CN104307038B - 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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- CN104307038B CN104307038B CN201310476339.4A CN201310476339A CN104307038B CN 104307038 B CN104307038 B CN 104307038B CN 201310476339 A CN201310476339 A CN 201310476339A CN 104307038 B CN104307038 B CN 104307038B
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- keratin
- solution
- gelatin
- albumen
- fibroin albumen
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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 42
- 229920000159 gelatin Polymers 0.000 title claims abstract description 42
- 239000008273 gelatin Substances 0.000 title claims abstract description 42
- 235000019322 gelatine Nutrition 0.000 title claims abstract description 42
- 235000011852 gelatine desserts Nutrition 0.000 title claims abstract description 42
- 239000002131 composite material Substances 0.000 title claims abstract description 6
- 238000002360 preparation method Methods 0.000 title claims description 13
- 108010022355 Fibroins Proteins 0.000 claims abstract description 60
- 108010076876 Keratins Proteins 0.000 claims abstract description 57
- 102000011782 Keratins Human genes 0.000 claims abstract description 57
- 150000001875 compounds Chemical class 0.000 claims abstract description 23
- 239000000243 solution Substances 0.000 claims description 75
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 37
- 239000007864 aqueous solution Substances 0.000 claims description 30
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 22
- 239000012141 concentrate Substances 0.000 claims description 18
- 239000012460 protein solution Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- 239000011259 mixed solution Substances 0.000 claims description 16
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 11
- 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
- 235000019333 sodium laurylsulphate Nutrition 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
- 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
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- -1 filters Substances 0.000 claims 1
- 239000003292 glue Substances 0.000 claims 1
- 125000005843 halogen group Chemical group 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 42
- 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
- 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 29
- 238000001962 electrophoresis Methods 0.000 description 28
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 21
- 238000001514 detection method Methods 0.000 description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- 239000000126 substance Substances 0.000 description 8
- 238000001035 drying Methods 0.000 description 7
- 238000004043 dyeing Methods 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
- 150000002148 esters Chemical class 0.000 description 7
- 230000037308 hair color Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 239000002453 shampoo Substances 0.000 description 7
- 238000003828 vacuum filtration Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 208000028484 Urethral disease Diseases 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
- 230000000694 effects Effects 0.000 description 2
- 229940056582 human hair preparation Drugs 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000010041 electrostatic spinning Methods 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 210000004247 hand Anatomy 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
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 229920002994 synthetic fiber Polymers 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, more particularly, to a kind of can be used for containing of urethra reconstruction
Albumen compound rest of gelatin and preparation method thereof.
Background technology
Because the complexity urethral disease that a variety of causes is led to is always one of a Urology Surgery clinical treatment difficult problem.
Although material obtains preferable effect in urethra reconstruction as an alternative for multiple autologous tissues, Comparatively speaking organize
Engineering technology 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 materials or
Material-research in urethra reconstruction for the seed cell complex is gradually expanded to clinical from laboratory.Wherein being no lack of has
The case of small range actual urethral disease clinical treatment, and obtain satisfying preliminary efficacy.
But with respect to being gradually improved of seed cell research, in urethral tissue engineering reconstruction, selected which kind of support
Material is ideal but to fail to reach common understanding all the time.At present mainly using two big class supports in the Tissue Engineering Study of urethra:
Natural de- cell collagen substrate (sis, bamg, acellular dermal matrix etc.) and synthetic material (polyglycolic acid, polylactic acid/
Co-glycolic acid).Although the former preparation is convenient, the production of suitable commercialization, due to have drawn from xenogenesis or even allosome more
Biological tissue, thus be constantly subjected to some scholars in terms of biological safety and query, it is difficult to be accepted by all patients.Simultaneously its
In space structure, biomechanics characteristic and promotion blood capillary and Premeabilisation of cells aspect also have inherent shortcoming simultaneously.The latter
Although the risk of biological safety aspect is fallen below minimum, the cell to surrounding for the metabolite in degradation process for the support
And microenvironment can cause inevitably to negatively affect, in addition synthetic class support is due to a lack of corresponding somatomedin,
Seed cell is promoted to stick, propagation aspect also has inborn deficiency.
Based on above-mentioned background, at present in this research field, the structure carrying out associated biomolecule timbering material using protein is just
Paid close attention to by increasing researcher.In existing report, carry out bioprotein support using fibroin albumen, human hair keratin
The report building has had successful story.But the mechanical characteristic of the simple proteins timbering material going out constructed by existing method is still no
Method is comparable with the timbering material of above-mentioned natural de- cell collagen substrate and synthetic.If by above-mentioned protein material
Mechanical property strengthened further, many at present operated using the method for electrostatic spinning.This operating technology is to setting
Standby requirement is higher, is related to the high enrichment of protein solution, its operation is also cumbersome simultaneously.
Comparatively speaking also have pointed out in external correlational study and multiple protein mixed, available different proteins because
The difference of space structure and lead to the different feature of mechanical property come finally prepared material mechanics strengthen.Although existing report
Utilize Crinis Carbonisatus reduced form keratin to be combined the report building timbering material with fibroin albumen in road, but its suitable concentration has been joined
Than still without deeply being probed into.Although the more single albumen in mechanical property of the material obtained by two kinds of mixed proteins props up simultaneously
Frame 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 such as Ji Niping in the past, or photo-crosslinking method was entered to timbering material
The further mechanics of row is modified.But the cross-linking agent involved by chemical crosslink technique may be follow-up to material et al. Ke and its table
The cell seeding in face brings certain negative effect.And the longer process time of optical cross-linking method may be to the stability of protein
Bring certain impact.
Content of the invention
It is an object of the invention to overcoming above-mentioned deficiency, provide a kind of albumen compound rest containing gelatin, its physics is special
Property more existing simple protein timbering material more excellent, thus be suitable for actual operation operation needs, finally improve urethra
The success rate of reconstruction.
The first aspect of the invention is to provide a kind of albumen containing gelatin that can be used for urethra reconstruction to be combined
Support, the composition material of described albumen compound rest includes fibroin albumen, keratin and gelatin.
Preferably, described fibroin albumen and keratic mass ratio are (1-10): (1-10), more preferably (1.5-9):
(1.5-9), more preferably (2-8): (2-8), more preferably (3-7): (3-7), such as 3:7,4:6,5:5,6:4 or 7:3.
Preferably, described gelatin and keratin are (0.1-5) with the mass ratio of fibroin albumen total amount: 10, more preferably
(0.5-4): 10, more preferably (0.8-3.5): 10, 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.
The second aspect of the invention is to provide a kind of preparation method of the above-mentioned albumen compound rest containing gelatin, including
Following steps:
Step 1, keratin solution and silk fibroin protein solution are mixed to get mixed liquid of protein, control fibroin albumen and angle egg
White mass ratio is (1-10): (1-10), and in mixed liquid of protein, keratin and the total concentration of fibroin albumen are 0.05-0.2g/
ml；
Step 2, gelatin is added in mixed liquid of protein, after gelatin is completely dissolved, by mixed solution liquid sealing and standing,
Until gelation, uncovered spontaneous evaporation, obtain albumen compound rest, wherein, described gelatin and keratin and fibroin albumen total amount
Mass ratio is (0.1-5): 10.
In step 1, described fibroin albumen and keratic mass ratio are preferably (1.5-9): (1.5-9), more preferably (2-
8): (2-8), 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, keratin and the total concentration of fibroin albumen are preferably 0.06-0.17g/ml, more excellent
Elect 0.07-0.15g/ml, more preferably 0.08-0.13g/ml, more preferably 0.09-0.11g/ml as, such as 0.1g/ml or
0.105g/ml.
In step 2, described gelatin and keratin are preferably (0.5-4) with the mass ratio of fibroin albumen total amount: 10, more preferably
For (0.8-3.5): 10, 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 and carries
Take in liquid, under 50-150 DEG C (preferably 70-130 DEG C, more preferably 80-120 DEG C, more preferably 100-110 DEG C), soak 10-
60min(is preferably 15-50min, more preferably 25-35min), filter, filtrate is dialysed, concentrate, obtain reduced form Crinis Carbonisatus angle
Albumen, wherein, the quality of human hair is (0.5-4g): (5-20ml) with the ratio of the volume of extracting solution.
It is further preferred that the quality of human hair is (1-3g): (8-15ml) with the ratio of the volume of extracting solution, more preferably
(1.5-2g): (10-12ml).
Preferably, described extracting solution is carbamide, sodium lauryl sulphate, na2s2o5Mixed aqueous solution.
It is further preferred that in described extracting solution, in every 100ml water, being preferably 35-60g containing 30-70g(, more preferably
For 45-50g) carbamide, 40-80g(be preferably 45-70g, more preferably 50-60g) sodium lauryl sulphate, 80-120g(be preferred
For 88-110g, more preferably 92-100g) na2s2o5.
In step 1, the preparation method of described fibroin albumen is as follows: boiled silk is dissolved in mx solution, filters, by filtrate
Dialysis, concentrate, obtain the fibroin albumen of purification, wherein, m be alkali metal (such as li, na, k etc.), x be halogen (such as f, cl,
Br, i etc.).
Preferably, in step 2, after gelation, uncovered spontaneous evaporation 8-16h(is preferably 10-14h, more preferably 11-12h)
Afterwards, pour anhydrous alcohol 0.5-2h(into and be preferably 0.8-1.5h, more preferably 1-1.2h) afterwards take out obtain albumen compound rest.
The albumen compound rest containing gelatin that the present invention provides is compounded with keratin, fibroin albumen, gelatin, physical strength
It is better than using the membrane structure material constructed by simple silk fibroin protein solution and simple proteins combination solution, more suitable for actual handss
The needs of art operation, the final success rate improving urethra reconstruction.
Brief description
The he colored graph of the albumen compound rest containing gelatin that Fig. 1 provides for the present invention.
Specific embodiment
With reference to the accompanying drawings, the invention will be further described in conjunction with specific embodiments, to more fully understand this
Bright.
Embodiment 1
1st, obtain the normal hair color of dye-free from barber shop, conventional shampoo cleans 1-2 time, is placed in ordinary room temperature
It is dried.
2nd, the hair after dried is soaked in 24 hours in ethyl acetate and the mixed solution of methanol, wherein acetic acid second
Ester is 3:1 with the volume ratio of methanol.
3rd, after hair after drying, fully shred, in 1.5g/10ml ratio add extracting solution (in every 1000ml water,
Dissolving 480.4g carbamide, 57.676g sodium lauryl sulphate sds, 95.045gna2s2o5) in, add in 100 DEG C of oil baths after submergence
Hot 30min.
4th, vacuum filtration, black extracting solution is placed in bag filter, and dialyse 5d, contains in aqueous solution around bag filter
The na of 0.1wt%2s2o5.
5th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 10000) in aqueous solution, concentrate 6 hours, adjust
Reduced form human hair keratin concentration be 0.1g/ml after, row sds protein electrophoresises detection prompting protein electrophoresises band in 45-60kd
Left and right, illustrates to obtain reduced form human hair keratin.
6th, the reduced form obtaining human hair keratin solution is preserved under 4 DEG C of environment.
7th, 100g lithium bromide is dissolved in 60ml water, is configured to lithium bromide water solution, agitating solution is simultaneously heated to 60 DEG C.
8th, by the boiled silk purchased from market 10g solution lithium bromide water solution, after 5h, multilamellar filtered through gauze, mistake
After filter, solution is placed in bag filter.
9th, dialyse 5d, the na containing 0.1wt% in aqueous solution around bag filter2s2o5, change water daily once.
10th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 20000) in aqueous solution, concentrate 6h, adjust to
The concentration of fibroin albumen is 0.1g/ml, and in 25-30kd, explanation obtains row sds protein electrophoresises detection prompting protein electrophoresises band
Fibroin albumen.
11st, the silk fibroin protein solution obtaining is preserved under 4 DEG C of environment.
12nd, by the reduced form obtaining human hair keratin solution and silk fibroin protein solution, 5:5 is mixed by volume.
13. take the mixed liquor 15ml that step 12 obtains, and are merged and are stirred and heated to 37 DEG C, add gelatin, control mixed
The concentration closing gelatin in liquid is 0.01g/ml.Insert after high-speed stirred 30s in square mould.
14. mixed solutions in square mould, add a cover by mould, places 12h hour, after band gelation, uncap under room temperature, from
So evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
The final material general appearance that obtains is the translucent film material of brown, and this material one eosin stains is pointed out in its he dyeing
Positive membranoid substance (shown in Fig. 1).
Through mechanical stretch detection, the maximum elongation rate 45.49% of this material, elastic modelling quantity 0.1mpa.
Embodiment 2
1st, obtain the normal hair color of dye-free from barber shop, conventional shampoo cleans 1-2 time, is placed in ordinary room temperature
It is dried.
2nd, the hair after dried is soaked in 24 hours in ethyl acetate and the mixed solution of methanol, wherein acetic acid second
Ester is 3:1 with the volume ratio of methanol.
3rd, after hair after drying, fully shred, in 1.5g/10ml ratio add extracting solution (in every 1000ml water,
Dissolving 480.4g carbamide, 57.676g sodium lauryl sulphate sds, 95.045gna2s2o5) in, add in 100 DEG C of oil baths after submergence
Hot 30min.
4th, vacuum filtration, black extracting solution is placed in bag filter, and dialyse 5d, contains in aqueous solution around bag filter
The na of 0.1wt%2s2o5.
5th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 10000) in aqueous solution, concentrate 6 hours, adjust
Reduced form human hair keratin concentration be 0.1g/ml after, row sds protein electrophoresises detection prompting protein electrophoresises band in 45-60kd
Left and right, illustrates to obtain reduced form human hair keratin.
6th, the reduced form obtaining human hair keratin solution is preserved under 4 DEG C of environment.
7th, 100g lithium bromide is dissolved in 60ml water, is configured to lithium bromide water solution, agitating solution is simultaneously heated to 60 DEG C.
8th, by the boiled silk purchased from market 10g solution lithium bromide water solution, after 5h, multilamellar filtered through gauze, mistake
After filter, solution is placed in bag filter.
9th, dialyse 5d, the na containing 0.1wt% in aqueous solution around bag filter2s2o5, change water daily once.
10th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 20000) in aqueous solution, concentrate 6h, adjust to
The concentration of fibroin albumen is 0.1g/ml, and in 25-30kd, explanation obtains row sds protein electrophoresises detection prompting protein electrophoresises band
Fibroin albumen.
11st, the silk fibroin protein solution obtaining is preserved under 4 DEG C of environment.
12nd, by the reduced form obtaining human hair keratin solution and silk fibroin protein solution, 5:5 is mixed by volume.
13. take the mixed liquor 15ml that step 12 obtains, and are merged and are stirred and heated to 37 DEG C, add gelatin, control mixed
The concentration closing gelatin in liquid is 0.02g/ml.Insert after high-speed stirred 30s in square mould.
14. mixed solutions in square mould, add a cover by mould, places 12h hour, after band gelation, uncap under room temperature, from
So evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
The final material general appearance that obtains is the translucent film material of brown, and this material one eosin stains is pointed out in its he dyeing
Positive membranoid substance.
Through mechanical stretch detection, the maximum elongation rate 55.29% of this material, elastic modelling quantity 0.117mpa.
Embodiment 3
1st, obtain the normal hair color of dye-free from barber shop, conventional shampoo cleans 1-2 time, is placed in ordinary room temperature
It is dried.
2nd, the hair after dried is soaked in 24 hours in ethyl acetate and the mixed solution of methanol, wherein acetic acid second
Ester is 3:1 with the volume ratio of methanol.
3rd, after hair after drying, fully shred, in 1.5g/10ml ratio add extracting solution (in every 1000ml water,
Dissolving 480.4g carbamide, 57.676g sodium lauryl sulphate sds, 95.045gna2s2o5) in, add in 100 DEG C of oil baths after submergence
Hot 30min.
4th, vacuum filtration, black extracting solution is placed in bag filter, and dialyse 5d, contains in aqueous solution around bag filter
The na of 0.1wt%2s2o5.
5th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 10000) in aqueous solution, concentrate 6 hours, adjust
Reduced form human hair keratin concentration be 0.1g/ml after, row sds protein electrophoresises detection prompting protein electrophoresises band in 45-60kd
Left and right, illustrates to obtain reduced form human hair keratin.
6th, the reduced form obtaining human hair keratin solution is preserved under 4 DEG C of environment.
7th, 100g lithium bromide is dissolved in 60ml water, is configured to lithium bromide water solution, agitating solution is simultaneously heated to 60 DEG C.
8th, by the boiled silk purchased from market 10g solution lithium bromide water solution, after 5h, multilamellar filtered through gauze, mistake
After filter, solution is placed in bag filter.
9th, dialyse 5d, the na containing 0.1wt% in aqueous solution around bag filter2s2o5, change water daily once.
10th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 20000) in aqueous solution, concentrate 6h, adjust to
The concentration of fibroin albumen is 0.1g/ml, and in 25-30kd, explanation obtains row sds protein electrophoresises detection prompting protein electrophoresises band
Fibroin albumen.
11st, the silk fibroin protein solution obtaining is preserved under 4 DEG C of environment.
12nd, by the reduced form obtaining human hair keratin solution and silk fibroin protein solution, 5:5 is mixed by volume.
13. take the mixed liquor 15ml that step 12 obtains, and are merged and are stirred and heated to 37 DEG C, add gelatin, control mixed
The concentration closing gelatin in liquid is 0.03g/ml.Insert after high-speed stirred 30s in square mould.
14. mixed solutions in square mould, add a cover by mould, places 12h hour, after band gelation, uncap under room temperature, from
So evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
The final material general appearance that obtains is the translucent film material of brown, and this material one eosin stains is pointed out in its he dyeing
Positive membranoid substance.
Through mechanical stretch detection, the maximum elongation rate 11.27% of this material, elastic modelling quantity 0.05mpa.
Embodiment 4
1st, obtain the normal hair color of dye-free from barber shop, conventional shampoo cleans 1-2 time, is placed in ordinary room temperature
It is dried.
2nd, the hair after dried is soaked in 24 hours in ethyl acetate and the mixed solution of methanol, wherein acetic acid second
Ester is 3:1 with the volume ratio of methanol.
3rd, after hair after drying, fully shred, in 1.5g/10ml ratio add extracting solution (in every 1000ml water,
Dissolving 480.4g carbamide, 57.676g sodium lauryl sulphate sds, 95.045gna2s2o5) in, add in 100 DEG C of oil baths after submergence
Hot 30min.
4th, vacuum filtration, black extracting solution is placed in bag filter, and dialyse 5d, contains in aqueous solution around bag filter
The na of 0.1wt%2s2o5.
5th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 10000) in aqueous solution, concentrate 6 hours, adjust
Reduced form human hair keratin concentration be 0.1g/ml after, row sds protein electrophoresises detection prompting protein electrophoresises band in 45-60kd
Left and right, illustrates to obtain reduced form human hair keratin.
6th, the reduced form obtaining human hair keratin solution is preserved under 4 DEG C of environment.
7th, 100g lithium bromide is dissolved in 60ml water, is configured to lithium bromide water solution, agitating solution is simultaneously heated to 60 DEG C.
8th, by the boiled silk purchased from market 10g solution lithium bromide water solution, after 5h, multilamellar filtered through gauze, mistake
After filter, solution is placed in bag filter.
9th, dialyse 5d, the na containing 0.1wt% in aqueous solution around bag filter2s2o5, change water daily once.
10th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 20000) in aqueous solution, concentrate 6h, adjust to
The concentration of fibroin albumen is 0.1g/ml, and in 25-30kd, explanation obtains row sds protein electrophoresises detection prompting protein electrophoresises band
Fibroin albumen.
11st, the silk fibroin protein solution obtaining is preserved under 4 DEG C of environment.
12nd, by the reduced form obtaining human hair keratin solution and silk fibroin protein solution, 3:7 is mixed by volume.
13. take the mixed liquor 15ml that step 12 obtains, and are merged and are stirred and heated to 37 DEG C, add gelatin, control mixed
The concentration closing gelatin in liquid is 0.02g/ml.Insert after high-speed stirred 30s in square mould.
14. mixed solutions in square mould, add a cover by mould, places 12h hour, after band gelation, uncap under room temperature, from
So evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
The final material general appearance that obtains is the translucent film material of brown, and this material one eosin stains is pointed out in its he dyeing
Positive membranoid substance.
Through mechanical stretch detection, the maximum elongation rate 42.06% of this material, elastic modelling quantity 0.09mpa.
Embodiment 5
1st, obtain the normal hair color of dye-free from barber shop, conventional shampoo cleans 1-2 time, is placed in ordinary room temperature
It is dried.
2nd, the hair after dried is soaked in 24 hours in ethyl acetate and the mixed solution of methanol, wherein acetic acid second
Ester is 3:1 with the volume ratio of methanol.
3rd, after hair after drying, fully shred, in 1.5g/10ml ratio add extracting solution (in every 1000ml water,
Dissolving 480.4g carbamide, 57.676g sodium lauryl sulphate sds, 95.045gna2s2o5) in, add in 100 DEG C of oil baths after submergence
Hot 30min.
4th, vacuum filtration, black extracting solution is placed in bag filter, and dialyse 5d, contains in aqueous solution around bag filter
The na of 0.1wt%2s2o5.
5th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 10000) in aqueous solution, concentrate 6 hours, adjust
Reduced form human hair keratin concentration be 0.1g/ml after, row sds protein electrophoresises detection prompting protein electrophoresises band in 45-60kd
Left and right, illustrates to obtain reduced form human hair keratin.
6th, the reduced form obtaining human hair keratin solution is preserved under 4 DEG C of environment.
7th, 100g lithium bromide is dissolved in 60ml water, is configured to lithium bromide water solution, agitating solution is simultaneously heated to 60 DEG C.
8th, by the boiled silk purchased from market 10g solution lithium bromide water solution, after 5h, multilamellar filtered through gauze, mistake
After filter, solution is placed in bag filter.
9th, dialyse 5d, the na containing 0.1wt% in aqueous solution around bag filter2s2o5, change water daily once.
10th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 20000) in aqueous solution, concentrate 6h, adjust to
The concentration of fibroin albumen is 0.1g/ml, and in 25-30kd, explanation obtains row sds protein electrophoresises detection prompting protein electrophoresises band
Fibroin albumen.
11st, the silk fibroin protein solution obtaining is preserved under 4 DEG C of environment.
12nd, by the reduced form obtaining human hair keratin solution and silk fibroin protein solution, 4:6 is mixed by volume.
13. take the mixed liquor 15ml that step 12 obtains, and are merged and are stirred and heated to 37 DEG C, add gelatin, control mixed
The concentration closing gelatin in liquid is 0.02g/ml.Insert after high-speed stirred 30s in square mould.
14. mixed solutions in square mould, add a cover by mould, places 12h hour, after band gelation, uncap under room temperature, from
So evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
The final material general appearance that obtains is the translucent film material of brown, and this material one eosin stains is pointed out in its he dyeing
Positive membranoid substance.
Through mechanical stretch detection, the maximum elongation rate 39.31% of this material, elastic modelling quantity 0.146mpa.
Embodiment 6
1st, obtain the normal hair color of dye-free from barber shop, conventional shampoo cleans 1-2 time, is placed in ordinary room temperature
It is dried.
2nd, the hair after dried is soaked in 24 hours in ethyl acetate and the mixed solution of methanol, wherein acetic acid second
Ester is 3:1 with the volume ratio of methanol.
3rd, after hair after drying, fully shred, in 1.5g/10ml ratio add extracting solution (in every 1000ml water,
Dissolving 480.4g carbamide, 57.676g sodium lauryl sulphate sds, 95.045gna2s2o5) in, add in 100 DEG C of oil baths after submergence
Hot 30min.
4th, vacuum filtration, black extracting solution is placed in bag filter, and dialyse 5d, contains in aqueous solution around bag filter
The na of 0.1wt%2s2o5.
5th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 10000) in aqueous solution, concentrate 6 hours, adjust
Reduced form human hair keratin concentration be 0.1g/ml after, row sds protein electrophoresises detection prompting protein electrophoresises band in 45-60kd
Left and right, illustrates to obtain reduced form human hair keratin.
6th, the reduced form obtaining human hair keratin solution is preserved under 4 DEG C of environment.
7th, 100g lithium bromide is dissolved in 60ml water, is configured to lithium bromide water solution, agitating solution is simultaneously heated to 60 DEG C.
8th, by the boiled silk purchased from market 10g solution lithium bromide water solution, after 5h, multilamellar filtered through gauze, mistake
After filter, solution is placed in bag filter.
9th, dialyse 5d, the na containing 0.1wt% in aqueous solution around bag filter2s2o5, change water daily once.
10th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 20000) in aqueous solution, concentrate 6h, adjust to
The concentration of fibroin albumen is 0.1g/ml, and in 25-30kd, explanation obtains row sds protein electrophoresises detection prompting protein electrophoresises band
Fibroin albumen.
11st, the silk fibroin protein solution obtaining is preserved under 4 DEG C of environment.
12nd, by the reduced form obtaining human hair keratin solution and silk fibroin protein solution, 6:4 is mixed by volume.
13. take the mixed liquor 15ml that step 12 obtains, and are merged and are stirred and heated to 37 DEG C, add gelatin, control mixed
The concentration closing gelatin in liquid is 0.02g/ml.Insert after high-speed stirred 30s in square mould.
14. mixed solutions in square mould, add a cover by mould, places 12h hour, after band gelation, uncap under room temperature, from
So evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
The final material general appearance that obtains is the translucent film material of brown, and this material one eosin stains is pointed out in its he dyeing
Positive membranoid substance (shown in Fig. 1).
Through mechanical stretch detection, the maximum elongation rate 52.43% of this material, elastic modelling quantity 0.202mpa.
Embodiment 7
1st, obtain the normal hair color of dye-free from barber shop, conventional shampoo cleans 1-2 time, is placed in ordinary room temperature
It is dried.
2nd, the hair after dried is soaked in 24 hours in ethyl acetate and the mixed solution of methanol, wherein acetic acid second
Ester is 3:1 with the volume ratio of methanol.
3rd, after hair after drying, fully shred, in 1.5g/10ml ratio add extracting solution (in every 1000ml water,
Dissolving 480.4g carbamide, 57.676g sodium lauryl sulphate sds, 95.045gna2s2o5) in, add in 100 DEG C of oil baths after submergence
Hot 30min.
4th, vacuum filtration, black extracting solution is placed in bag filter, and dialyse 5d, contains in aqueous solution around bag filter
The na of 0.1wt%2s2o5.
5th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 10000) in aqueous solution, concentrate 6 hours, adjust
Reduced form human hair keratin concentration be 0.1g/ml after, row sds protein electrophoresises detection prompting protein electrophoresises band in 45-60kd
Left and right, illustrates to obtain reduced form human hair keratin.
6th, the reduced form obtaining human hair keratin solution is preserved under 4 DEG C of environment.
7th, 100g lithium bromide is dissolved in 60ml water, is configured to lithium bromide water solution, agitating solution is simultaneously heated to 60 DEG C.
8th, by the boiled silk purchased from market 10g solution lithium bromide water solution, after 5h, multilamellar filtered through gauze, mistake
After filter, solution is placed in bag filter.
9th, dialyse 5d, the na containing 0.1wt% in aqueous solution around bag filter2s2o5, change water daily once.
10th, after completing dialysis, dialysis band is placed in 20wt%peg(molecular weight 20000) in aqueous solution, concentrate 6h, adjust to
The concentration of fibroin albumen is 0.1g/ml, and in 25-30kd, explanation obtains row sds protein electrophoresises detection prompting protein electrophoresises band
Fibroin albumen.
11st, the silk fibroin protein solution obtaining is preserved under 4 DEG C of environment.
12nd, by the reduced form obtaining human hair keratin solution and silk fibroin protein solution, 7:3 is mixed by volume.
13. take the mixed liquor 15ml that step 12 obtains, and are merged and are stirred and heated to 37 DEG C, add gelatin, control mixed
The concentration closing gelatin in liquid is 0.02g/ml.Insert after high-speed stirred 30s in square mould.
14. mixed solutions in square mould, add a cover by mould, places 12h hour, after band gelation, uncap under room temperature, from
So evaporation 12h, takes out after pouring ethanol 1h into, obtains albumen compound rest.
The final material general appearance that obtains is the translucent film material of brown, and this material one eosin stains is pointed out in its he dyeing
Positive membranoid substance.
Through mechanical stretch detection, the maximum elongation rate 39.81% of this material, elastic modelling quantity 0.148mpa.
Above the specific embodiment of the present invention is described in detail, but it has been intended only as example, the present invention has not limited
It is formed on particular embodiments described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and
Substitute also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should cover within the scope of the invention.
Claims (7)
1. a kind of albumen compound rest containing gelatin is it is characterised in that composition material includes fibroin albumen, keratin and bright
Glue；
Described keratin is reduced form human hair keratin；
Described fibroin albumen and keratic mass ratio are (1-10): (1-10)；
Described gelatin and keratin are (0.1-5) with the mass ratio of fibroin albumen total amount: 10.
2. albumen compound rest according to claim 1 is it is characterised in that described fibroin albumen and keratic mass ratio
For (3-7): (3-7).
3. albumen compound rest according to claim 1 is it is characterised in that described gelatin and keratin are total with fibroin albumen
The mass ratio of amount is (1-3): 10.
4. a kind of preparation method of the albumen compound rest containing gelatin as described in any one in claim 1-3, it is special
Levy and be, comprise the following steps:
Step 1, keratin solution and silk fibroin protein solution are mixed to get mixed liquid of protein, control fibroin albumen and keratic
Mass ratio is (1-10): (1-10), and in mixed liquid of protein, keratin and the total concentration of fibroin albumen are 0.05-0.2g/ml；
Step 2, gelatin is added in mixed liquid of protein, after gelatin is completely dissolved, by mixed solution liquid sealing and standing, until
Gelation, uncovered spontaneous evaporation, obtain albumen compound rest, wherein, the quality of described gelatin and keratin and fibroin albumen total amount
Than for (0.1-5): 10.
5. preparation method according to claim 4, it is characterised in that described keratin is reduced form human hair keratin, is made
Preparation Method is as follows:
Human hair is placed in extracting solution, at 50-150 DEG C, soaks 10-60min, filter, filtrate is dialysed, concentrate, reduced
Type human hair keratin, wherein, the quality of human hair is (0.5-4g): (5-20ml) with the ratio of the volume of extracting solution.
6. preparation method according to claim 5 it is characterised in that described extracting solution be carbamide, sodium lauryl sulphate,
na2s2o5Mixed aqueous solution, wherein, in every 100ml water, containing 30-70g carbamide, 40-80g sodium lauryl sulphate, 80-
120g na2s2o5.
7. preparation method according to claim 4 is it is characterised in that the preparation method of described fibroin albumen is as follows:
Boiled silk is dissolved in mx solution, filters, filtrate is dialysed, concentrate, obtain the fibroin albumen of purification,
Wherein, m is alkali metal, and x is halogen.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN1887362A (en) * | 2006-07-13 | 2007-01-03 | 苏州大学 | Cell culturing rack material and its prepn |
| CN102688525A (en) * | 2012-05-07 | 2012-09-26 | 东南大学 | Bio-macromolecular hydrogel 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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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN1887362A (en) * | 2006-07-13 | 2007-01-03 | 苏州大学 | Cell culturing rack material and its prepn |
| CN102688525A (en) * | 2012-05-07 | 2012-09-26 | 东南大学 | Bio-macromolecular hydrogel and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Porous keratin scaffold–promising biomaterial for tissue engineering and drug delivery;Balaji Srinivasan等;《Journal of Biomedical Materials Research Part B: Applied Biomaterials》;20100131;第92B卷(第1期);5-12 * |
| 蚕丝蛋白和明胶复合组织工程材料的组织相容性研究;杨照等;《华南国防医学杂志》;20110228;第25卷(第1期);44-47 * |
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