CN107485728A - A kind of collagen fibrous proteins compound hemostatic plaster and preparation method thereof - Google Patents
A kind of collagen fibrous proteins compound hemostatic plaster and preparation method thereof Download PDFInfo
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- CN107485728A CN107485728A CN201610442708.1A CN201610442708A CN107485728A CN 107485728 A CN107485728 A CN 107485728A CN 201610442708 A CN201610442708 A CN 201610442708A CN 107485728 A CN107485728 A CN 107485728A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/38—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/44—Medicaments
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
- A61L2300/254—Enzymes, proenzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/418—Agents promoting blood coagulation, blood-clotting agents, embolising agents
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Abstract
The present invention proposes a kind of collagen fibrous proteins compound hemostatic plaster and preparation method thereof, freeze-drying forms collagen fibrous proteins compound hemostatic plaster after the collagen solution of mammal is mixed in proportion with fibrinogen, the factor extracted from mammalian plasma, for covering burn, operation, wound, skin or internal organ Wound Defect caused by disease, induction wound repair, hemostasis, absorb wound exudate, prevent tissue adhesion.
Description
Technical field
The present invention relates to a kind of bleeding-stopping dressing and preparation method thereof, for covering burn, operation, wound, disease cause
Skin, internal organ Wound Defect covering and hemostasis, induce wound repair.Belong to biomedical engineering and material science and technology neck
Domain.
Background technology
Autologous thrombin includes endogenous blood coagulation and external source coagulation pathway.Intrinsic coagulation pathway refers to that the clotting factor of participation is complete
Portion carrys out autoblood (endogenous).Intrinsic coagulation pathway refers to activate from factor XI, plasma thromboplastin antecedent I, to the process of tenase.Work as vascular wall
Damage, subendothelial tissue exposure, negatively charged subendothelial collagen fiber contacts with clotting factor, and factor XI, plasma thromboplastin antecedent I is i.e. therewith
With reference to being activated in HK and PK presence as XIIa.Under conditions of independent of calcium ion, Factor XIIa swashs factor XI, plasma thromboplastin antecedent
It is living.In the presence of calcium ion, the XIa of activation have activated factors IX again.Single IXa activity factors X effect is at a fairly low, it
1: 1 compound, also known as factor X multienzyme complexs are combined to form with VIIIa.This reaction must also have Ca2+ and PL common
Participate in.Exogenous cruor pathway：The clotting factor for referring to participate in not is all present in blood, also external clotting factor
Participate in hemostasis.This process is started, the process being activated to factor X from tissue factor exposed to blood.Clinically with solidifying
The timing of hemase original reflects the situation of exogenous cruor pathway.Tissue factor is the one kind being present in various kinds of cell plasma membrane
Specific transmembrane albumen.After tissue damage, the factor is discharged, in the presence of calcium ion, it forms 1 together with factor Ⅴ II:
1 compound.Time needed for extrinsic coagulation is short, is swift in response.Exogenous cruor pathway is mainly suppressed by tissue factor approach
Thing (TFPI) is adjusted.TFPI is a kind of glycoprotein being present in human normal plasma and blood platelet and vascular endothelial cell.It is logical
Cross and combine to form compound with factor Xa or factor VIIa-tissue factor-factor Xa and come inhibiting factor Xa or factor VIIa-tissue
The activity of the factor.But when by larger wound, autologous thrombin is often not enough to stop blooding rapidly, it is necessary to which additional hemostatic material is
Bleeding can be controlled rapidly, avoid excessive blood loss.
Currently used Absorbable hemostatic material has Fibrin Glue, gelfoam, collagen Haikang, zeolite powder, more
Following one or more be present in peptide, oxycellulose, microfibrillar collagen, chitosan and Sorbsan etc., these hemostatic materials
Defect：1. anthemorrhagic speed is slow, effect is poor；2. influenceing postoperative suture, increase new surface of a wound bleeding；3. natural degradation is unable to,
Residual is formed in vivo；4. anaphylactogen reaction etc. be present.For hemostatic mechanism is accelerates the material that blood clot is formed, it is lacked
Point is that they are only effective to the less bleeding of flow velocity, therefore must coordinate hemostasis clamp during use, and this allows for above-mentioned hemostatic material
Material usually needs surgeon's look-ahead amount of bleeding or repeated multiple times uses haemostatic clamp when in use.Collagen protein sponge,
Fibrin ferment and biological fibrin glue (fibrin ferment and fibrinogen share) are respectively used to clinical existing decades, collagen protein sponge
Hydrophily it is poor, it is also bad to the adhesiveness of the surface of a wound, therefore anthemorrhagic performance does not protrude；Thrombin powder is also only capable of oozing for the hemostasis surface of a wound
Blood, bleeding caused by larger angiorrhoxis can not stop；Adhesion of the biological fibrin glue to the surface of a wound is worse, the jelly and water of formation
Peeled off afterwards from the surface of a wound, therefore haemostatic effect is not as good as collagen sponge.As various countries' medical field will to hemostatic material anthemorrhagic performance
The raising asked, exploitation haemostatic effect more preferably material is imperative, can be a variety of according to the different performance of various hemostatic materials, use
Method associated with hemostatic material, make the more preferable anthemorrhagic performance of materials serve.The present inventor was proposed collagen in 2004
Sponge, fibrin ferment and fibrinogen combine to form a kind of absorbable fibre albumen hemostatic adhesive bandage (patent No.：ZL
200410033680.3, authorized on January 4th, 2006), substantially increase the hydrophily of hemostatic material, the adhesion to the surface of a wound
Power and haemostatic effect, can stop blooding massive haemorrhage, and the notable innovative point of the invention is that fibrin ferment and fibrinogen are blended in into one
Rise without playing biochemical reaction, until blood coagulation biochemical reaction just occurs after contact wound bed fluid, but the pure wine of the mixed method used
Essence makees solvent, by fibrinogen with thrombin powder system into suspension, then by suspension on collagen sponge, this side
Although method solves the problem for avoiding two kinds of powders from reacting and fail before the surface of a wound is sticked, but undercompounding be present, in glue
Olynthus surface distributed is uneven, production when waste it is big, lyophilized after powder is easy to fall off, absolute alcohol makes fibrinogen and fibrin ferment
The defects of partial inactivation.Avoiding these defects from dissolving above two powder with water can just be sufficiently mixed, uniform and efficient
Ground is coated on collagen protein sponge, but how to avoid two kinds of powders from reacting and fail before the surface of a wound is sticked is to need to solve
Key issue.The present invention same blood plasma step by step arithmetic fibrinogen and fibrin ferment, save blood plasma；Dexterously by blood coagulation
Proenzyme and fibrinogen mixture directly mix in proportion with collagen, fully can dissolve and avoid before the surface of a wound is sticked
Reacted with fibrinogen, while eliminate lyophilized thrombin original and fibrinogen respectively again and be then applied to collagen egg
A series of first-class cumbersome steps of Bai Haimian.The collagen fibrous proteins compound hemostatic so produced, which is attached to when sticking the surface of a wound, to be passed through
Into fibrin ferment a series of Coagulation tests are occurred for PROTHROMBIN ACTIVATOR by Inner sources exogenous cruor pathway.
Test data shows that 1g ice can about produce the steam of 10000 liters of volumes in 10Pa, in time produce these
Raw steam excludes, and relies solely on vavuum pump and is unable to reach requirement, therefore, in the structure of freeze dryer, devises steam
Condenser (cold-trap), less than .45 DEG C of low temperature conduction oil is flowed in the coil pipe of cold-trap.Vapor caused by product distillation leads to
Cross vacuum suction and enter cold-trap, on the condenser coil of cold-trap, vapor runs into the ice that low temperature is condensed into solid-state again.Certainly, exist
In freeze-drying process, in addition to vacuum should be controlled, the heat that system provides should also be controlled, it is ensured that vapor caused by distillation
It is in a basic balance with the condensation rate of vapor, maintain system vacuum to be in a controllable and effective state.In sublimation stage, by
In the continuous distillation of ice, the vacuum of drying box is constantly rising, therefore, lasting, stable vacuum suction and cold-trap
Water vapor condensation to act on maintenance to the vacuum of drying box be highly important.We by the three-phase diagram of water (although this
When system be not in real pure water state, the three-phase diagram of pure water still has reference significance) it is observed that when the pressure of drying box
Power rise to a certain extent when, the ice in product is not just gone further distillation approach, but the generation water that can liquefy, and the water of generation can will be dry
Dry is partly dissolved, and blocks distillation passage, and the product for causing to have freezed caves in, and can seriously cause the generation of spray bottle, these are final
The exceeded of the moisture of finished product will be caused, freezes failure.Whether vacuum is necessarily more low better during vacuum freeze drying
During vacuum freeze drying, the transmission of heat has three kinds of approach：Conduction, convection current, radiation.If vacuum is too high, convection current is just
It can slow down, the transmission of heat can also be reduced, and the whole freeze-drying cycle can be extended.According to correlative study and practice summary, glue
Vacuum degree control is advantageous for rapid freeze-drying between 0.25-0.3mbar in former albumen freeze-drying process.Vacuum freeze drying mistake
The technological parameter such as Cheng Zhong, vacuum, temperature interacts, and the present inventor explores small in pre-freeze 3 in repeatedly lyophilized practice
When after heat conduction oil temperature risen to 30 DEG C, can be distilled rapidly in the case of ensureing that collagen ice crystal is infusible.In freeze-drying process
Middle aeration mainly makes heating quicker, as long as will not be too fast because of heating up, heat be caused bottom to melt, reached in congregate
To the purpose of our aerations.Because we influence after all the still influence of temperature, aeration by aeration to rate of sublimation
Purpose be intended to that our setting value can be reached as early as possible, ensureing that aeration is not above saturated vapor pressure, product does not have
It is as far as possible miserable more on the premise of thawing, product is rapidly heated, improve rate of sublimation.The inventors discovered that in pre-freeze
Opening ginseng air valve while vacuumizing is opened after 3 hours and is advantageous to product rapid freeze-drying.During by changing above-mentioned vacuum, ginseng gas
Between, heat up moment and the moment of vacuumizing so that the collagen lyophilized time, (pre-freeze 4 hours, vacuum was maintained at by routine
0.05mbar, -45 DEG C insulation vacuumize 24 hours after be then turned on join gas, by 24 hours progressively by 0 DEG C to 30 DEG C) 72 hours
Foreshorten to 24 hours, every batch of product is lyophilized save 48 hours time, save electric energy 2880KWh (with a power 60KW, 10 squares
Rice freeze dryer calculates).Temperature will be increased above ambient temperature before sample export, in case sample gets damp again after going out dry storehouse.
The content of the invention
The present invention proposes a kind of collagen fibrous proteins compound hemostatic plaster and preparation method thereof, by the collagen of mammal
Freeze-drying forms collagen fibre after solution mixes in proportion with fibrinogen, the factor extracted from mammalian plasma
Fibrillarin compound hemostatic plaster, for covering burn, operation, wound, skin or internal organ Wound Defect caused by disease, induction
Wound repair, hemostasis, absorb wound exudate, prevent tissue adhesion.
Specifically comprise the following steps：
A, the extraction of fibrinogen
- 20 to -25 DEG C 95% of cold ethanol is added in mammalian plasma and (adds second by 10-15% volume ratio
Alcohol) sediment is produced afterwards, precipitation is collected by centrifugation, supernatant is used to extract factor.With 0-4 DEG C of 0.3-0.5% Nacl water
Solution washing precipitate.Once dissolve：The Nacl that a lytic agent 1.3-1.5% is added into the sediment by washing is water-soluble
Centrifugation removes insoluble matter collection supernatant after liquid is heated to 36-38 DEG C of abundant dissolving while stirring；Secondary precipitation：It is upper after separation
- 20 to -25 DEG C 95% cold ethanol (adding ethanol by 10-15% volume ratio) precipitation fibrinogen is added in clear liquid, from
The heart goes supernatant collection to precipitate.Secondary washing：Washed with the 0-4 DEG C of 0.3-0.5% Nacl aqueous solution, sediment is collected by centrifugation
As pure fibrinogen.As the sediment after secondary washing adds the lytic agent 1.3-1.5% Nacl aqueous solution
Being heated to 36-38 DEG C while stirring can not fully dissolve, then repeats above-mentioned dissolving and washing step again, until no longer occurring not
Molten thing.The sediment for finally washing to obtain is pure fibrinogen.
B, the extraction of factor
BaCl is slowly added to while stirring in the supernatant that said extracted crosses fibrinogen2Solution, containing anti-in blood plasma
Solidifying agent trisodium citrate, BaCl2Barium citrate is produced with trisodium citrate, barium citrate can adsorb factor and together sink
Form sediment.With 5L physiological saline suspension barium citrate-PCC, with 1mol/L acid for adjusting pH to 1.8, now barium citrate
Resolve into Ba2+And citrate ions, factor dissociate.Obtained sediment is centrifuged with the precipitation obtained after brine
As pure factor.
C, the preparation of collagen fibrous proteins compound hemostatic plaster
First freeze dryer cold-trap is started and cooled, treats that temperature drops to less than -50 DEG C, by being added in every L collagen solutions
The fibrinogen and factor extracted in 2-4L blood plasma pours into stainless steel disc immediately after being sufficiently mixed spreads out thick into 3-6mm
Thin layer, dry storehouse is placed into, each flaggy temp probe is immersed in above-mentioned mixed solution, before opening freeze dryer immediately after the door of pass
Case cools；Preceding case cools 3-4 hours, and can open vavuum pump below each -25 DEG C of flaggy displays temperature vacuumizes, while by vacuum
Degree is arranged to 0.25-0.3mbar, is 25-30 DEG C by the temperature setting of conduction oil, opens ginseng air valve.It is small persistently to vacuumize 10-12
When after by the temperature setting of conduction oil be 35-40 DEG C, being persistently evacuated to each flaggy temperature display can close for more than 25 DEG C
Freeze dryer, go out dry storehouse.Spongy collagen fibrous proteins compound hemostatic plaster after export is cut into sealed bundle after dimension
Dress, it is collagen fibrous proteins compound hemostatic plaster product to send outside after irradiation sterilization.
The specific embodiment of the present invention is described below：
Embodiment 1
A, the extraction of fibrinogen
- 20 DEG C 95% of cold ethanol (adding ethanol by 10% volume ratio) is added in Swine plasma and produces sediment afterwards,
Precipitation is collected by centrifugation, supernatant is used to extract factor.With 0 DEG C 0.3% of Nacl aqueous solution washing precipitates.It is once molten
Solution：The Nacl aqueous solution that a lytic agent 1.3% is added into the sediment by washing is heated to 36 DEG C fully while stirring
Centrifugation removes insoluble matter and collects supernatant after dissolving；Secondary precipitation：- 20 DEG C 95% of cold second is added in supernatant after separation
Alcohol (adding ethanol by 10% volume ratio) precipitation fibrinogen, centrifugation go supernatant collection to precipitate.Secondary washing：With 0 DEG C
0.3% Nacl aqueous solution washing, it is pure fibrinogen that sediment, which is collected by centrifugation,.Such as the sediment after secondary washing
The Nacl aqueous solution of lytic agent 1.3% of addition is heated to 36 DEG C while stirring fully to be dissolved, then repeats again above-mentioned
Dissolving and washing step, until no longer there is insoluble matter.The sediment for finally washing to obtain is pure fibrinogen.
B, the extraction of factor
BaCl is slowly added to while stirring in the supernatant that said extracted crosses fibrinogen2Solution, containing anti-in blood plasma
Solidifying agent trisodium citrate, BaCl2Barium citrate is produced with trisodium citrate, barium citrate can adsorb factor and together sink
Form sediment.With 5L physiological saline suspension barium citrate-PCC, with 1mol/L acid for adjusting pH to 1.8, now barium citrate
Resolve into Ba2+And citrate ions, factor dissociate.Obtained sediment is centrifuged with the precipitation obtained after brine
As pure factor.
C, the preparation of collagen fibrous proteins compound hemostatic plaster
First freeze dryer cold-trap is started and cooled, treats that temperature drops to less than -50 DEG C, is added by every L porcine collagens solution
Enter the fibrinogen extracted in 2L Swine plasmas and factor be sufficiently mixed after to pour into stand in stainless steel disc immediately thick into 3mm
Thin layer, dry storehouse is placed into, each flaggy temp probe is immersed in above-mentioned mixed solution, before opening freeze dryer immediately after the door of pass
Case cools；Preceding case cools 3 hours, and can open vavuum pump below each -25 DEG C of flaggy displays temperature vacuumizes, while by vacuum
0.25mbar is arranged to, is 25 DEG C by the temperature setting of conduction oil, opens ginseng air valve.By conduction oil after persistently vacuumizing 10 hours
Temperature setting be 35 DEG C, freeze dryer can be closed by being persistently evacuated to each flaggy temperature display as more than 25 DEG C, go out dry storehouse.
Packed after spongy collagen fibrous proteins compound hemostatic plaster after export is cut into dimension, after sending irradiation sterilization outside
As collagen fibrous proteins compound hemostatic plaster product.
Embodiment 2
A, the extraction of fibrinogen
- 21 DEG C 95% of cold ethanol (adding ethanol by 11% volume ratio) is added in ox blood slurry and produces sediment afterwards,
Precipitation is collected by centrifugation, supernatant is used to extract factor.With 1 DEG C 0.4% of Nacl aqueous solution washing precipitates.It is once molten
Solution：The Nacl aqueous solution that a lytic agent 1.4% is added into the sediment by washing is heated to 37 DEG C fully while stirring
Centrifugation removes insoluble matter and collects supernatant after dissolving；Secondary precipitation：- 21 DEG C 95% of cold second is added in supernatant after separation
Alcohol (adding ethanol by 11% volume ratio) precipitation fibrinogen, centrifugation go supernatant collection to precipitate.Secondary washing：With 1 DEG C
0.4% Nacl aqueous solution washing, it is pure fibrinogen that sediment, which is collected by centrifugation,.Such as the sediment after secondary washing
The Nacl aqueous solution of lytic agent 1.4% of addition is heated to 37 DEG C while stirring fully to be dissolved, then repeats again above-mentioned
Dissolving and washing step, until no longer there is insoluble matter.The sediment for finally washing to obtain is pure fibrinogen.
B, the extraction of factor
BaCl is slowly added to while stirring in the supernatant that said extracted crosses fibrinogen2Solution, containing anti-in blood plasma
Solidifying agent trisodium citrate, BaCl2Barium citrate is produced with trisodium citrate, barium citrate can adsorb factor and together sink
Form sediment.With 5L physiological saline suspension barium citrate-PCC, with 1mol/L acid for adjusting pH to 1.8, now barium citrate
Resolve into Ba2+And citrate ions, factor dissociate.Obtained sediment is centrifuged with the precipitation obtained after brine
As pure factor.
C, the preparation of collagen fibrous proteins compound hemostatic plaster
First freeze dryer cold-trap is started and cooled, treats that temperature drops to less than -50 DEG C, is added by every L bovine collagens protein solution
Entering the fibrinogen that extracts and factor in 3L ox bloods slurry, to pour into stand in stainless steel disc after being sufficiently mixed immediately thick into 4mm
Thin layer, dry storehouse is placed into, each flaggy temp probe is immersed in above-mentioned mixed solution, before opening freeze dryer immediately after the door of pass
Case cools；Preceding case cools 4 hours, and can open vavuum pump below each -25 DEG C of flaggy displays temperature vacuumizes, while by vacuum
0.27mbar is arranged to, is 26 DEG C by the temperature setting of conduction oil, opens ginseng air valve.By conduction oil after persistently vacuumizing 11 hours
Temperature setting be 38 DEG C, freeze dryer can be closed by being persistently evacuated to each flaggy temperature display as more than 25 DEG C, go out dry storehouse.
Packed after spongy collagen fibrous proteins compound hemostatic plaster after export is cut into dimension, after sending irradiation sterilization outside
As collagen fibrous proteins compound hemostatic plaster product.
Embodiment 3
A, the extraction of fibrinogen
- 23 DEG C 95% of cold ethanol (adding ethanol by 13% volume ratio) is added in sheep blood plasma and produces sediment afterwards,
Precipitation is collected by centrifugation, supernatant is used to extract factor.With 2 DEG C 0.4% of Nacl aqueous solution washing precipitates.It is once molten
Solution：The Nacl aqueous solution that a lytic agent 1.5% is added into the sediment by washing is heated to 38 DEG C fully while stirring
Centrifugation removes insoluble matter and collects supernatant after dissolving；Secondary precipitation：- 23 DEG C 95% of cold second is added in supernatant after separation
Alcohol (adding ethanol by 13% volume ratio) precipitation fibrinogen, centrifugation go supernatant collection to precipitate.Secondary washing：With 2 DEG C
0.4% Nacl aqueous solution washing, it is pure fibrinogen that sediment, which is collected by centrifugation,.Such as the sediment after secondary washing
The Nacl aqueous solution of lytic agent 1.5% of addition is heated to 38 DEG C while stirring fully to be dissolved, then repeats again above-mentioned
Dissolving and washing step, until no longer there is insoluble matter.The sediment for finally washing to obtain is pure fibrinogen.
B, the extraction of factor
BaCl is slowly added to while stirring in the supernatant that said extracted crosses fibrinogen2Solution, containing anti-in blood plasma
Solidifying agent trisodium citrate, BaCl2Barium citrate is produced with trisodium citrate, barium citrate can adsorb factor and together sink
Form sediment.With 5L physiological saline suspension barium citrate-PCC, with 1mol/L acid for adjusting pH to 1.8, now barium citrate
Resolve into Ba2+And citrate ions, factor dissociate.Obtained sediment is centrifuged with the precipitation obtained after brine
As pure factor.
C, the preparation of collagen fibrous proteins compound hemostatic plaster
First freeze dryer cold-trap is started and cooled, treats that temperature drops to less than -50 DEG C, is added by every L sheep collagen solution
Enter the fibrinogen extracted in 4L sheep blood plasmas and factor be sufficiently mixed after to pour into stand in stainless steel disc immediately thick into 5mm
Thin layer, dry storehouse is placed into, each flaggy temp probe is immersed in above-mentioned mixed solution, before opening freeze dryer immediately after the door of pass
Case cools；Preceding case cools 4 hours, and can open vavuum pump below each -25 DEG C of flaggy displays temperature vacuumizes, while by vacuum
0.28mbar is arranged to, is 27 DEG C by the temperature setting of conduction oil, opens ginseng air valve.By conduction oil after persistently vacuumizing 12 hours
Temperature setting be 37 DEG C, freeze dryer can be closed by being persistently evacuated to each flaggy temperature display as more than 25 DEG C, go out dry storehouse.
Packed after spongy collagen fibrous proteins compound hemostatic plaster after export is cut into dimension, after sending irradiation sterilization outside
As collagen fibrous proteins compound hemostatic plaster product.
Embodiment 4
A, the extraction of fibrinogen
- 24 DEG C 95% of cold ethanol (adding ethanol by 14% volume ratio) is added in horse blood plasma and produces sediment afterwards,
Precipitation is collected by centrifugation, supernatant is used to extract factor.With 3 DEG C 0.3% of Nacl aqueous solution washing precipitates.It is once molten
Solution：The Nacl aqueous solution that a lytic agent 1.5% is added into the sediment by washing is heated to 38 DEG C fully while stirring
Centrifugation removes insoluble matter and collects supernatant after dissolving；Secondary precipitation：- 24 DEG C 95% of cold ethanol is added in supernatant after separation
(adding ethanol by 14% volume ratio) precipitation fibrinogen, centrifugation go supernatant collection to precipitate.Secondary washing：With 3 DEG C
0.3% Nacl aqueous solution washing, it is pure fibrinogen that sediment, which is collected by centrifugation,.Such as the sediment after secondary washing
The Nacl aqueous solution of lytic agent 1.5% of addition is heated to 38 DEG C while stirring fully to be dissolved, then repeats again above-mentioned
Dissolving and washing step, until no longer there is insoluble matter.The sediment for finally washing to obtain is pure fibrinogen.
B, the extraction of factor
BaCl is slowly added to while stirring in the supernatant that said extracted crosses fibrinogen2Solution, containing anti-in blood plasma
Solidifying agent trisodium citrate, BaCl2Barium citrate is produced with trisodium citrate, barium citrate can adsorb factor and together sink
Form sediment.With 5L physiological saline suspension barium citrate-PCC, with 1mol/L acid for adjusting pH to 1.8, now barium citrate
Resolve into Ba2+And citrate ions, factor dissociate.Obtained sediment is centrifuged with the precipitation obtained after brine
As pure factor.
C, the preparation of collagen fibrous proteins compound hemostatic plaster
First freeze dryer cold-trap is started and cooled, treats that temperature drops to less than -50 DEG C, is added by every L horse collagens protein solution
Enter the fibrinogen extracted in 4L horse blood plasma and factor be sufficiently mixed after to pour into stand in stainless steel disc immediately thick into 6mm
Thin layer, dry storehouse is placed into, each flaggy temp probe is immersed in above-mentioned mixed solution, before opening freeze dryer immediately after the door of pass
Case cools；Preceding case cools 3.5 hours, and can open vavuum pump below each -25 DEG C of flaggy displays temperature vacuumizes, while by vacuum
Degree is arranged to 0.3mbar, is 30 DEG C by the temperature setting of conduction oil, opens ginseng air valve.It will be led after persistently vacuumizing 10.5 hours
The temperature setting of deep fat is 36 DEG C, and freeze dryer can be closed for more than 25 DEG C by being persistently evacuated to each flaggy temperature display, goes out to freeze
Dry storehouse.Packed after spongy collagen fibrous proteins compound hemostatic plaster after export is cut into dimension, send irradiation outside
It is collagen fibrous proteins compound hemostatic plaster product after sterilizing.
Embodiment 5
A, the extraction of fibrinogen
- 24 DEG C 95% of cold ethanol (adding ethanol by 14% volume ratio) is added in Swine plasma and produces sediment afterwards,
Precipitation is collected by centrifugation, supernatant is used to extract factor.With 3 DEG C 0.3% of Nacl aqueous solution washing precipitates.It is once molten
Solution：The Nacl aqueous solution that a lytic agent 1.5% is added into the sediment by washing is heated to 38 DEG C fully while stirring
Centrifugation removes insoluble matter and collects supernatant after dissolving；Secondary precipitation：- 24 DEG C 95% of cold ethanol is added in supernatant after separation
(adding ethanol by 14% volume ratio) precipitation fibrinogen, centrifugation go supernatant collection to precipitate.Secondary washing：With 3 DEG C
0.3% Nacl aqueous solution washing, it is pure fibrinogen that sediment, which is collected by centrifugation,.Such as the sediment after secondary washing
The Nacl aqueous solution of lytic agent 1.5% of addition is heated to 38 DEG C while stirring fully to be dissolved, then repeats again above-mentioned
Dissolving and washing step, until no longer there is insoluble matter.The sediment for finally washing to obtain is pure fibrinogen.
B, the extraction of factor
BaCl is slowly added to while stirring in the supernatant that said extracted crosses fibrinogen2Solution, containing anti-in blood plasma
Solidifying agent trisodium citrate, BaCl2Barium citrate is produced with trisodium citrate, barium citrate can adsorb factor and together sink
Form sediment.With 5L physiological saline suspension barium citrate-PCC, with 1mol/L acid for adjusting pH to 1.8, now barium citrate
Resolve into Ba2+And citrate ions, factor dissociate.Obtained sediment is centrifuged with the precipitation obtained after brine
As pure factor.
C, the preparation of collagen fibrous proteins compound hemostatic plaster
First freeze dryer cold-trap is started and cooled, treats that temperature drops to less than -50 DEG C, is added by every L porcine collagens solution
Enter the fibrinogen extracted in 2.5L Swine plasmas and factor be sufficiently mixed after pour into stainless steel disc stand immediately into 3.5mm
Thick thin layer, is placed into dry storehouse, and each flaggy temp probe is immersed in above-mentioned mixed solution, and unlatching immediately is lyophilized after closing door
Case cools before machine；Preceding case cools 3.5 hours, and can open vavuum pump below each -25 DEG C of flaggy displays temperature vacuumizes, and simultaneously will
Vacuum is arranged to 0.29mbar, is 30 DEG C by the temperature setting of conduction oil, opens ginseng air valve.After persistently vacuumizing 10.5 hours
It is 40 DEG C by the temperature setting of conduction oil, freeze dryer can be closed for more than 25 DEG C by being persistently evacuated to each flaggy temperature display,
Go out dry storehouse.Pack, send outside after spongy collagen fibrous proteins compound hemostatic plaster after export is cut into dimension
It is collagen fibrous proteins compound hemostatic plaster product after irradiation sterilization.
Claims (8)
1. a kind of collagen fibrous proteins compound hemostatic plaster and preparation method thereof, it is characterized in that glue of the hemostatic adhesive bandage by mammal
Former protein solution is freeze-dried shape after being mixed in proportion with fibrinogen, the factor extracted from mammalian plasma
Into.
2. collagen as claimed in claim 1, fibrinogen, factor are mixed uniformly in proportion, and not stratified.
3. the extraction of fibrinogen as claimed in claim 1 is to add the mammalian plasma of anti-coagulants as raw material, addition-
20 to -25 DEG C 95% cold ethanol (adding ethanol by 10-15% volume ratio) caused sediment, then with 0-4 DEG C afterwards
0.3-0.5% Nacl aqueous solution washing precipitates.Once dissolve：Add lytic agent 1.3-1.5%'s into sediment
Centrifugation removes insoluble matter collection supernatant after the Nacl aqueous solution is heated to 36-38 DEG C of abundant dissolving while stirring；Secondary precipitation：Point
- 20 to -25 DEG C 95% cold ethanol (adding ethanol by 10-15% volume ratio) precipitation fiber egg is added in supernatant from after
Bai Yuan, centrifugation go supernatant collection to precipitate.Secondary washing：Washed, be collected by centrifugation with the 0-4 DEG C of 0.3-0.5% Nacl aqueous solution
Sediment.Secondary dissolving：Add secondary lytic agent (0.75% Nacl+0.01mol/L trisodium citrates-citrate buffer solution,
PH7.0), 36-38 DEG C is heated to while stirring to be completely dissolved, removal insoluble matter is then centrifuged if any insoluble matter.Will be secondary molten
It is stand-by into powder to solve liquid freeze-drying.
4. the extraction of factor as claimed in claim 1 be in the supernatant that said extracted crosses fibrinogen while stirring
It is slowly added to BaCl2Solution, trisodium citrate containing anti-coagulants in blood plasma, BaCl2Barium citrate, lemon are produced with trisodium citrate
Sour barium can adsorb factor and together precipitate.With 5L physiological saline suspension barium citrate-PCC, use
Ba is resolved into 1mol/L acid for adjusting pH to 1.8, now barium citrate2+And citrate ions, factor dissociate.What centrifugation obtained
Sediment is pure factor with the precipitation obtained after brine.
5. the ratio of three kinds of compositions as described in right 2 is that the fiber of 2-4L blood plasma extraction is added in every liter of (L) collagen solution
Proteinogen and factor are sufficiently mixed.
6. the freezing dry process of collagen fibrous proteins compound hemostatic plaster as claimed in claim 1 is：First freeze dryer cold-trap is opened
Dynamic cooling, treat that temperature drops to less than -50 DEG C, collagen solution, fibrinogen and factor be sufficiently mixed after immediately
Stand in stainless steel disc is poured into, into thin layer thick 3-6mm, to be placed into dry storehouse, each flaggy temp probe is immersed into above-mentioned mixed solution
In, front box of freeze dryer cooling is opened immediately after closing door；Preceding case cools 3-4 hours, below each -25 DEG C of flaggy displays temperature
Open vavuum pump to vacuumize, while vacuum be arranged to 0.25-0.3mbar, be 25-30 DEG C by the temperature setting of conduction oil,
Open ginseng air valve.It is 35-40 DEG C persistently to vacuumize the temperature setting of conduction oil after 10-12 hours, is persistently evacuated to each plate
Layer temperature display, which is more than 25 DEG C, can close freeze dryer, go out dry storehouse.
7. acid as claimed in claim 3 is acetic acid, one kind in hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, citric acid.
8. mammal as claimed in claim 1 is one kind of ox, sheep, horse, pig.
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