CN1228095C - Method for eliminating antigen from animal fur - Google Patents
Method for eliminating antigen from animal fur Download PDFInfo
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- CN1228095C CN1228095C CNB2004100225054A CN200410022505A CN1228095C CN 1228095 C CN1228095 C CN 1228095C CN B2004100225054 A CNB2004100225054 A CN B2004100225054A CN 200410022505 A CN200410022505 A CN 200410022505A CN 1228095 C CN1228095 C CN 1228095C
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Abstract
The present invention discloses a method for eliminating antigenic substances from animal fur. The present invention is characterized in that a supercritical fluid processor and chemical reagents are made use of, and antigenic substances are quickly, effectively and thoroughly eliminated from animal fur on the premise that the three-dimensional histological structure of the animal fur remains unchanged. The processed animal fur is suitable for a plurality of needs of medical application, and the present invention develops a new method for the medical application of the animal fur.
Description
One, technical field
The present invention relates to a kind of method of removing antigenic substance in the animal skins, belong to the manufacture field of bio-medical material.
Two, background technology
We know that collagen is extended familys in the collagen, mainly is I type and III Collagen Type VI, is the main component of mammalian body inner structure tissue, constitute the protein of human body about 30%.It is reported that up to now, the collagen-type of having found has 18 kinds or 19 kinds (Yan Longfei, Sun Zhirong " protein molecular structure " Beijing: publishing house of Tsing-Hua University, 1999).Put it briefly, Biological properties of collagen mainly shows following 4 aspects, that is: (1) low antigenicity; (2) absorbed characteristic in human body; (3) inducing cell propagation, growth; (4) promote platelet to condense.(but defend China, Wang Kunyu, Ceng Rui etc., the medical application of collagen and development prospect thereof, " biomedical engineering and clinical " .2004,8 (1): 45-48)
The animal dermal collagen is a kind of natural biologic material, has better biocompatibility, more weak antigenicity, and good hydrophilic property, and the adsorption of pair cell is strong.Because itself is exactly a kind of extracellular matrix components, also have the effect of inducing cell propagation and differentiation concurrently.Therefore, for a long time, people utilize the performance characteristics of animal dermal collagen, research and develop out a series of collagen base biological medical materials, as biological dressing, artificial skin, biological medicinal membrane, sthptic sponge, acellular dermal matrix material, Injectable Collagen and tissue engineering bracket material or the like, the part collagen base biological medical material has been applied to clinical, is proved to be to have certain curative effect.
Facts have proved that the greatest problem that collagen base biological medical material faced is that biocompatibility is difficult to control in manufacture process, thereby, the collagen base biological medical material reliability in use and the problem of safety easily caused.For example, a kind of Injectable Collagen that is used for the reduce wrinkle beauty treatment produced in USA is exactly because there is during clinical practice 20% people irritated and can not open up markets.As previously mentioned, collagen has low antigenicity, but this low antigenicity also can influence the biocompatibility of material under certain condition, and hyperacute rejection takes place.This may be exactly that the reliability and the safety of collagen base biological medical material up to now remains insoluble root place.
So-called antigen is meant that one group can be comprised micromolecule such as polypeptide, oligosaccharide and lipidic acid by the organic substance of T or B cell recognition, its chemical constituent often be different from host cell self chemical composition and can be by T and B cell recognition.
We found out qualitatively already that the antigenic substance that exists mainly was in animal skins: the epidermis cell of (1) animal skins, other cell component and fragment thereof; (2) " the end peptide " in the tropocollagen molecule, i.e. amino terminal in the polypeptide chain of tropocollagen molecule (N end) and carboxyl terminal (C end).These two zones belong to unspiralized region, have very big species variation, play main immunogenicity effect.This immunogenicity effect may can show when preparation collagen solution or membrane material, and in the acellular dermal matrix material because generally there is not " end peptide " structure (being actually the end peptide is not " activated "), and do not have any expression; (3) cell component between the animal dermal collagen fiber; (4) other small-molecule substance mainly comprises the catabolite of noncollagen protein or some xenobiotics etc.Because the existence of these antigenic substances has caused the reliability of collagen base biological medical material and safety extremely unstable, thereby, seriously limited the application and the development of collagen base biological medical material.
As mentioned above, thoroughly remove the antigen in the animal skins collagen, just equaled to open an important treasure-house of biomaterial resource, its prospect is estimated all within reason in any case.What is particularly worth mentioning is that along with deepening continuously of stem cell and applied research thereof, animal skins will play an increasingly important role as the desirable feedstock of collagen base biological medical material.
The relevant in the past report of removing antigenic substance in the animal skins generally only limits to take off cytosis.This cytosis of taking off mainly depends on NaCl, surfactant and certain special enzyme and realizes.This method is commonly used to prepare the acellular dermal matrix material.When needs adopt the method for acidolysis or enzymolysis to extract collagen, in the peptide chain of the collagen after the hydrolysis, may comprise the segment that has " end peptide ", this segment may have immunogenicity, thereby, belong to antigenic substance.The existence of this antigenic substance becomes the reliability of collagen base biological medical material and the basic reason that safety is under suspicion repeatedly.
Prior art has only considered to be present in a kind of antigenic substance---the cell in the animal skins, and does not consider the existence and the harm thereof of other antigenic substance comprehensively, and following deficiency is still arranged:
(1) method for removing cells complexity, and do not have controllability;
(2) be unsuitable for suitability for industrialized production;
(3) target is single.
Three, summary of the invention
The purpose of this invention is to provide a kind of method of removing antigenic substance in the animal skins at the deficiency of prior art, be characterized in utilizing treatment with supercritical fluid device and some chemical reagent, construct under the constant prerequisite in the three-dimensional tissue that keeps animal skins, thoroughly remove the antigenic substance that is present in the animal skins, lay a good foundation for utilizing animal skins collagen to prepare safe and reliable bio-medical material.
Purpose of the present invention is realized that by following technical measures wherein said raw material umber is parts by weight except that specifying.
Remove the method for the antigenic substance in the animal skins:
(1) choose healthy animal, through butchering, peel skin, get 100 parts of its new Cortex Dictamnis, put into rotary drum, add 28-36 ℃ of water 200-500 of temperature part, penetrating agent JFC 5-30 part is rotated 30-120min, controls dried waste liquid.Adding temperature again is 28-36 ℃ of water 200-500 part, builds the rotary drum door, rotates 30-120min.So repeatable operation is 3-5 time.Then, fully centrifuge dewatering is used in washing.
(2) from centrifuge, take out animal skins, put into the treatment with supercritical fluid device, add peregal 5-20 part, detergent 5-30 part and penetrating agent JFC 3-10 part, under temperature 0-40 ℃, the condition of pressure 7.0-30.0Mpa, handle 20-240min.
(3) in the treatment with supercritical fluid device, take out animal skins, put into rotary drum, add normal-temperature water 200-500 part, build the rotary drum door, rotate 30-120min, control dried waste liquid.Add room temperature 200-500 part again, build the rotary drum door, rotate 30-120min.So repeatable operation is 3-4 time.Fully after the washing, use centrifuge dewatering.
(4) take out animal skins from centrifuge, put into the treatment with supercritical fluid device, add compound enzyme collagen inorganic agent E-895 2-10 part, peregal 3-15 part under temperature 0-30 ℃, the condition of pressure 7.2-25.0Mpa, is handled 30-120min.Add sodium sulfide 0.5-8 part, sodium hydroxide 0.5-5 part and calcium oxide 5-10 part, under temperature 0-30 ℃, the condition of pressure 7.2-25.0Mpa, handle 40-90min.Add enzyme-added auxiliary liming agent EA 0.3-5 weight portion, under temperature 0-30 ℃, the condition of pressure 7.2-25.0Mpa, handle 30-240min.
(5) in the treatment with supercritical fluid device, take out animal skins, put into rotary drum, add normal-temperature water 200-500 part, build drum door, rotate 30-120min.Control dried waste liquid.Add normal-temperature water 200-500 part again, build the rotary drum door, rotate 30-120min.Then, fully washing.Adding temperature is 28-34 ℃ of water 200-400 part, ammonium chloride or ammonium sulfate 1.0-8.0 part, sulphuric acid 0.1-0.8 part and peregal 0.2-1.0 part, rotates 40-180min.Control dried waste liquid.Add normal-temperature water 200-500 part, build the rotary drum door, rotate 30-120min, control dried waste liquid.Add normal-temperature water 200-500 part again, build the rotary drum door, rotate 30-120min.Fully after the washing, animal skins is taken out, use centrifuge dewatering.
(6) take a sample from the animal skins after dehydration, detect, judge the removing effect of antigenic substance in the animal skins.After reaching requirement, can carry out subsequent operation according to a conventional method.
Among the present invention used animal skins be fresh virus-free infection, do not have dermatosis and do not have the animal skins of medical history, if pig, cattle, sheep domestic animal skin then should be chosen pig, cattle, the sheep domestic animal of not using forage feed.
Used rotary drum is suspension type wood rotary drum or rustless steel rotary drum among the present invention.
The method that the present invention adopts observation by light microscope to detect is differentiated the removing effect of antigenic substance in the animal skins, concrete grammar is: at first, randomly draw testing sample, the material of getting 15mm * 15mm size from testing sample is put into fixedly 24h of 10% neutral formalin solution as sample; Secondly, carry out tissue slice with freezing microtome, slice thickness is 0.8 μ m; Once more, adopt the plain method of Garapa that it is dyeed, gummy sealing is used in dehydration then; At last, carrying out tissue slice at the high optics microscope observes.Materials with hide glue fibril takes on a red color, and nucleus is crineous, and it is yellow that epidermis is, and it is yellow that muscle is.Collagen through the present invention handles should only contain collagen fiber, takes on a red color.Do not see that crineous and yellow, a show color are qualified samples so under light microscopic, observe.
The animal skins collagen that the present invention handled is mainly used in:
1. prepare 3D-SC artificial skin material.
Behind acidolysis or the enzymolysis as the raw material of collagen base biological medical material (as medical collagen film, sutures, sthptic sponge, Injectable Collagen etc.).
3. preparation tissue engineering bracket material.
4. prepare the tissue reconstruction host material.
The present invention has the following advantages:
1. under the prerequisite of three-dimensional tissue's structure of keeping animal skins collagen, antigenic substance and other all non-collagen tissues such as interfibrillar substance, cell and fragment thereof of animal skins collagen have been removed fast, efficiently.Animal skins after treatment is fit to multiple use.
2. method is simple, rapidly and efficiently, haves laid a good foundation for preparing a series of bio-medical materials with good biocompatibility, suitable biodegradation rate.
3. open up the brand-new road that animal skins collagen medical science is utilized, can avoid adopting animal tendon, Mus tail tendon etc. to extract the difficulty and the problem of the existing shortage of resources of collagen.
4. for antigenic removing in other class Collagen Type VI provides reliable reference method, this method also can promote the use of the removing of the antigenic substance in the collagen of other type.
5. have remarkable economic efficiency, social benefit and environmental benefit.
Four, description of drawings
Fig. 1 is one of light microscopic figure of collagen histological structure
As seen from Figure 1, the materials with hide glue fibril good dispersion, clean, free from foreign meter between materials with hide glue fibril, materials with hide glue fibril braiding rule, it is qualified to confirm as.
Fig. 2 be the collagen histological structure light microscopic figure two
As seen from Figure 2, the materials with hide glue fibril good dispersion, clean, free from foreign meter between materials with hide glue fibril, the materials with hide glue fibril braiding is comparatively loose, and it is qualified to confirm as.
Fig. 3 be the collagen histological structure light microscopic figure three
As seen from Figure 3, the materials with hide glue fibril good dispersion, but contain more impurity between materials with hide glue fibril, confirm as defective.
Fig. 4 be the collagen histological structure light microscopic figure four
As seen from Figure 4, the materials with hide glue fibril good dispersion, but contain small amount of impurities between materials with hide glue fibril, still confirm as defective.
Five, the specific embodiment
Below by embodiment the present invention is described particularly; have and to be pointed out that at this present embodiment only is used for the present invention is further specified; can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the foregoing invention content.
Embodiment 1
(1) choose healthy pig, through butchering, peel skin, get fresh porcine skin 100kg, put into rotary drum, the adding temperature is 28 ℃ water 220kg, and penetrating agent JFC 8kg builds the rotary drum door, rotates 60min, controls dried waste liquid.Adding temperature again is 28 ℃ of water 220kg, builds the rotary drum door, rotates 60min.So repeatable operation is 3 times.Then, fully centrifuge dewatering is used in washing.
(2) from centrifuge, take out Corii Sus domestica, put into the treatment with supercritical fluid device, add paregal O 8kg, detergent 5kg and penetrating agent JFC 3kg, under the condition of 5 ℃ of temperature, pressure 9.0Mpa, handle 60min.
(3) in the treatment with supercritical fluid device, take out Corii Sus domestica, put into rotary drum, add normal-temperature water 220kg, build the rotary drum door, rotate 60min, control dried waste liquid.Add normal-temperature water 220kg again, build the rotary drum door, rotate 60min.So repeatable operation is 3 times.Fully after the washing, use centrifuge dewatering.
(4) from centrifuge, take out Corii Sus domestica, put into supercritical processors, add compound enzyme collagen inorganic agent E-8953kg, peregal 6kg, under the condition of 5 ℃ of temperature, pressure 8.0Mpa, handle 60min.Add sodium sulfide 2.0kg, sodium hydroxide 0.8kg and calcium oxide 4.0kg, under the condition of 5 ℃ of temperature, pressure 8.0Mpa, handle 40min.Add enzyme-added auxiliary liming agent EA 1.5kg, under the condition of 5 ℃ of temperature, pressure 8.0Mpa, handle 120min.
(5) in the treatment with supercritical fluid device, take out Corii Sus domestica, put into rotary drum, add normal-temperature water 220kg, build the rotary drum door, rotate 60min.Control dried waste liquid.Add normal-temperature water 220kg again, build the rotary drum door, rotate 60min.Fully washing then.Adding temperature is 28 ℃ of water 220kg, and ammonium chloride (or ammonium sulfate) 2.5kg, sulphuric acid 0.2kg and paregal O 0.5kg rotate 90min.Control dried waste liquid, add normal-temperature water 220kg, build the rotary drum door, rotate 60min, control dried waste liquid.Add normal-temperature water 220kg again, build the rotary drum door, rotate 50min.Fully after the washing, Corii Sus domestica is taken out, use centrifuge dewatering.
(6) from the sampling of the Corii Sus domestica after dehydration, detection, judge antigenic removing effect in the Corii Sus domestica.After reaching requirement, can carry out subsequent operation according to a conventional method.
Embodiment 2
(1) choose healthy goat, through butchering, peel skin, get fresh goat skin 100kg, put into rotary drum, the adding temperature is 32 ℃ water 350kg, and penetrating agent JFC 16kg builds the rotary drum door, rotates 75min, controls dried waste liquid.Adding temperature again is 32 ℃ of water 350kg, builds the rotary drum door, rotates 75min.So repeatable operation is 4 times.Then, fully centrifuge dewatering is used in washing.
(2) from centrifuge, take out goat skin, put into the treatment with supercritical fluid device, add peregal 12kg, detergent 7.5kg and penetrating agent JFC 6.0kg, under the condition of 22 ℃ of temperature, pressure 18.0Mpa, handle 150min.
(3) in the treatment with supercritical fluid device, take out goat skin, put into rotary drum, add normal-temperature water 350kg, build the rotary drum door, rotate 75min, control dried waste liquid.Add normal-temperature water 350kg again, build the rotary drum door, rotate 75min.So repeatable operation is 4 times.Fully after the washing, use centrifuge dewatering.
(4) from centrifuge, take out goat skin, put into supercritical processors, add compound enzyme collagen inorganic agent E-895 6.0kg, peregal 9.0kg, under the condition of 15 ℃ of temperature, pressure 16.0Mpa, handle 90min.Add sodium sulfide 4.5kg, sodium hydroxide 3.0kg and calcium oxide 8.0kg, under the condition of 15 ℃ of temperature, pressure 16.0Mpa, handle 60min.Add enzyme-added auxiliary liming agent EA 2.5kg, under the condition of 15.0 ℃ of temperature, pressure 16.0Mpa, handle 150min.
(5) in the treatment with supercritical fluid device, take out goat skin, put into rotary drum, add normal-temperature water 350kg, build the rotary drum door, rotate 75min.Control dried waste liquid.Add normal-temperature water 350kg again, build the rotary drum door, rotate 75min.Fully washing then.Adding temperature is 300 parts in 31 ℃ of water, and ammonium chloride (or ammonium sulfate) 4.5kg, sulphuric acid 0.45kg and peregal 0.7kg rotate 110min.Control dried waste liquid.Add normal-temperature water 350kg, build the rotary drum door, rotate 75min, control dried waste liquid.Add normal-temperature water 350kg again, build the rotary drum door, rotate 75min.Fully after the washing, goat skin is taken out, use centrifuge dewatering.
(6) from the sampling of the goat skin after dehydration, detection, judge antigenic removing effect in the goat skin.After reaching requirement, can carry out subsequent operation according to a conventional method.
Embodiment 3
(1) choose healthy cattle, through butchering, peel skin, get fresh cattle hide 100kg, put into rotary drum, the adding temperature is 36 ℃ water 500kg, and penetrating agent JFC 30kg builds the rotary drum door, rotates 120min, controls dried waste liquid.Adding temperature again is 36 ℃ of water 500kg, builds the rotary drum door, rotates 120min.So repeatable operation is 5 times.Then, fully centrifuge dewatering is used in washing.
(2) from centrifuge, take out cattle hide, put into the treatment with supercritical fluid device, add peregal 20kg, detergent 30kg and penetrating agent JFC 10kg, under the condition of 40 ℃ of temperature, pressure 25.0Mpa, handle 240min.
(3) in the treatment with supercritical fluid device, take out cattle hide, put into rotary drum, add normal-temperature water 500kg, build the rotary drum door, rotate 120min, control dried waste liquid.Add normal-temperature water 500kg again, build the rotary drum door, rotate 120min.So repeatable operation is 5 times.Fully after the washing, use centrifuge dewatering.
(4) from centrifuge, take out cattle hide, put into supercritical processors, add compound enzyme collagen inorganic agent E-895 10.0kg, peregal 15.0kg, under the condition of 30 ℃ of temperature, pressure 25.0Mpa, handle 120min.Add sodium sulfide 8.0kg, sodium hydroxide 5.0kg and calcium oxide 10.0kg, under the condition of 30 ℃ of temperature, pressure 25.0Mpa, handle 90min.Add enzyme-added auxiliary liming agent EA 5.0kg, under the condition of 30 ℃ of temperature, pressure 25.0Mpa, handle 240min.
(5) in the treatment with supercritical fluid device, take out cattle hide, put into rotary drum, add normal-temperature water 500kg, build the rotary drum door, rotate 120min.Control dried waste liquid.Add normal-temperature water 500kg again, build the rotary drum door, rotate 120min.Fully washing then.Adding temperature is 34 ℃ of water 400kg, and ammonium chloride (or ammonium sulfate) 8.0kg, sulphuric acid 0.80kg and peregal 1.0kg rotate 180min.Control dried waste liquid, add normal-temperature water 500kg, build the rotary drum door, rotate 120min, control dried waste liquid.Add normal-temperature water 500kg again, build the rotary drum door, rotate 120min.Fully after the washing, cattle hide is taken out, use centrifuge dewatering.
(6) from the sampling of the cattle hide after dehydration, detection, judge antigenic removing effect in the cattle hide.After reaching requirement, can carry out subsequent operation according to a conventional method.
Used compound enzyme collagen inorganic agent is that Chengdu enlightening Australia collagen Hitek Ltd produces, enzyme-added auxiliary liming agent EA produces for the auxiliary reagent factory, Shanghai for the chemical plant production of Guangdong Pericarpium Citri tangerinae leather, peregal, detergent and penetrating agent JFC among the present invention.All the other chemical reagent are commercially available analysis pure preparation.
Claims (4)
1. method of removing antigenic substance in the animal skins is characterized in that:
(1) chooses healthy animal,, peel skin through butchering, get fresh animal skin 100 weight portions, put into rotary drum, adding temperature is 28-36 ℃ of water 200-500 weight portion, penetrating agent JFC 5-30 weight portion rotates 30-120min, controls dried waste liquid, adding temperature again is 28-36 ℃ of water 200-500 weight portion, builds the rotary drum door, rotates 30-120min, so repeatable operation is 3-5 time, then, and fully washing, use centrifuge dewatering
(2) from centrifuge, take out animal skins, put into the treatment with supercritical fluid device, add peregal 5-20 weight portion, detergent 5-30 weight portion and penetrating agent JFC 3-10 weight portion, under temperature 0-40 ℃, the condition of pressure 7.0-30.0Mpa, handle 20-240min,
(3) in the treatment with supercritical fluid device, take out animal skins, put into rotary drum, add normal-temperature water 200-500 weight portion, build the rotary drum door, rotate 30-120min, control dried waste liquid, add normal-temperature water 200-500 weight portion again, build the rotary drum door, rotate 30-120min, so repeatable operation is 3-5 time, fully after the washing, use centrifuge dewatering
(4) take out animal skins from centrifuge, put into the treatment with supercritical fluid device, add compound enzyme collagen inorganic agent E-8952-10 weight portion, peregal 3-15 weight portion under temperature 0-30 ℃, the condition of pressure 7.2-25.0Mpa, is handled 30-120min; Add sodium sulfide 0.5-8 weight portion, sodium hydroxide 0.5-5 weight portion and calcium oxide 0.5-10 weight portion, under temperature 0-30 ℃, the condition of pressure 7.2-25.0Mpa, handle 40-90min; Add enzyme-added auxiliary liming agent EA 0.3-5 weight portion, under temperature 0-30 ℃, the condition of pressure 7.2-25.0Mpa, handle 30-240min,
(5) in the treatment with supercritical fluid device, take out animal skins, put into rotary drum, add normal-temperature water 200-500 weight portion, build the rotary drum door, rotate 30-120min, control dried waste liquid, add normal-temperature water 200-500 weight portion again, build the rotary drum door, rotate 30-120min, then, fully washing, adding temperature is 28-34 ℃ of water 200-400 weight portion, ammonium sulfate or ammonium chloride 1.0-8.0 weight portion, sulphuric acid 0.1-0.8 weight portion and peregal 0.2-1.0 weight portion, rotate 40-180min, control dried waste liquid, add normal-temperature water 200-500 weight portion, build the rotary drum door, rotate 30-120min, control dried waste liquid, add normal-temperature water 200-500 weight portion again, build the rotary drum door, rotate 30-120min, fully after the washing, animal skins is taken out, use centrifuge dewatering
(6) from the animal skins sampling after dehydration, detect, judge the removing effect of antigenic substance in the animal skins, reach requirement after, can carry out subsequent operation according to a conventional method.
2. according to the method for antigenic substance in the described removing animal skins of claim 1, it is characterized in that animal skins be fresh virus-free infection, do not have dermatosis and do not have the animal skins of medical history.
3. according to the method for antigenic substance in the described removing animal skins of claim 1, it is characterized in that animal skins is not use the pig of forage feed, cattle, sheep domestic animal.
4. according to the method for antigenic substance in the described removing animal skins of claim 1, it is characterized in that used rotary drum is suspension type wood rotary drum or rustless steel rotary drum.
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| CNB2004100225054A CN1228095C (en) | 2004-05-12 | 2004-05-12 | Method for eliminating antigen from animal fur |
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| CNB2004100225054A CN1228095C (en) | 2004-05-12 | 2004-05-12 | Method for eliminating antigen from animal fur |
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| CN102580153B (en) * | 2012-03-09 | 2013-11-06 | 潘银根 | Method for preparing allograft acellular dermal matrixes |
| CN103263694B (en) * | 2013-05-14 | 2014-12-03 | 北京华信佳音医疗科技发展有限责任公司 | Collagen-based dura and preparation method thereof |
| ES2842505T3 (en) * | 2016-01-08 | 2021-07-14 | Acro Biomedical Company Ltd | Acellular corneas, their production methods and their uses |
| TWI712687B (en) * | 2018-09-11 | 2020-12-11 | 亞果生醫股份有限公司 | Acellular organs, and methods of producing the same |
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