platelets in 1d, 2d, 3d, 4d and 5d（5μL）And A23187 processing is added under normal temperature
Wash blood platelet（5μL）, it is separately added into after the μ L of Annexin V-FITC 5 and 150 μ 1 × Loading of L Buffer mixings, room
Warm lucifuge is incubated 15 minutes, and 350 μ 1 × Loading of L Buffer are added before upper machine.Finally carry out flow cytometry analysis.
Western Blot detect that washing blood platelet is divided into 3 groups under Bcl-xl protein expressions, aseptic condition：（Ⅰ）It is untreated
Group,（Ⅱ）Add Forskolin,（Ⅲ）Add DMSO（0.5%）, by three kinds of blood platelets at 0 DEG C, 4 DEG C, 10 DEG C, 22 DEG C and 37 DEG C
Deng preserving 5 days in 5 temperature, three kinds of blood platelets are respectively taken in 1d, 2d, 3d, 4d and 5d（50μL）And added under normal temperature at A23187
The fresh wash blood platelet of reason（50μL）, add isometric lysate（Containing PMSF）30 min, 3500 r/min centrifugations are split on ice
2 min, take supernatant to add 4x sample-loading buffers and-mercaptoethanol, boil 10min.Sample carries out SDS-PAGE, is transferred to
Pvdf membrane.TBST washes film, anti-Bcl-xl monoclonal antibodies after the closing of 5% milk（1:1000 dilutions）And anti-β-actin monoclonals are anti-
Body（1:1000 dilutions）4 DEG C of night incubations.The goat anti-rabbit antibodies of horseradish peroxidase-labeled after washing film through TBST（1:8000
Dilution）It is incubated at room temperature 1h.TBST washes film and using ECL methods detection Bcl-xl and β-actin bands again.
Data statistic analysis, all experimental results are at least repeated 3 times.Experimental data is carried out using the softwares of SPSS 16.0
Statistical analysis, experimental data represents that the comparison between different groups is united using one-way ANOVA with mean ± standard deviation
Meter analysis, P<0.05 indicates significant difference.
Embodiment
Under aseptic condition, washing blood platelet is divided into three groups, untreated fish group, 5 μM of Forsklin groups of addition, addition 0.5%
DMSO groups；Three groups of blood platelets are divided equally, is preserved 5 days in 0 DEG C, 4 DEG C, 10 DEG C, 22 DEG C, 37 DEG C, 5 μ L is respectively taken daily, with normal temperature
The μ L of washing blood platelet 5 for adding A23187 processing are control.
Fig. 1 is that Flow cytometry PS of washing blood platelet preservation five days after Forskolin and DMSO processing turns up
As a result.Using DMSO groups as control, the PS percentages that turn up are represented with mean ± standard deviation, and above experiment is repeated 5 times, P<0.05；With
DMSO groups blood platelet is contrasted, and the Forskolin groups washing blood platelet PS percentages that turn up are reduced in same time and Conservation environment, and
Reduce all the more obvious with time lengthening, the 5th day Forskolin turns up work to the washing blood platelet PS being stored in each temperature environment
Statistical significance is respectively provided with difference；As a result show that Forskolin is inhibited to washing blood platelet apoptosis.
Fig. 2 is five days anti-apoptotic proteins Bcl-xl of blood platelet preservation after the detection distinct methods processing of Western Blot methods
Expression, wherein in each figure（-）Fresh untreated washing blood platelet is represented, the fresh wash blood that A representatives are handled through A23187 is small
Plate, 0,4,10,22,37 represent correspondence storage temperature respectively（It is repeated 3 times）；Show that different temperatures washing blood platelet has different journeys
Apoptosis is spent, and Forskolin turns up inhibited to blood platelet PS.Western Blot results are shown, with control group phase
Than same time same temperature Forskolin groups washing blood platelet Bcl-xl expression quantity rises, and shows that Forskolin has upper
Adjust the effect of washing blood platelet BCL-xl expression.
Fig. 3 is washing blood platelet anti-apoptotic proteins Bcl-xl expression after the detection two methods processing of Western blot methods,
（A）Express and change for continuous 5 days anti-apoptotic proteins Bcl-xl,（B）For the 1st day and the 5th day washing blood platelet anti-apoptotic proteins
Bcl-xl expression changes（It is repeated 3 times）；It can be seen that 22 DEG C of DMSO groups washing blood platelet preserves continuous 5 days anti-apoptotic proteins Bcl-
Xl is expressed to be decreased obviously with the extension expression of time, and Forskolin groups washing blood platelet had no bright on the 1st day compared with the 5th day
It is aobvious to decline（Fig. 3 B）, show that Forskolin plays facilitation to washing blood platelet anti-apoptotic.
Blood platelet apoptosis is to store the main cause that blood platelet generating function is disorderly and is quickly removed, mitochondrial membrane potential
The intrinsic program apoptosis of depolarising regulation and control is an irreversible process.Fig. 4 is washing blood platelet △ Ψ m depolarising knots
Fruit is schemed；Fig. 5 turns up result figure for washing blood platelet PS；Fig. 6 is flow cytometry analysis result figure；Wherein 3 in Fig. 4, Fig. 5 ×
108/ mL washings blood platelet respectively with 25uM H89,5uM 22 DEG C of pretreatments of forskin and DMSO internal references it is different when
Between point, the JC-1 that Fig. 4 is added makes its final concentration of 2 μ g/mL, and lucifuge is incubated 10 min；Fig. 5's and 5 μ g/mL
Lactadherin lucifuges are incubated 30 minutes, and the blood platelet of different disposal goes to pole through flow cytomery mitochondrial transmembrane potentials
Change the percentage of (Fig. 4) and the positive blood platelets (Fig. 5) of PS, as a result represented with mean ± standard deviation, 3 × 10 in Fig. 68/ mL is washed
Wash blood platelet to be incubated 72 hours with 25uM H89,5uM 22 DEG C of forskin and DMSO internal references respectively, blood platelet is labeled
Fed back by tail vein in Mice Body, whole blood is collected in orbital vein blood sampling, flow cytometry analysis detects the blood platelet of mark,
It is more than experiment in triplicate；Protein kinase A is added when being as a result shown in platelet reserve（PKA）Activator, according to the present invention,
PKA activator substantially suppresses the generation of blood platelet apoptosis；Not only further checking PKA adjusts the work of blood platelet apoptosis to these data
With, and also indicate that PKA is located at the upstream that mitochondrial depolarization adjusts Apoptosis.
Fig. 7 is the platelet electrophoresis figure after H89, forskin and DMSO are handled；Washing blood platelet uses 37.5 uM respectively
H89, pre-process 160 minutes under 10 uM forskin and DMSO internal reference normal temperature, hematoblastic plasmosin is extracted respectively
And mitochondrial protein, western blot testing goal albumen, the amount of Image J software analysis destination proteins, count four realities
Test and result is shown with mean ± standard deviation；Fig. 8 is the electrophoretogram after the platelet lysates of pretreatment, 17,000 4 DEG C of g centrifugations 10
Minute, obtained supernatant and corresponding antibodies are incubated precipitates overnight, are incubated 2 hours with protein A/G+4 DEG C of sepharose 4B
Afterwards, protein hybridization is used for after pearl is eluted, four experiments of statistical analysis to show result, * P with mean ± standard deviation< 0.05,
**P < 0.01；Fig. 9 is combined for BAD with Bcl-xL in mitochondria coexists figure；Figure 10 is expression figures of the BAD on mitochondria.Grind
Study carefully discovery, PKA activators forskolin can strengthen the serine phosphorylation level in the sites of Bad 155, and regulate and control 14-3-3 eggs
In vain with anti-apoptotic proteins Bcl-xL binding, and then regulating cell apoptosis.Co-immunoprecipitation result shows H89 or Forsklin
Under stimulation, the effect between Bcl-xL and BAD is significantly reduced or strengthened, and the combination between 14-3-3 and BAD is but in contrast；
Coexisted in addition, BAD is combined with Bcl-xL in mitochondria, H89 can strengthen and forskolin can reduce BAD on mitochondria
Expression, these as shown by data PKA slows down hematoblastic apoptosis by regulating and controlling serine phosphorylation.
The washing blood platelet of people（3×108/mL）Respectively with 5uM 22 DEG C of pretreatments 96 of forskin and DMSO internal references
Hour, detect ristomycin (Figure 11) and collagen using platelet aggregation instrument（Figure 12）The platelet aggregation of induction.As a result table
Bright, forskin can keep platelet aggregation.Mouse washs blood platelet（3×108/mL）Respectively with 25uM H89,
5uM 22 DEG C of forskin and DMSO internal references are incubated 96 hours, are fed back after blood platelet is labeled through tail vein in Mice Body,
Mouse mesenteric, after FeCl3 damages, the situation of record thrombosis and fluorescence labeling blood platelet in thrombus（Figure 13）,
As a result show, forskin can keep platelet adhesion reaction and form thrombus function.
Change Forsklin group additions, such as 2uM, 8uM；22 DEG C are incubated 72 hours, quiet through tail after blood platelet is labeled
Arteries and veins is fed back in Mice Body, and whole blood is collected in orbital vein blood sampling, and the blood platelet of flow cytometry analysis detection mark, experiment is repeated
More than three times；As a result show blood platelet survival rate more than 70%；10 DEG C are incubated 72 hours, and survival rate reaches more than 82%, 120
Hour reaches more than 71%；4 DEG C, 15 DEG C are incubated 72 hours, and survival rate reaches 77%, more than 78%.22 DEG C pre-process 96 hours,
Ristomycin and collagen-induced platelet aggregation are detected using platelet aggregation instrument, is as a result shown, forskin can be kept
Platelet aggregation, PAR all reaches 70%, 4 DEG C, 15 DEG C of incubations reach 75%；10 DEG C pre-process 96 hours, it is 140 small
When, ristomycin and collagen-induced platelet aggregation are detected using platelet aggregation instrument, is as a result shown, forskin can
Platelet aggregation is kept, PAR all reaches 81%, 68%.
Replacing forskin using Amrinone, meglumine adenosine cycle phosphate is used for hematoblastic preservation, adds 5uM, 22 DEG C of incubations
72 hours, fed back after blood platelet is labeled through tail vein in Mice Body, whole blood, flow cytometer point are collected in orbital vein blood sampling
More than the blood platelet of analysis detection mark, experiment in triplicate, blood platelet survival rate is as a result shown more than 70%, 10 DEG C are incubated 72
Hour, survival rate reaches more than 82%, and 15 DEG C are incubated 72 hours, and survival rate reaches more than 75%.
Washing blood platelet is divided into 3 groups by the present invention：（Ⅰ）Untreated fish group,（Ⅱ）Add protein kinase A activator such as
Forskolin,（Ⅲ）DMSO is added, three kinds of blood platelets are preserved 5 in 5 temperature such as 0 DEG C, 4 DEG C, 10 DEG C, 22 DEG C and 37 DEG C
My god.The detection for index of correlation being carried out to each sample in continuous five days.Mitochondrial membrane potential is detected using Flow Cytometry（△Ψm）
Depolarising and phosphatidylserine（PS）The change turned up.Using Western Blot detection washing blood platelet apoptosis indexs
Caspase-3 and Bcl-xl expression.As a result show：Same time and at a temperature of, untreated fish group washing blood platelet
Be stored within continuous 5 days in five temperature occur mitochondrial membrane potential depolarising and phosphatidylserine turn up, Forskolin can
Suppress phosphatidylserine to turn up；When only storage temperature is different, 10 DEG C of washing blood platelet anti-apoptotic proteins are stored in
Bcl-x expression, which has no, to be decreased obviously, but Forskolin up-regulation anti-apoptotic proteins Bcl-xl expression；Show Forskolin to blood
Platelet plays a part of suppressing apoptosis, and extension preserves the hematoblastic time, can further suppress to wither with 10 DEG C of environment synergies
Die albumen caspase-3 activation and up-regulation anti-apoptotic proteins Bcl-xl expression.
Blood platelet apoptosis limits its life-span, and blood platelet apoptosis caused by many diseases can result in decrease of platelet
Disease, but the startup of blood platelet apoptosis and regulation mechanism are not illustrated also completely at present.Technique according to the invention scheme, improves storage
Blood platelet PKA activity is deposited, slows down the startup endogenic programmed death of blood platelet, it is to avoid cause thrombopenia in vivo.
Importantly, the present invention is not only able to the blood platelet of protection storage and can improve what internal a variety of pathological stimulis were caused
Platelet injury, extends the hematoblastic life-span；As a result confirm that the present invention rises to hematoblastic stabilization under pathological and physiological condition very big
Effect.The serine residue enhancing in 155 sites that phosphoBad pro apoptotic protein is passed through using technical scheme, PKA
And 14-3-3 combination, so as to promote anti-apoptotic proteins Bcl-xL releases to suppress blood platelet apoptosis, suppresses Bcl- in blood platelet
XL degraded, prevents from storing blood platelet apoptosis, it is to avoid external blood platelet apoptosis and internal Acute thrombocytopenia, can
Protect blood platelet from it is a variety of stimulate induction apoptosis, can animal improve peripheral blood in hematoblastic quantity.Storage blood platelet is made
Into damage seriously limit clinical practice of the blood platelet in thrombopenia, this is a generally acknowledged global problem.Wither
It is the main cause for causing storage to damage to die, and is attempted to hematoblastic to mitigate by the enzyme for suppressing apoptotic proteins and correlation
Lesion is stored, but does not up to the present obtain substantial progress.Technical scheme disclosed by the invention is a kind of improvement
The new strategy of blood platelet life cycle is stored, as a result shows that the present invention determines hematoblastic life-span and existence by modulating apoptosis, it is right
Improving platelet reserve, the hematoblastic clinical practice of raising has far reaching significance.
Claims (10)
1. protein kinase A activator is suppressing blood platelet apoptosis, is keeping biologically active pdgf, extension platelet survival time or blood
Application in platelet preservation.
2. protein kinase A activator is preparing the application during blood platelet preserves addition reagent.
3. the application according to claims 1 or 2, it is characterised in that the hematoblastic storage temperature is 4~15 DEG C.
4. application according to claim 3, it is characterised in that the hematoblastic storage temperature is 10 DEG C.
5. the application according to claims 1 or 2, it is characterised in that the protein kinase A activator includes di(2-ethylhexyl)phosphate fat
Enzyme inhibitor, adenyl cyclase activator, adenosine cyclophosphate, adenosine cyclophosphate derivative.
6. application according to claim 5, it is characterised in that the Pimobendane include PDE without
Machine thing inhibitor, PDE organic matter inhibitor；It is inorganic that the adenyl cyclase activator includes adenyl cyclase
Thing activator, adenyl cyclase organic matter activator；The adenosine cyclophosphate derivative is included using adenosine cyclophosphate as nucleus
Trim.
7. a kind of addition reagent preserved for blood platelet, it is characterised in that described addition reagent includes protein kinase A and swashed
Agent living.
8. a kind of blood platelet store method, it is characterised in that protein kinase A activator is added in blood platelet, then preserved.
9. blood platelet store method according to claim 8, it is characterised in that the temperature of the preservation is 4~15 DEG C.
10. a kind of preparation method for the reagent for extending the blood platelet holding time, it is characterised in that by protein kinase A activator system
It is standby to obtain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710158221.5A CN107047538B (en) | 2017-03-16 | 2017-03-16 | Application of protein kinase A activator in platelet preservation and platelet preservation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710158221.5A CN107047538B (en) | 2017-03-16 | 2017-03-16 | Application of protein kinase A activator in platelet preservation and platelet preservation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107047538A true CN107047538A (en) | 2017-08-18 |
| CN107047538B CN107047538B (en) | 2021-04-13 |
Family
ID=59620856
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710158221.5A Active CN107047538B (en) | 2017-03-16 | 2017-03-16 | Application of protein kinase A activator in platelet preservation and platelet preservation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107047538B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108159421A (en) * | 2018-02-05 | 2018-06-15 | 苏州大学 | Purposes of the phosphatidylserine blocking agent in preparing treatment platelet counts and reducing relevant disease drug |
| WO2018137396A1 (en) * | 2017-01-25 | 2018-08-02 | 苏州大学 | Use of protein kinase a activator and inhibitor in preparation of drugs for treating diseases associated with changes in platelet counts |
| CN113016780A (en) * | 2021-02-24 | 2021-06-25 | 中国人民解放军空军军医大学 | Application of Vx702 in preparation of platelet in-vitro preservative and related platelets |
| CN113712023A (en) * | 2021-04-02 | 2021-11-30 | 苏州大学 | Method for preserving blood platelets |
| CN114667996A (en) * | 2022-03-14 | 2022-06-28 | 苏天生命科技(苏州)有限公司 | Application of mitophagy activator in platelet preservation |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919614A (en) * | 1994-10-19 | 1999-07-06 | Lifecell Corporation | Composition comprising three platelet lesion inhibitors for platelet storage |
| CA2623666A1 (en) * | 2005-09-26 | 2007-04-05 | Lifecell Corporation | Dry platelet composition |
| CN105050390A (en) * | 2012-12-19 | 2015-11-11 | 先进保存技术股份有限公司 | Platelet concentrate preservation method |
| CN106342788A (en) * | 2011-04-14 | 2017-01-25 | 特兰斯迈迪茨公司 | Organ care solution for ex-vivo machine perfusion of donor lungs |
-
2017
- 2017-03-16 CN CN201710158221.5A patent/CN107047538B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5919614A (en) * | 1994-10-19 | 1999-07-06 | Lifecell Corporation | Composition comprising three platelet lesion inhibitors for platelet storage |
| CA2623666A1 (en) * | 2005-09-26 | 2007-04-05 | Lifecell Corporation | Dry platelet composition |
| EP1929289B1 (en) * | 2005-09-26 | 2018-02-21 | Lifecell Corporation | Dry platelet composition |
| CN106342788A (en) * | 2011-04-14 | 2017-01-25 | 特兰斯迈迪茨公司 | Organ care solution for ex-vivo machine perfusion of donor lungs |
| CN105050390A (en) * | 2012-12-19 | 2015-11-11 | 先进保存技术股份有限公司 | Platelet concentrate preservation method |
Non-Patent Citations (1)
| Title |
|---|
| 赵丽丽等: ""抑制蛋白激酶A活性诱导血小板凋亡及其制剂的研究"", 《第十五届中国中西医结合学会微循环专业委员会暨第二届中国微循环学会痰瘀专业委员会学术会议资料汇编》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018137396A1 (en) * | 2017-01-25 | 2018-08-02 | 苏州大学 | Use of protein kinase a activator and inhibitor in preparation of drugs for treating diseases associated with changes in platelet counts |
| CN108159421A (en) * | 2018-02-05 | 2018-06-15 | 苏州大学 | Purposes of the phosphatidylserine blocking agent in preparing treatment platelet counts and reducing relevant disease drug |
| CN113016780A (en) * | 2021-02-24 | 2021-06-25 | 中国人民解放军空军军医大学 | Application of Vx702 in preparation of platelet in-vitro preservative and related platelets |
| CN113712023A (en) * | 2021-04-02 | 2021-11-30 | 苏州大学 | Method for preserving blood platelets |
| CN114667996A (en) * | 2022-03-14 | 2022-06-28 | 苏天生命科技(苏州)有限公司 | Application of mitophagy activator in platelet preservation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107047538B (en) | 2021-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107047538A (en) | Application and blood platelet store method of the protein kinase A activator in blood platelet preservation | |
| Kim et al. | The platelet response to hypotonic shock. Its value as an indicator of platelet viability after storage | |
| Xu et al. | Hydroxyl radical inhibits sarcoplasmic reticulum Ca2+-ATPase function by direct attack on the ATP binding site | |
| Pasdois et al. | Extent of mitochondrial hexokinase II dissociation during ischemia correlates with mitochondrial cytochrome c release, reactive oxygen species production, and infarct size on reperfusion | |
| EP0281621B1 (en) | Glucose free media for storing blood platelets | |
| JP5450958B2 (en) | Compositions and methods for prolonging platelet survival | |
| US4447415A (en) | Plasma-free medium for platelet storage | |
| JP5123663B2 (en) | Compositions and methods for prolonging platelet survival | |
| RU2397641C2 (en) | Solution for perfusion and/or preservation of organs and ways of its application | |
| JP5552231B2 (en) | Compositions and methods for prolonging platelet survival | |
| JP2008529550A (en) | Compositions and methods for the preservation of red blood cells | |
| Wang et al. | Shenfu injection suppresses apoptosis by regulation of Bcl-2 and caspase-3 during hypoxia/reoxygenation in neonatal rat cardiomyocytes in vitro | |
| EP2536416B1 (en) | Arginine-containing storage and/or rejuvenating compositions and methods for storing or rejuvenating red blood cells | |
| US20100004342A1 (en) | LFA-1 Inhibitors and Use Thereof | |
| Ramkissoon | Macroscopic identification of early myocardial infarction by dehydrogenase alterations | |
| Rodiño-Janeiro et al. | Glycated human serum albumin induces NF-κB activation and endothelial nitric oxide synthase uncoupling in human umbilical vein endothelial cells | |
| Åkerblom et al. | Studies on citrate‐phosphate‐dextrose (CPD) blood supplemented with adenine | |
| Fabiani et al. | Chromosomal aberrations and neutrophil activation induced by reperfusion in the ischaemic human heart | |
| BERENS et al. | Pentoxifylline in the isolated perfused rat kidney | |
| Dales et al. | Cytological aspects of haemoglobin and chlorocruorin synthesis in polychaete annelids | |
| MX2010011050A (en) | Methods of regulating actin cytoskeletal rearrangement and intercellular gap formation. | |
| Roland et al. | Effect of hydroxyethylrutosides on hypoxial-induced neutrophil adherence to umbilical vein endothelium | |
| JPWO2018151243A1 (en) | Erythrocyte protective agent | |
| CN108210930B (en) | Application of flavonoid compound or composition thereof in protecting erythrocytes from damage | |
| Radice et al. | Hyperbaric oxygen worsens myocardial low flow ischemia-reperfusion injury in isolated rat heart |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |