CN103785060B - Fish skin collagen support loading epidermal growth factors and preparation method thereof - Google Patents
Fish skin collagen support loading epidermal growth factors and preparation method thereof Download PDFInfo
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- CN103785060B CN103785060B CN201410078358.6A CN201410078358A CN103785060B CN 103785060 B CN103785060 B CN 103785060B CN 201410078358 A CN201410078358 A CN 201410078358A CN 103785060 B CN103785060 B CN 103785060B
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Abstract
The invention discloses a fish skin collagen support loading epidermal growth factors and a preparation method thereof. The fish skin collagen support is composed of cross-linked fish skin collagen support bodies and nanometer particles of the load epidermal growth factors. The preparation method comprises the steps that after dewatering, degreasing, enzyme treatment and dialysis is conducted on fish skin, the cross-linked fish skin collagen support bodies are prepared, then the cross-linked fish skin collagen support bodies are soaked in a solution of the nanometer particles of the load epidermal growth factors, and the fish skin collagen support loading the epidermal growth factors is obtained after freezing and drying. The support has a slow releasing effect on the loaded factors, and prolongs the acting time of the epidermal growth factors at a trauma position. The fish skin collagen support obtained through the method has the advantages of being good in biocompatibility and biodegradability, free of immunogenicity and the like, is harmless to a wound surface, can effectively induce wound surface healing and is a good medical material.
Description
Technical field
The invention belongs to technical field of medical instruments, be specifically related to a kind ofly be used for the treatment of fish skin collagen support of the loading liquifier skin growth factor of diabetic ulcer and preparation method thereof.
Background technology
Along with living standard raising, life pattern change and social senilization, onset diabetes rate rises just year by year.Diabetic ulcer is one of its common complication, it is blood vessel and neuropathy caused by diabetes, cause the abnormal change of skin, clinical manifestation is local erosion, ulcer, necrosis, wound surface is prolonged does not heal, be one of diabetics severe complication, treatment can cause Pathological dissemination, operation amputation (toe) not in time, even threat to life.Diabetic ulcer is recurrent exerbation often, protracted course of disease, forms obstinate dermal chronic ulcer, the health of serious harm diabetic, reduces quality of life, brings pressure and the burden of great social economy aspect to patient.Therefore, the treatment of diabetic ulcer is one of important topic of urgent clinical needs solution.
Epidermal growth factor (epidermal growth factor, EGF) is a kind of polypeptide factor found the sixties in 20th century.HEGF (human epidermal growth factor, hEGF) extensively human multiple tissue is present in, have and promote keratinocyte, fibroblast, smooth muscle cell and the isocellular growth of endotheliocyte, studying and show in external model is in vitro that one effectively stimulates reparative factor, has the biologic activity promoting cell proliferation and tissue regeneration widely.Oneself develops recombinant human epidermal growth factor (recombinant human epidermal growth factor at present, rhEGF), there is the effect of asking when promoting epidermal growth, acceleration wound healing, shortening healing, and nontoxic, without chemistry and physical stimulation, mucosa better tolerance.In recent years about the applied research of epidermal growth factor is subject to the extensive concern of Chinese scholars, existing data proves that rhEGF has good therapeutical effect to the multiple organ injury such as lung, liver, kidney, gastrointestinal and body surface wound, burn, ulcer etc.
At present; the mode for the treatment of diabetic ulcer is mainly at wound surface local directly supplemented with exogenous epidermal growth factor; but there is following shortcoming in this kind of administering mode: epidermal growth factor diffusion is fast and the half-life is shorter; if carrier-free is protected; easily lost biologic activity by diluting or degrade; show as epidermal growth factor water preparation that local uses just to be destroyed by the proteasome degradation in wound fluid in several minutes of skin wound in contact, or to run off from wound surface very soon.For overcoming the above problems, epidermal growth factor is made slow released nano microsphere by the present invention, is carried on simultaneously and has in the fish skin collagen membrane support of good biological performance and space structure.Slow released nano microsphere is prepared from by the nanometer particle load epidermal growth factor adopting heparin and the chitosan with antibacterial action to form.
Collagen protein is the abundantest structural protein of animal body intensive amount, it has unique triple-helix structure, there is the features such as low antigenicity, avirulence, good biocompatibility and biodegradability, be widely used in the medical domains such as tissue engineering bracket material, sthptic sponge, medicine control slow-released system, cosmetic surgery.Current industry and the collagen applied clinically are all that this type of collagen exists by the self-contained infectious disease of animal as the risk of the virus contamination such as bovine spongiform encephalopathy, foot and mouth disease from terrestrial animal as extracted cattle, sheep, pig etc.External restriction uses cattle source property raw material production medical apparatus and instruments and medicine, and China also nonimportation cattle source property gelatin is used for the Capsules of pharmaceutical production.On the other hand, the people looking up to the area such as Islam, Islam repel and refusal uses the gelatin produced with Corii Sus domestica or Os Sus domestica and the pharmaceutical products produced with it.Except having good characteristic same with terrestrial animal collagen, there is not infectious disease and the problem of religion of above-mentioned infecting both domestic animals and human in fish skin collagen, and has the advantages such as raw material sources is extensive, cheap and easy to get.
Summary of the invention
The object of the present invention is to provide fish skin collagen support of a kind of loading liquifier skin growth factor and preparation method thereof, this fish skin collagen support is a kind ofly used for the treatment of diabetic ulcer, good to body nonhazardous, pliability and resistance to enzymatic, to have good biocompatibility and tissue repair performance skin tissue engineering scaffold.
For achieving the above object, the present invention adopts following technical scheme:
A kind of fish skin collagen support of loading liquifier skin growth factor is made up of the nanoparticle of the fish skin collagen support be cross-linked and loading liquifier skin growth factor.
Preparation method comprises the following steps:
(1) fish skin is scaled, and shreds clean, in 10 ~ 20 times of raw materials heavy be 0.5 ~ 3%H containing volume fraction
2o
2naOH solution in stir and soak 24-36 h, clear water is washed till neutrality; Continue to add aqueous isopropanol and stir 4 h, purification washes 3 times, then adds sodium chloride solution and stir 12 h, centrifugal, removes waste liquid, obtains pretreated broken fish skin;
(2) be that 1:5-1:10 mix with deionized water with mass ratio by pretreated broken fish skin, homogenate, adds pepsin, pH=2.0-3.0 is adjusted with hydrochloric acid, enzymolysis 16-24 h, filters, centrifugal 20 min of 8000-20000 rpm, in supernatant, add sodium chloride to final salinity is 0.9 mol/L, saltout, centrifugal 20 min of 8000 rpm, abandoning supernatant, the 0.5 mol/L acetic acid solution adding 10 times of volumes dissolves, and crosses and filters insoluble impurities; Then dialyse 12h in 0.1 mol/L acetic acid solution of 10 times of volumes, and every 3 h change a solution, finally extremely neutral with pure water dialysis, and lyophilization is crosslinked, purified water soaking and washing 5-10 time, and namely lyophilization obtains fish skin collagen support again;
(3) under stirring condition, the chitosan of pH=6.0-acetic acid solution instillation is contained in the heparin solution of epidermal growth factor, rate of addition 10-40 drips/min, mixing speed is 500-700 rpm, obtains the chitosan-heparin nano-particle solution of loading liquifier skin growth factor;
(4) the fish skin collagen support chitosan-heparin nano-particle solution of loading liquifier skin growth factor is pressed 2ml/cm
2ratio infiltrates, and lyophilization, obtains the fish skin collagen support of described loading liquifier skin growth factor.
The molecular weight of described chitosan is 8000-20000, and deacetylation is 70-90%.
Described fish skin is the fish skin of Hypophthalmichthys molitrix, Ctenopharyngodon idellus, Mylopharyngodon piceus, Aristichthys nobilis or shark.
Described in step (1), NaOH solution concentration is 0.01 ~ 0.05mol/L, and the volume fraction of described aqueous isopropanol is 10 ~ 20%, and the mass fraction of sodium chloride solution is 1.5 ~ 2.5%.
Step (2) described pepsin accounts for 0.5 ~ 2.5% of fish skin quality, and hydrolysis temperature is 4-8 DEG C; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution that crosslinked cross-linking agent used is the glutaraldehyde solution of volume fraction 0.2-0.3%, the D-ribose solution of volume fraction 0.2-0.3% or concentration are 40-60 mM, crosslinking time 10-24 h.
Step (3) chitosan-acetic acid solution concentration is 1-4 mg/ml, and heparin solution concentration is 0.5-2 mg/ml, and the load capacity of the chitosan-heparin nanoparticle of obtained loading liquifier skin growth factor is 3000 IU/ml.
Remarkable advantage of the present invention is: the collagen that the present invention adopts fish skin to originate, avoid the infectious disease such as bovine spongiform encephalopathy, the foot and mouth disease virus that the animal origin collagens such as cattle, sheep, pig may bring on the one hand, can effectively utilize fish production leftover bits and pieces on the other hand, create huge economic benefit; Prepared loading liquifier skin growth factor fish skin collagen support effectively eliminates the non-helical end peptide of tropocollagen molecule, therefore no antigen, and biocompatibility is good; Introduce epidermal growth factor in the bracket, can effectively induce wound site autogenous cell to grow, wound healing; The growth factor slow-release technology adopted, can make epidermal growth factor sustained release, efficiently solves the epidermal growth factor problems such as the half-life is too short in vivo.
Accompanying drawing explanation
Fig. 1 is the cross section field scanning Electronic Speculum figure of loading liquifier skin growth factor fish skin collagen support.
Fig. 2 is diabetic ulcer rat model wound repair experimental result.
Detailed description of the invention
embodiment 1: the preparation of fish skin collagen support
(1) fish skin is thawed, shred;
(2) after cleaning with distilled water, the volume fraction adding its quality 20 times is the hydrogen peroxide of 1% and the mixed solution of 0.01 mol/L sodium hydroxide, mechanical agitation 24 h, and every 8 h change primary alkali solution;
(3) add the aqueous isopropanol that volume fraction is 10%, mechanical agitation 4 h, purified water is cleaned;
(4) add the sodium chloride solution of 2.5%, mechanical agitation 12 h, purified water is cleaned;
(5) add 10 volume purified water, adjust pH=2.0-3.0 with hydrochloric acid, add the pepsin of fish skin quality 2.5%, at 4-8 DEG C, centrifugal 20 min of enzymolysis 16-24 h, 8000-20000 rpm, get supernatant;
(6) in above-mentioned supernatant, add a certain amount of sodium chloride to final salinity 0.9mol/L, saltout, centrifugal 20 min of 8000 rpm, abandoning supernatant;
(7) add the acetic acid of 0.5 mol/L of 10 times of volumes, cross and filter insoluble impurities.Then dialyse 12 h in 0.1 mol/L acetic acid solution of 10 times of volumes, and every 3 h change a solution, finally extremely neutral with pure water dialysis, lyophilization;
(8) crosslinked 24 h in 0.25% glutaraldehyde solution, purified water soaking and washing, again lyophilizing.
embodiment 2: the preparation of the nanoparticle of loading liquifier skin growth factor
(1) chitosan is dissolved in 1% acetic acid, dialyse 4 days, lyophilization, obtains the chitosan of purification;
(2) claim 0.2 g chitosan to be dissolved in 5 ml1% acetic acid, 0.1mol/LNaOH adjust ph to 6.0, be settled to 100 ml, obtaining concentration is 2 mg/ml chitosan solutions;
(3) compound concentration is 1 mg/ml heparin solution, with this solution for mother solution, and the heparin mixed liquor of preparation containing epidermal growth factor 10500 IU/ml;
(4) at 4 DEG C, above-mentioned for 2ml 1mg/mL heparin mixed liquor dropwise instills in 5 ml 2 mg/ml chitosan solutions under stirring by electricity, drips speed and drips for 10-40 per minute, can obtain the nanoparticle of finely dispersed loading liquifier skin growth factor.
embodiment 3: the preparation of loading liquifier skin growth factor fish skin collagen support
By the nanoparticle solution immersion of the fish skin collagen support of preparation in embodiment 1 with loading liquifier skin growth factor, ratio is 2 ml/cm
2; Infiltration is placed on-20 DEG C.Vacuum lyophilization, to obtain final product.
embodiment 4: loading liquifier skin growth factor fish skin collagen stent in the treatment diabetic ulcer rat model
(1) preparation of animal model: get male Wistar rat 40, after normal diet adaptability feeds 1 week, on an empty stomach, tail vein injection 1% streptozotocin (1% STZ) 45 mg/kg, detect blood glucose after 1 week, be considered as modeling success with blood glucose >=16. 7mmol/L.
(2) grouping and Ulcer Models preparation: random selecting 30 rat models are divided into 3 groups by blood glucose and body weight stratified random: load EGF collagen scaffold group, load nano particle collagen scaffold (without EGF) group, model control group.3 treated animals are anesthesia on an empty stomach, and hair is shaved at back, routine disinfection, and with card punch in rat back left of spine same position punching (f 2 cm), dark and fascia layer, with sterile gauze wrapping after hemostasis, freely drinks water and feeding.After 3 d, ulcer wound surface has granulation tissue to start growth, and Ulcer Models is successfully prepared.
(3) animal process: 3 groups of single cages of rat are fed, and routine disinfection before operation, 0.9% sodium chloride injection rinses wound surface.Loading liquifier skin growth factor collagen scaffold group, load nano particle collagen scaffold (without EGF) are organized and are sticked loading liquifier skin growth factor collagen scaffold and load nano particle collagen scaffold (without EGF) outside ulcer place respectively, model control group does not stick process, and above 3 treated animals are all fixed with sterile gauze and wrapped up.
(4) animal is observed: 3 groups of rats all detect blood glucose in 1,3,5,7,14 d tail venous blood samplings and carry out wound surface photograph simultaneously, and appliance computer image analysis system processes, and calculates the size of wound surface.
(5) data statistics: statistical method data represent with mean ± standard deviation, adopting SPSS software to carry out t inspection, is that data have significant difference with p<0.05.
(6) result of the test: result shows that this support has good therapeutical effect to diabetic ulcer rat model, compare with model control group and all have height statistical significance (p<0.01) the 7th, 14 day difference, can obviously promote that skin histology heals, accelerate healing rate.The results are shown in Figure 2.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (6)
1. a preparation method for the fish skin collagen support of loading liquifier skin growth factor, is characterized in that: support is made up of the nanoparticle of the fish skin collagen support be cross-linked and loading liquifier skin growth factor;
Fish skin collagen support is infiltrated, the fish skin collagen support of the loading liquifier skin growth factor described in obtained by freeze drying by the nano-particle solution of loading liquifier skin growth factor; Comprise the following steps:
(1) fish skin is scaled, and shreds clean, in 10 ~ 20 times of raw materials heavy be 0.5 ~ 3%H containing volume fraction
2o
2naOH solution in stir and soak 24-36 h, clear water is washed till neutrality; Continue to add aqueous isopropanol and stir 4 h, purification washes 3 times, then adds sodium chloride solution and stir 12 h, centrifugal, removes waste liquid, obtains pretreated broken fish skin;
(2) be that 1:5-1:10 mix with deionized water with mass ratio by pretreated broken fish skin, homogenate, adds pepsin, pH=2.0-3.0 is adjusted with hydrochloric acid, enzymolysis 16-24 h, filters, centrifugal 20 min of 8000-20000 rpm, in supernatant, add sodium chloride to final salinity is 0.9 mol/L, saltout, centrifugal 20 min of 8000 rpm, abandoning supernatant, the 0.5 mol/L acetic acid solution adding 10 times of volumes dissolves, and crosses and filters insoluble impurities; Then dialyse 12h in 0.1 mol/L acetic acid solution of 10 times of volumes, and every 3 h change a solution, finally extremely neutral with pure water dialysis, and lyophilization is crosslinked, purified water soaking and washing 5-10 time, and namely lyophilization obtains fish skin collagen support again;
(3) under stirring condition, the chitosan of pH=6.0-acetic acid solution instillation is contained in the heparin solution of epidermal growth factor, rate of addition 10-40 drips/min, mixing speed is 500-700 rpm, obtains the chitosan-heparin nano-particle solution of loading liquifier skin growth factor;
(4) the fish skin collagen support chitosan-heparin nano-particle solution of loading liquifier skin growth factor is pressed 2ml/cm
2ratio infiltrates, and lyophilization, obtains the fish skin collagen support of described loading liquifier skin growth factor.
2. the preparation method of the fish skin collagen support of loading liquifier skin growth factor according to claim 1, is characterized in that: the molecular weight of described chitosan is 8000-20000, and deacetylation is 70-90%.
3. the preparation method of the fish skin collagen support of loading liquifier skin growth factor according to claim 1, is characterized in that: described fish skin is the fish skin of Hypophthalmichthys molitrix, Ctenopharyngodon idellus, Mylopharyngodon piceus, Aristichthys nobilis or shark.
4. the preparation method of the fish skin collagen support of loading liquifier skin growth factor according to claim 1, it is characterized in that: described in step (1), NaOH solution concentration is 0.01 ~ 0.05mol/L, the volume fraction of described aqueous isopropanol is 10 ~ 20%, and the mass fraction of sodium chloride solution is 1.5 ~ 2.5%.
5. the preparation method of the fish skin collagen support of loading liquifier skin growth factor according to claim 1, is characterized in that: step (2) described pepsin accounts for 0.5 ~ 2.5% of fish skin quality, and hydrolysis temperature is 4-8 DEG C; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution that crosslinked cross-linking agent used is the glutaraldehyde solution of volume fraction 0.2-0.3%, the D-ribose solution of volume fraction 0.2-0.3% or concentration are 40-60 mM, crosslinking time 10-24 h.
6. the preparation method of the fish skin collagen support of loading liquifier skin growth factor according to claim 1, it is characterized in that: step (3) chitosan-acetic acid solution concentration is 1-4 mg/ml, heparin solution concentration is 0.5-2 mg/ml, and the load capacity of the chitosan-heparin nanoparticle of obtained loading liquifier skin growth factor is 3000 IU/ml.
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| CN104758983B (en) * | 2015-04-10 | 2017-01-18 | 福州大学 | Preparation method and application of bFGF-loaded fish collagen-based composite material |
| CN106390209A (en) * | 2016-08-31 | 2017-02-15 | 奥美医疗用品股份有限公司 | Silk fibroin biological material compounded with epidermal growth factor and preparation method of silk fibroin biological material |
| CN106880871B (en) * | 2017-01-18 | 2019-12-13 | 烟台正海生物科技股份有限公司 | Collagen dermal material for promoting endometrial repair and preparation method thereof |
| CN108355171B (en) * | 2018-04-09 | 2021-05-14 | 青岛海洋生物医药研究院 | Acellular dermal matrix guided tissue regeneration membrane material and preparation method and application thereof |
| CN111840650B (en) * | 2020-07-28 | 2022-03-18 | 中国医学科学院生物医学工程研究所 | Collagen-based modified citrus pectin composite material and preparation method and application thereof |
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| CN1382806A (en) * | 2002-05-15 | 2002-12-04 | 中国科学院上海有机化学研究所 | Collagen used for deep-sea fish skin tissue engineering and its preparing process |
| CN1511592A (en) * | 2002-12-30 | 2004-07-14 | 中国皮革和制鞋工业研究院 | Construction method for skin tissue engineering rack containing epidermal growth factor |
| CN101361988A (en) * | 2008-09-12 | 2009-02-11 | 西南交通大学 | Preparation method of blood vessel support or cardiac valve surface coating with good biocompatibility |
| CN101905038A (en) * | 2010-05-21 | 2010-12-08 | 中国医学科学院生物医学工程研究所 | Growth factor-loaded collagen group composite material as well as preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1382806A (en) * | 2002-05-15 | 2002-12-04 | 中国科学院上海有机化学研究所 | Collagen used for deep-sea fish skin tissue engineering and its preparing process |
| CN1511592A (en) * | 2002-12-30 | 2004-07-14 | 中国皮革和制鞋工业研究院 | Construction method for skin tissue engineering rack containing epidermal growth factor |
| CN101361988A (en) * | 2008-09-12 | 2009-02-11 | 西南交通大学 | Preparation method of blood vessel support or cardiac valve surface coating with good biocompatibility |
| CN101905038A (en) * | 2010-05-21 | 2010-12-08 | 中国医学科学院生物医学工程研究所 | Growth factor-loaded collagen group composite material as well as preparation method and application thereof |
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