CN104307038A - Gelatin-containing protein composite scaffold and preparation method thereof - Google Patents
Gelatin-containing protein composite scaffold and preparation method thereof Download PDFInfo
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- CN104307038A CN104307038A CN201310476339.4A CN201310476339A CN104307038A CN 104307038 A CN104307038 A CN 104307038A CN 201310476339 A CN201310476339 A CN 201310476339A CN 104307038 A CN104307038 A CN 104307038A
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- keratin
- gelatin
- solution
- albumen
- fibroin
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 49
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 49
- 108010010803 Gelatin Proteins 0.000 title claims abstract description 43
- 229920000159 gelatin Polymers 0.000 title claims abstract description 43
- 239000008273 gelatin Substances 0.000 title claims abstract description 43
- 235000019322 gelatine Nutrition 0.000 title claims abstract description 43
- 235000011852 gelatine desserts Nutrition 0.000 title claims abstract description 43
- 239000002131 composite material Substances 0.000 title claims abstract description 6
- 238000002360 preparation method Methods 0.000 title claims description 12
- 108010022355 Fibroins Proteins 0.000 claims abstract description 60
- 108010076876 Keratins Proteins 0.000 claims abstract description 58
- 102000011782 Keratins Human genes 0.000 claims abstract description 58
- 150000001875 compounds Chemical class 0.000 claims abstract description 27
- 239000000243 solution Substances 0.000 claims description 90
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 51
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 32
- 235000019333 sodium laurylsulphate Nutrition 0.000 claims description 32
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 22
- 239000012460 protein solution Substances 0.000 claims description 18
- 239000011734 sodium Substances 0.000 claims description 17
- 239000007864 aqueous solution Substances 0.000 claims description 16
- 239000011259 mixed solution Substances 0.000 claims description 16
- 239000004141 Sodium laurylsulphate Substances 0.000 claims description 11
- 235000013877 carbamide Nutrition 0.000 claims description 11
- 239000004202 carbamide Substances 0.000 claims description 11
- 238000001704 evaporation Methods 0.000 claims description 10
- 230000008020 evaporation Effects 0.000 claims description 10
- 238000001879 gelation Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 4
- 230000002269 spontaneous effect Effects 0.000 claims description 3
- 229910052783 alkali metal Inorganic materials 0.000 claims description 2
- 150000001340 alkali metals Chemical group 0.000 claims description 2
- -1 filter Substances 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 125000005843 halogen group Chemical group 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 43
- 210000003708 urethra Anatomy 0.000 abstract description 9
- 239000012528 membrane Substances 0.000 abstract description 2
- 238000001356 surgical procedure Methods 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 42
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 42
- 238000000502 dialysis Methods 0.000 description 35
- 238000001962 electrophoresis Methods 0.000 description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- 238000001035 drying Methods 0.000 description 14
- 239000000126 substance Substances 0.000 description 8
- 238000004140 cleaning Methods 0.000 description 7
- 239000000975 dye Substances 0.000 description 7
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 7
- 230000003203 everyday effect Effects 0.000 description 7
- 238000001914 filtration Methods 0.000 description 7
- 230000037308 hair color Effects 0.000 description 7
- 238000010438 heat treatment Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000002453 shampoo Substances 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- 238000003828 vacuum filtration Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 229920000954 Polyglycolide Polymers 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000004633 polyglycolic acid Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 229910004879 Na2S2O5 Inorganic materials 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 208000028484 Urethral disease Diseases 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 238000010041 electrostatic spinning Methods 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 229940056582 human hair preparation Drugs 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
Abstract
The invention provides a gelatin-containing protein composite scaffold. The gelatin-containing protein composite scaffold is made from silk fibroin, keratin and gelatin. A mass ratio of the silk fibroin to the keratin is preferably (1-10):(1-10); and a mass ratio of the gelatin to a total amount of the keratin and the silk fibroin is preferably (0.1-5):10. The gelatin-containing protein composite scaffold provided by the invention compounds the silk fibroin, the keratin and the gelatin, has physical strength superior to that of a membrane structural material constructed by using a silk fibroin solution and a pure protein combined solution, is suitable for requirements of actual surgery operations, and finally increases success rate of urethra repair and reconstruction.
Description
Technical field
The present invention relates to a kind of albumen compound rest, particularly relate to a kind of albumen compound rest containing gelatin that may be used for urethra reconstruction and preparation method thereof.
Background technology
The complexity urethral disease caused due to a variety of causes is always a difficult problem in Urology Surgery clinical treatment.Although multiple autologous tissue as an alternative material obtains desirable effect in urethra reconstruction, but Comparatively speaking tissue engineering technique does not have the inherent shortcoming of the former " sacrifice normal structure is cost; with operation wound repair tissue defect ", thus more can be used as the Main way of following urethra reconstruction development.In existing domestic and foreign literature report for various tissue engineering material or the material-research of seed cell complex in urethra reconstruction gradually from laboratory to clinical expansion.Wherein be no lack of the case having actual urine tract disease clinical treatment among a small circle, and obtain the satisfied preliminary efficacy of another people.
But relative to being gradually improved of seed cell research, in urethral tissue engineering reconstruction, select ideal but the failing all the time of which kind of timbering material to reach common understanding.Main employing two large class supports in the Tissue Engineering Study of urethra at present: natural de-cell collagen substrate (SIS, BAMG, acellular dermal matrix etc.) and synthetic material (polyglycolic acid, polylactic acid/polyglycolic acid copolymer).Although the former is easy to prepare, be applicable to commercial production, due to xenogenesis and even the allosome biological tissue of having drawn from more, be thus subject in biological safety always some scholars query, be difficult to accept by all patients.Simultaneously it is at space structure, biomechanics characteristic and promote that blood capillary and Premeabilisation of cells aspect also also exist inherent shortcoming simultaneously.Although the risk of biological safety aspect to drop to minimum by the latter, but the metabolite of support in degradation process can cause inevitable negative effect to the cell of surrounding and microenvironment, synthetic class support for want of corresponding somatomedin in addition, stick at promotion seed cell, also there is inborn deficiency propagation aspect.
Based on above-mentioned background, at present in this research field, the structure utilizing protein to carry out associated biomolecule timbering material is just receiving the concern of increasing researcher.In existing report, the report utilizing fibroin albumen, human hair keratin to carry out bioprotein support structure has successful story.But the mechanical characteristic of the simple proteins timbering material gone out constructed by existing method still cannot be comparable with the timbering material of above-mentioned natural de-cell collagen substrate and synthetic.If the mechanical property of above-mentioned protein material will be strengthened further, the method for electrostatic spinning that adopts operates more at present.This operating technology is higher to equipment requirements, relates to the high enrichment of protein solution simultaneously, and its operation is also more loaded down with trivial details.
Comparatively speaking also proposed multiple protein to mix in external correlational study, different proteins can be utilized to cause because of the difference of space structure the different feature of mechanical property finally to prepare the mechanics strengthening of material.Although utilize Crinis Carbonisatus reduced form keratin to be combined the report building timbering material with fibroin albumen existing in existing report, its suitable concentration proportioning is not still deeply probed into.Although the material more single albumen support in mechanical property simultaneously obtained by two kinds of mixed proteins improves to some extent, but still there is a certain distance with natural acellular matrix support.
For the problems referred to above, once adopted chemical cross-linking agent as Ji Niping, or photo-crosslinking method carry out further mechanics modification to timbering material in the past.But the cross-linking agent involved by chemical crosslink technique may bring certain negative effect to the cell seeding on the follow-up et al. Ke of material and its surface.And optical cross-linking method longer processing time may bring certain impact to the stability of protein.
Summary of the invention
The object of the invention is to overcome above-mentioned deficiency, a kind of albumen compound rest containing gelatin is provided, its physical characteristic more existing simple protein timbering material is more excellent, thus is suitable for the needs of actual operation operation, the final success rate improving urethra reconstruction.
First aspect of the present invention is to provide a kind of albumen compound rest containing gelatin that may be used for urethra reconstruction, and the composition material of described albumen compound rest comprises fibroin albumen, keratin and gelatin.
Preferably, described fibroin albumen and keratic mass ratio are (1-10): (1-10), are more preferably (1.5-9): (1.5-9), are more preferably (2-8): (2-8), be more preferably (3-7): (3-7), such as 3:7,4:6,5:5,6:4 or 7:3.
Preferably, the mass ratio of described gelatin and keratin and fibroin albumen total amount is (0.1-5): 10, is more preferably (0.5-4): 10, is more preferably (0.8-3.5): 10, be more preferably (1-3): 10, such as 1.5:10,2:10,2.5:10 or 2.8:10.
Wherein, described keratin is preferably reduced form human hair keratin.
Second aspect of the present invention is to provide a kind of preparation method of the above-mentioned albumen compound rest containing gelatin, comprises the following steps:
Step 1, is mixed to get mixed liquid of protein by keratin solution and silk fibroin protein solution, and control fibroin albumen and keratic mass ratio are (1-10): (1-10), and in mixed liquid of protein, the total concentration of keratin and fibroin albumen is 0.05-0.2g/mL;
Step 2, joins gelatin in mixed liquid of protein, after gelatin dissolves completely, mixed solution liquid sealing is left standstill, until gelation, uncovered spontaneous evaporation, obtain albumen compound rest, wherein, the mass ratio of described gelatin and keratin and fibroin albumen total amount is (0.1-5): 10.
In step 1, described fibroin albumen and keratic mass ratio are preferably (1.5-9): (1.5-9), is more preferably (2-8): (2-8), is more preferably (3-7): (3-7), such as 3:7,4:6,5:5,6:4 or 7:3.
In step 1, in mixed liquid of protein, the total concentration of keratin and fibroin albumen is preferably 0.06-0.17g/mL, is more preferably 0.07-0.15g/mL, is more preferably 0.08-0.13g/mL, is more preferably 0.09-0.11g/mL, such as 0.1g/mL or 0.105g/mL.
In step 2, the mass ratio of described gelatin and keratin and fibroin albumen total amount is preferably (0.5-4): 10, is more preferably (0.8-3.5): 10, is more preferably (1-3): 10, such as 1.5:10,2:10,2.5:10 or 2.8:10
In step 1, described keratin is preferably reduced form human hair keratin, and preparation method is as follows: human hair is placed in extracting solution, 50-150 DEG C (is preferably 70-130 DEG C, be more preferably 80-120 DEG C, be more preferably 100-110 DEG C) under soak 10-60min(and be preferably 15-50min, be more preferably 25-35min), filter, filtrate is dialysed, concentrated, obtain reduced form human hair keratin, wherein, the quality of human hair is (0.5-4g) with the ratio of the volume of extracting solution: (5-20mL).
Further preferably, the quality of human hair is (1-3g) with the ratio of the volume of extracting solution: (8-15mL), is more preferably (1.5-2g): (10-12mL).
Preferably, described extracting solution is carbamide, sodium lauryl sulphate, Na
2s
2o
5mixed aqueous solution.
Further preferably, in described extracting solution, in every 100mL water, 35-60g is preferably containing 30-70g(, being more preferably 45-50g) carbamide, 40-80g(be preferably 45-70g, be more preferably 50-60g) sodium lauryl sulphate, 80-120g(be preferably 88-110g, is more preferably 92-100g) Na2S2O5.
In step 1, the preparation method of described fibroin albumen is as follows: be dissolved in by boiled silk in MX solution, filters, filtrate is dialysed, concentrated, obtain the fibroin albumen of purification, wherein, M is alkali metal (such as Li, Na, K etc.), X is halogen (such as F, Cl, Br, I etc.).
Preferably, in step 2, after gelation, uncovered spontaneous evaporation 8-16h(is preferably 10-14h, is more preferably 11-12h) after, pour anhydrous alcohol 0.5-2h(into and be preferably 0.8-1.5h, be more preferably 1-1.2h) afterwards take out obtain albumen compound rest.
Albumen compound rest containing gelatin provided by the invention is compounded with keratin, fibroin albumen, gelatin, physical strength is better than and utilizes simple silk fibroin protein solution and the membrane structure material constructed by simple proteins combination solution, be more suitable for the needs of actual operation operation, the final success rate improving urethra reconstruction.
Accompanying drawing explanation
Fig. 1 is the HE colored graph of the albumen compound rest containing gelatin provided by the invention.
Detailed description of the invention
With reference to the accompanying drawings, the invention will be further described in conjunction with specific embodiments, to understand the present invention better.
Embodiment 1
1, in barber shop, obtain the normal hair color of dye-free, conventional shampoo cleaning 1-2 time, is placed in ordinary room temperature and carries out drying.
2, the hair after dried to be soaked in the mixed solution of ethyl acetate and methanol 24 hours, wherein the volume ratio of ethyl acetate and methanol is 3:1.
3, after hair after drying, fully shred, it is added extracting solution in 1.5g/10ml ratio and (in every 1000ml water, dissolves 480.4g carbamide, 57.676g sodium lauryl sulphate SDS, 95.045gNa
2s
2o
5) in, at 100 DEG C of oil bath heating 30min after submergence.
4, vacuum filtration, is placed in bag filter by black extracting solution, dialysis 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5.
5, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 10000) aqueous solution, concentrated 6 hours, regulate reduced form human hair keratin concentration be after 0.1g/mL, row SDS protein electrophoresis detects prompting protein electrophoresis band at about 45-60kD, illustrates and obtains reduced form human hair keratin.
6, the reduced form human hair keratin solution obtained is preserved under 4 DEG C of environment.
7, be dissolved in 60ml water by 100g lithium bromide, be mixed with lithium bromide water solution, agitating solution is heated to 60 DEG C simultaneously.
8, by the boiled silk 10g solution lithium bromide water solution purchased on market, after 5h, multilamellar filtered through gauze, after filtering, solution is placed in bag filter.
9, dialyse 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5, change water every day once.
10, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 20000) aqueous solution, concentrated 6h, the concentration being adjusted to fibroin albumen is 0.1g/mL, and row SDS protein electrophoresis detects prompting protein electrophoresis band at about 25-30kD, illustrates and obtains fibroin albumen.
11, the silk fibroin protein solution obtained is preserved under 4 DEG C of environment.
12, by the reduced form human hair keratin solution obtained and silk fibroin protein solution by volume 5:5 mix.
13. get the mixed liquor 15ml that step 12 obtains, and are merged and stir and be heated to 37 DEG C, add gelatin, and the concentration controlling gelatin in mixed liquor is 0.01g/mL.Insert after high-speed stirred 30s in square mould.
14. mixed solutions are in square mould, and mould is added a cover, and ambient temperatare puts 12h hour, and after band gelation, uncap, natural evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
Final acquisition material general appearance is the translucent film material of brown, and its HE dyes and points out positive membranoid substance of this material one eosin stains (shown in Fig. 1).
Detect through mechanical stretch, the maximum elongation rate 45.49% of this material, elastic modelling quantity 0.1Mpa.
Embodiment 2
1, in barber shop, obtain the normal hair color of dye-free, conventional shampoo cleaning 1-2 time, is placed in ordinary room temperature and carries out drying.
2, the hair after dried to be soaked in the mixed solution of ethyl acetate and methanol 24 hours, wherein the volume ratio of ethyl acetate and methanol is 3:1.
3, after hair after drying, fully shred, it is added extracting solution in 1.5g/10ml ratio and (in every 1000ml water, dissolves 480.4g carbamide, 57.676g sodium lauryl sulphate SDS, 95.045gNa
2s
2o
5) in, at 100 DEG C of oil bath heating 30min after submergence.
4, vacuum filtration, is placed in bag filter by black extracting solution, dialysis 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5.
5, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 10000) aqueous solution, concentrated 6 hours, regulate reduced form human hair keratin concentration be after 0.1g/mL, row SDS protein electrophoresis detects prompting protein electrophoresis band at about 45-60kD, illustrates and obtains reduced form human hair keratin.
6, the reduced form human hair keratin solution obtained is preserved under 4 DEG C of environment.
7, be dissolved in 60ml water by 100g lithium bromide, be mixed with lithium bromide water solution, agitating solution is heated to 60 DEG C simultaneously.
8, by the boiled silk 10g solution lithium bromide water solution purchased on market, after 5h, multilamellar filtered through gauze, after filtering, solution is placed in bag filter.
9, dialyse 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5, change water every day once.
10, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 20000) aqueous solution, concentrated 6h, the concentration being adjusted to fibroin albumen is 0.1g/mL, and row SDS protein electrophoresis detects prompting protein electrophoresis band at about 25-30kD, illustrates and obtains fibroin albumen.
11, the silk fibroin protein solution obtained is preserved under 4 DEG C of environment.
12, by the reduced form human hair keratin solution obtained and silk fibroin protein solution by volume 5:5 mix.
13. get the mixed liquor 15ml that step 12 obtains, and are merged and stir and be heated to 37 DEG C, add gelatin, and the concentration controlling gelatin in mixed liquor is 0.02g/mL.Insert after high-speed stirred 30s in square mould.
14. mixed solutions are in square mould, and mould is added a cover, and ambient temperatare puts 12h hour, and after band gelation, uncap, natural evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
Final acquisition material general appearance is the translucent film material of brown, and its HE dyes and points out this material one eosin stains positive membranoid substance.
Detect through mechanical stretch, the maximum elongation rate 55.29% of this material, elastic modelling quantity 0.117Mpa.
Embodiment 3
1, in barber shop, obtain the normal hair color of dye-free, conventional shampoo cleaning 1-2 time, is placed in ordinary room temperature and carries out drying.
2, the hair after dried to be soaked in the mixed solution of ethyl acetate and methanol 24 hours, wherein the volume ratio of ethyl acetate and methanol is 3:1.
3, after hair after drying, fully shred, it is added extracting solution in 1.5g/10ml ratio and (in every 1000ml water, dissolves 480.4g carbamide, 57.676g sodium lauryl sulphate SDS, 95.045gNa
2s
2o
5) in, at 100 DEG C of oil bath heating 30min after submergence.
4, vacuum filtration, is placed in bag filter by black extracting solution, dialysis 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5.
5, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 10000) aqueous solution, concentrated 6 hours, regulate reduced form human hair keratin concentration be after 0.1g/mL, row SDS protein electrophoresis detects prompting protein electrophoresis band at about 45-60kD, illustrates and obtains reduced form human hair keratin.
6, the reduced form human hair keratin solution obtained is preserved under 4 DEG C of environment.
7, be dissolved in 60ml water by 100g lithium bromide, be mixed with lithium bromide water solution, agitating solution is heated to 60 DEG C simultaneously.
8, by the boiled silk 10g solution lithium bromide water solution purchased on market, after 5h, multilamellar filtered through gauze, after filtering, solution is placed in bag filter.
9, dialyse 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5, change water every day once.
10, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 20000) aqueous solution, concentrated 6h, the concentration being adjusted to fibroin albumen is 0.1g/mL, and row SDS protein electrophoresis detects prompting protein electrophoresis band at about 25-30kD, illustrates and obtains fibroin albumen.
11, the silk fibroin protein solution obtained is preserved under 4 DEG C of environment.
12, by the reduced form human hair keratin solution obtained and silk fibroin protein solution by volume 5:5 mix.
13. get the mixed liquor 15ml that step 12 obtains, and are merged and stir and be heated to 37 DEG C, add gelatin, and the concentration controlling gelatin in mixed liquor is 0.03g/mL.Insert after high-speed stirred 30s in square mould.
14. mixed solutions are in square mould, and mould is added a cover, and ambient temperatare puts 12h hour, and after band gelation, uncap, natural evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
Final acquisition material general appearance is the translucent film material of brown, and its HE dyes and points out this material one eosin stains positive membranoid substance.
Detect through mechanical stretch, the maximum elongation rate 11.27% of this material, elastic modelling quantity 0.05Mpa.
Embodiment 4
1, in barber shop, obtain the normal hair color of dye-free, conventional shampoo cleaning 1-2 time, is placed in ordinary room temperature and carries out drying.
2, the hair after dried to be soaked in the mixed solution of ethyl acetate and methanol 24 hours, wherein the volume ratio of ethyl acetate and methanol is 3:1.
3, after hair after drying, fully shred, it is added extracting solution in 1.5g/10ml ratio and (in every 1000ml water, dissolves 480.4g carbamide, 57.676g sodium lauryl sulphate SDS, 95.045gNa
2s
2o
5) in, at 100 DEG C of oil bath heating 30min after submergence.
4, vacuum filtration, is placed in bag filter by black extracting solution, dialysis 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5.
5, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 10000) aqueous solution, concentrated 6 hours, regulate reduced form human hair keratin concentration be after 0.1g/mL, row SDS protein electrophoresis detects prompting protein electrophoresis band at about 45-60kD, illustrates and obtains reduced form human hair keratin.
6, the reduced form human hair keratin solution obtained is preserved under 4 DEG C of environment.
7, be dissolved in 60ml water by 100g lithium bromide, be mixed with lithium bromide water solution, agitating solution is heated to 60 DEG C simultaneously.
8, by the boiled silk 10g solution lithium bromide water solution purchased on market, after 5h, multilamellar filtered through gauze, after filtering, solution is placed in bag filter.
9, dialyse 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5, change water every day once.
10, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 20000) aqueous solution, concentrated 6h, the concentration being adjusted to fibroin albumen is 0.1g/mL, and row SDS protein electrophoresis detects prompting protein electrophoresis band at about 25-30kD, illustrates and obtains fibroin albumen.
11, the silk fibroin protein solution obtained is preserved under 4 DEG C of environment.
12, by the reduced form human hair keratin solution obtained and silk fibroin protein solution by volume 3:7 mix.
13. get the mixed liquor 15ml that step 12 obtains, and are merged and stir and be heated to 37 DEG C, add gelatin, and the concentration controlling gelatin in mixed liquor is 0.02g/mL.Insert after high-speed stirred 30s in square mould.
14. mixed solutions are in square mould, and mould is added a cover, and ambient temperatare puts 12h hour, and after band gelation, uncap, natural evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
Final acquisition material general appearance is the translucent film material of brown, and its HE dyes and points out this material one eosin stains positive membranoid substance.
Detect through mechanical stretch, the maximum elongation rate 42.06% of this material, elastic modelling quantity 0.09Mpa.
Embodiment 5
1, in barber shop, obtain the normal hair color of dye-free, conventional shampoo cleaning 1-2 time, is placed in ordinary room temperature and carries out drying.
2, the hair after dried to be soaked in the mixed solution of ethyl acetate and methanol 24 hours, wherein the volume ratio of ethyl acetate and methanol is 3:1.
3, after hair after drying, fully shred, it is added extracting solution in 1.5g/10ml ratio and (in every 1000ml water, dissolves 480.4g carbamide, 57.676g sodium lauryl sulphate SDS, 95.045gNa
2s
2o
5) in, at 100 DEG C of oil bath heating 30min after submergence.
4, vacuum filtration, is placed in bag filter by black extracting solution, dialysis 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5.
5, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 10000) aqueous solution, concentrated 6 hours, regulate reduced form human hair keratin concentration be after 0.1g/mL, row SDS protein electrophoresis detects prompting protein electrophoresis band at about 45-60kD, illustrates and obtains reduced form human hair keratin.
6, the reduced form human hair keratin solution obtained is preserved under 4 DEG C of environment.
7, be dissolved in 60ml water by 100g lithium bromide, be mixed with lithium bromide water solution, agitating solution is heated to 60 DEG C simultaneously.
8, by the boiled silk 10g solution lithium bromide water solution purchased on market, after 5h, multilamellar filtered through gauze, after filtering, solution is placed in bag filter.
9, dialyse 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5, change water every day once.
10, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 20000) aqueous solution, concentrated 6h, the concentration being adjusted to fibroin albumen is 0.1g/mL, and row SDS protein electrophoresis detects prompting protein electrophoresis band at about 25-30kD, illustrates and obtains fibroin albumen.
11, the silk fibroin protein solution obtained is preserved under 4 DEG C of environment.
12, by the reduced form human hair keratin solution obtained and silk fibroin protein solution by volume 4:6 mix.
13. get the mixed liquor 15ml that step 12 obtains, and are merged and stir and be heated to 37 DEG C, add gelatin, and the concentration controlling gelatin in mixed liquor is 0.02g/mL.Insert after high-speed stirred 30s in square mould.
14. mixed solutions are in square mould, and mould is added a cover, and ambient temperatare puts 12h hour, and after band gelation, uncap, natural evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
Final acquisition material general appearance is the translucent film material of brown, and its HE dyes and points out this material one eosin stains positive membranoid substance.
Detect through mechanical stretch, the maximum elongation rate 39.31% of this material, elastic modelling quantity 0.146Mpa.
Embodiment 6
1, in barber shop, obtain the normal hair color of dye-free, conventional shampoo cleaning 1-2 time, is placed in ordinary room temperature and carries out drying.
2, the hair after dried to be soaked in the mixed solution of ethyl acetate and methanol 24 hours, wherein the volume ratio of ethyl acetate and methanol is 3:1.
3, after hair after drying, fully shred, it is added extracting solution in 1.5g/10ml ratio and (in every 1000ml water, dissolves 480.4g carbamide, 57.676g sodium lauryl sulphate SDS, 95.045gNa
2s
2o
5) in, at 100 DEG C of oil bath heating 30min after submergence.
4, vacuum filtration, is placed in bag filter by black extracting solution, dialysis 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5.
5, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 10000) aqueous solution, concentrated 6 hours, regulate reduced form human hair keratin concentration be after 0.1g/mL, row SDS protein electrophoresis detects prompting protein electrophoresis band at about 45-60kD, illustrates and obtains reduced form human hair keratin.
6, the reduced form human hair keratin solution obtained is preserved under 4 DEG C of environment.
7, be dissolved in 60ml water by 100g lithium bromide, be mixed with lithium bromide water solution, agitating solution is heated to 60 DEG C simultaneously.
8, by the boiled silk 10g solution lithium bromide water solution purchased on market, after 5h, multilamellar filtered through gauze, after filtering, solution is placed in bag filter.
9, dialyse 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5, change water every day once.
10, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 20000) aqueous solution, concentrated 6h, the concentration being adjusted to fibroin albumen is 0.1g/mL, and row SDS protein electrophoresis detects prompting protein electrophoresis band at about 25-30kD, illustrates and obtains fibroin albumen.
11, the silk fibroin protein solution obtained is preserved under 4 DEG C of environment.
12, by the reduced form human hair keratin solution obtained and silk fibroin protein solution by volume 6:4 mix.
13. get the mixed liquor 15ml that step 12 obtains, and are merged and stir and be heated to 37 DEG C, add gelatin, and the concentration controlling gelatin in mixed liquor is 0.02g/mL.Insert after high-speed stirred 30s in square mould.
14. mixed solutions are in square mould, and mould is added a cover, and ambient temperatare puts 12h hour, and after band gelation, uncap, natural evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
Final acquisition material general appearance is the translucent film material of brown, and its HE dyes and points out positive membranoid substance of this material one eosin stains (shown in Fig. 1).
Detect through mechanical stretch, the maximum elongation rate 52.43% of this material, elastic modelling quantity 0.202Mpa.
Embodiment 7
1, in barber shop, obtain the normal hair color of dye-free, conventional shampoo cleaning 1-2 time, is placed in ordinary room temperature and carries out drying.
2, the hair after dried to be soaked in the mixed solution of ethyl acetate and methanol 24 hours, wherein the volume ratio of ethyl acetate and methanol is 3:1.
3, after hair after drying, fully shred, it is added extracting solution in 1.5g/10ml ratio and (in every 1000ml water, dissolves 480.4g carbamide, 57.676g sodium lauryl sulphate SDS, 95.045gNa
2s
2o
5) in, at 100 DEG C of oil bath heating 30min after submergence.
4, vacuum filtration, is placed in bag filter by black extracting solution, dialysis 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5.
5, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 10000) aqueous solution, concentrated 6 hours, regulate reduced form human hair keratin concentration be after 0.1g/mL, row SDS protein electrophoresis detects prompting protein electrophoresis band at about 45-60kD, illustrates and obtains reduced form human hair keratin.
6, the reduced form human hair keratin solution obtained is preserved under 4 DEG C of environment.
7, be dissolved in 60ml water by 100g lithium bromide, be mixed with lithium bromide water solution, agitating solution is heated to 60 DEG C simultaneously.
8, by the boiled silk 10g solution lithium bromide water solution purchased on market, after 5h, multilamellar filtered through gauze, after filtering, solution is placed in bag filter.
9, dialyse 5d, the Na containing 0.1wt% in bag filter ambient water solution
2s
2o
5, change water every day once.
10, after completing dialysis, dialysis band is placed in 20wt%PEG(molecular weight 20000) aqueous solution, concentrated 6h, the concentration being adjusted to fibroin albumen is 0.1g/mL, and row SDS protein electrophoresis detects prompting protein electrophoresis band at about 25-30kD, illustrates and obtains fibroin albumen.
11, the silk fibroin protein solution obtained is preserved under 4 DEG C of environment.
12, by the reduced form human hair keratin solution obtained and silk fibroin protein solution by volume 7:3 mix.
13. get the mixed liquor 15ml that step 12 obtains, and are merged and stir and be heated to 37 DEG C, add gelatin, and the concentration controlling gelatin in mixed liquor is 0.02g/mL.Insert after high-speed stirred 30s in square mould.
14. mixed solutions are in square mould, and mould is added a cover, and ambient temperatare puts 12h hour, and after band gelation, uncap, natural evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
Final acquisition material general appearance is the translucent film material of brown, and its HE dyes and points out this material one eosin stains positive membranoid substance.
Detect through mechanical stretch, the maximum elongation rate 39.81% of this material, elastic modelling quantity 0.148Mpa.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (10)
1. the albumen compound rest containing gelatin, it is characterized in that, composition material comprises fibroin albumen, keratin and gelatin.
2. albumen compound rest according to claim 1, is characterized in that, described fibroin albumen and keratic mass ratio are (1-10): (1-10).
3. albumen compound rest according to claim 2, is characterized in that, described fibroin albumen and keratic mass ratio are (3-7): (3-7).
4. albumen compound rest according to claim 1, is characterized in that, the mass ratio of described gelatin and keratin and fibroin albumen total amount is (0.1-5): 10.
5. albumen compound rest according to claim 4, is characterized in that, the mass ratio of described gelatin and keratin and fibroin albumen total amount is (1-3): 10.
6. albumen compound rest according to claim 1, is characterized in that, described keratin is reduced form human hair keratin.
7., as a preparation method for the albumen compound rest containing gelatin in claim 1-6 as described in any one, it is characterized in that, comprise the following steps:
Step 1, is mixed to get mixed liquid of protein by keratin solution and silk fibroin protein solution, and control fibroin albumen and keratic mass ratio are (1-10): (1-10), and in mixed liquid of protein, the total concentration of keratin and fibroin albumen is 0.05-0.2g/mL;
Step 2, joins gelatin in mixed liquid of protein, after gelatin dissolves completely, mixed solution liquid sealing is left standstill, until gelation, uncovered spontaneous evaporation, obtain albumen compound rest, wherein, the mass ratio of described gelatin and keratin and fibroin albumen total amount is (0.1-5): 10.
8. preparation method according to claim 7, is characterized in that, described keratin is reduced form human hair keratin, and preparation method is as follows:
Human hair is placed in extracting solution, soaks 10-60min at 50-150 DEG C, filter, filtrate dialysed, concentrated, obtain reduced form human hair keratin, wherein, the quality of human hair is (0.5-4g) with the ratio of the volume of extracting solution: (5-20mL).
9. preparation method according to claim 8, is characterized in that, described extracting solution is carbamide, sodium lauryl sulphate, Na
2s
2o
5mixed aqueous solution, wherein, in every 100mL water, containing 30-70g carbamide, 40-80g sodium lauryl sulphate, 80-120gNa
2s
2o
5.
10. preparation method according to claim 7, is characterized in that, the preparation method of described fibroin albumen is as follows:
Be dissolved in by boiled silk in MX solution, filter, filtrate dialysed, concentrated, obtain the fibroin albumen of purification, wherein, M is alkali metal, and X is halogen.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105457095A (en) * | 2015-12-22 | 2016-04-06 | 傅泽红 | Keratin/silk fibroin composite dense membrane and preparing method thereof |
| CN105524472A (en) * | 2015-12-22 | 2016-04-27 | 傅泽红 | Keratin/silk fibroin blended membrane and preparation method thereof |
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| US20030007991A1 (en) * | 1998-09-25 | 2003-01-09 | Masters David B. | Devices including protein matrix materials and methods of making and using thereof |
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| US20030007991A1 (en) * | 1998-09-25 | 2003-01-09 | Masters David B. | Devices including protein matrix materials and methods of making and using thereof |
| CN1274567A (en) * | 1999-05-25 | 2000-11-29 | 中国人民解放军第一军医大学 | Making process of artificial tendon made of human hair keratin and technology thereof |
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Cited By (2)
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| CN105457095A (en) * | 2015-12-22 | 2016-04-06 | 傅泽红 | Keratin/silk fibroin composite dense membrane and preparing method thereof |
| CN105524472A (en) * | 2015-12-22 | 2016-04-27 | 傅泽红 | Keratin/silk fibroin blended membrane and preparation method thereof |
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