CN107343967B - A kind of preparation method of peptide growth factor modification hair sill - Google Patents
A kind of preparation method of peptide growth factor modification hair sill Download PDFInfo
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- CN107343967B CN107343967B CN201610298484.1A CN201610298484A CN107343967B CN 107343967 B CN107343967 B CN 107343967B CN 201610298484 A CN201610298484 A CN 201610298484A CN 107343967 B CN107343967 B CN 107343967B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
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Abstract
The invention discloses a kind of preparation methods of peptide growth factor modification hair sill.This method includes the following steps: that hair is placed in the aqueous solution of alcohol by (1) and impregnates, and takes out hair and is cleaned and dried；Hair after drying is placed in the aqueous solution of acid and is impregnated, taken out hair and cleaned and dried, obtain hair sill；(2) under the action of initiator, step (1) the hair sill and polypeptide nerve growth factor react in water, obtain the peptide growth factor modification hair sill.The method of the present invention is surface-treated hair surface using the mild corrosiveness of diluted acid, and hair surface is made to expose carboxylic group, convenient for the grafting multiple polypeptides factor.For hair material as self accessory organ, materials are convenient.Hair sill provides a supporting role for peptide growth factor, and peptide growth factor is released, and provides peptide growth factor needed for entire regenerative process for defective tissue.
Description
Technical field
The present invention relates to the preparation sides of bioengineering field more particularly to a kind of peptide growth factor modification hair sill
Method.
Background technique
Peptide growth factor can be generated by internal many cells, have important physiological and pathological and therapeutic effect.It such as participates in
Adjust immune response and inflammatory reaction；And the combination of cell surface receptor eventually leads to intracellular rna and albumen synthesis mode
Change, to influence cell behavior；It regenerates and repairs after organism embryo generation, development, differentiation and the various tissue damages of human body
It plays an important role during multiple.Between the various factors by mutual induction, interactive adjustment to cell function generate collaboration, be added or
Antagonism.Half-life period is extremely short in vivo for polypeptide factor, therefore needs to be loaded on biomaterial, when extending its effect in vivo
Between.
Hair is as autologous material, and materials are convenient, and excellent in mechanical performance, obtains extensively in the application of bioengineering field
Concern.Hair is made of hair shaft, hair root and hair follicle three parts.Hair shaft refers to the part for being exposed to skin surface, is the appendicle of skin.
It is made of epithelial cells, main component is keratin.It has hair medulla, hair cortex and hair cuticle to constitute.Hair medulla is located at hair
Axis position is sent out, is formed by 1-2 row's cuboidal epithelium file, nutriment is mainly drawn from scalp, for natural on-off cycles of hair growth.Hair
Cortex accounts for the 75% to 90% of hair, is color, elasticity, intensity, the important component of thickness and buckling type for determining hair,
It is made of soft keratin；Hair cuticle, also known as epidermis are the outermost layers of hair, determine hair appearance and glossy degree.
It can resist external irritant, protect cortex and inhibit the evaporation of moisture.Part of the hair in skin is known as hair root, equally
With three hair medulla, hair cortex and hair cuticle parts.It expands in spherical, title ball top hair root end.The latter is located in hair follicle, in
Indent is entreated, hair follicle papilla is connect.
Fractions --- the keratin inside hair is paid close attention in existing research mostly.Keratin is extracted from hair, is led to
Hemostatic function can be promoted by crossing encapsulating or the hydrogel that is made of surface grafting polypeptide growth factor, improve the heart of cardiac
Dirty function promotes axon regeneration etc..These researchs can not be directly using hair as the carrier of peptide growth factor.
Summary of the invention
The object of the present invention is to provide a kind of preparation methods of peptide growth factor modification hair sill, and this method is by head
Breaking-out is the carrier of peptide growth factor, remains the overall structure of hair, and materials are convenient.
A kind of preparation method of peptide growth factor modification hair sill provided by the invention, includes the following steps:
(1) hair is placed in the aqueous solution of alcohol and is impregnated, taken out hair and cleaned and dried；By the hair after drying
It is placed in the aqueous solution of acid and impregnates, take out hair and cleaned and dried, obtain hair sill；
(2) under the action of initiator, step (1) the hair sill and peptide growth factor occur instead in water
It answers, obtains the peptide growth factor modification hair sill.
The processing of above-mentioned preparation method, step (1) can be under the premise of guaranteeing hair structure integrality, by hair surface
Carboxylic group exposure.
In step (1), the length of the hair can be 0.5~1cm, concretely 1cm.
In the aqueous solution of the alcohol, the mass concentration of alcohol can be 75%.The volume of the aqueous solution of the alcohol not by
Limitation can guarantee to submerge hair.
The time impregnated in the aqueous solution of alcohol can be 30~90min (such as 60min), and the temperature of immersion can be 20~37
DEG C (such as 37 DEG C).
Cleaning after taking out in the aqueous solution of alcohol can be washing, and distilled water specifically can be used.
The drying can be drying, and the temperature of the drying can be 20~37 DEG C (such as 37 DEG C), and the time can be 10~20min
(such as 15min).
In the aqueous solution of the acid, sour mass concentration can be 1%~15%, concretely 10%.The hair and institute
The mass volume ratio for stating the aqueous solution of acid can be (1~10) mg:10mL, concretely 8mg:10mL.
In the aqueous solution of the acid, acid can be hydrochloric acid, sulfuric acid, nitric acid or acetic acid.
Time for impregnating can be 5~15min in the aqueous solution of acid, concretely 10min, the temperature of immersion can for 20~
37 DEG C, concretely 37 DEG C.
Cleaning after taking out in the aqueous solution of acid can be washing, and distilled water specifically can be used.
The drying can be freeze-drying；The time of the drying can be 24~48h, concretely 36h.
Above-mentioned preparation method, in step (2), the reaction is the carboxylic of exposure in hair sill surface in step (1)
The cross-linking reaction that amino in base and polypeptide nerve growth factor occurs, polypeptide nerve growth factor are grafted on hair.
The initiator, the hair sill, the peptide growth factor and the water ratio can be (1~100)
Mg:(10~200) ng:(1~10) ng:1mL, concretely 0.1mg:0.8mg:5ng:1mL.
The peptide growth factor includes but is not limited to: nerve growth factor, brain-derived neurotrophic factor, neurotrophy
Element -3, Neurotrophin-4, neurenergen -5, neurotrophin-6, neurenergen -7, basic fibroblast growth because
Son, acid fibroblast growth factor, epidermal growth factor, erythropoietin(EPO), spongiocyte strain derived neurotrophic because
Son, ciliary neurotrophic factor, motor neuron nutrition factor -1, motor neuron nutrition factor -2, Bone Morphogenetic Protein, Nogo
Antibody or chondroitin sulfate antibody.
The initiator can be that Geniposide, glutaraldehyde or mass ratio are 1:(1~10) n-hydroxysuccinimide (NHS)
With 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC), concretely mass ratio is the N- hydroxyl amber of 1:1
Amber acid imide (NHS) and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC).
The water concretely distilled water.
The reaction can carry out under stirring conditions, and the revolving speed of the stirring can be 200~500rpm, such as 400rpm；
The temperature of the reaction can be 0~4 DEG C, and concretely 4 DEG C, the time can be 24~48h, concretely 48h.
The invention has the following beneficial effects:
(1) preparation method of peptide growth factor modification hair sill of the present invention, includes the following steps: correct using acid
Hair, which carries out processing, makes hair surface expose carboxyl, obtains hair sill；Under the action of initiator, the hair sill with
Peptide growth factor reacts, and the peptide growth factor modification hair sill can be obtained.The method of the present invention utilizes dilute
The mild corrosiveness of acid is surface-treated hair surface, so that hair surface is exposed carboxylic group, both ensure that in this way
The complete structure of hair, and carboxylic group is made to be convenient for crosslinking instead under initiator effect with the amino of peptide growth factor
It answers, obtains the hair sill of peptide growth factor modification.
(2) good biocompatibility and widespread popularity.Hair surface exposes carboxylic group, a variety of more convenient for being grafted
Peptide factor.For hair material as self accessory organ, materials are convenient.And hair sill can provide for peptide growth factor
Supporting role releases peptide growth factor, for defective tissue provide bypolypeptide growth needed for entire regenerative process because
Son has widespread popularity.
Detailed description of the invention
Fig. 1 is the surface topography (scanning electron microscopy of hair material original sample, hair sill, NGF modification hair sill
Mirror photo).Figure 1A is the surface topography of hair material original sample；Figure 1B is the surface topography of hair sill；Fig. 1 C is NGF modification
The surface topography of hair sill.
Fig. 2 is the infrared spectrum of hair material original sample, hair sill, NGF modification hair sill.Fig. 2A is hair material
Expect the infrared spectrum of original sample；Fig. 2 B is the infrared spectrum of hair sill；Fig. 2 C is the INFRARED SPECTRUM that NGF modifies hair sill
Figure.
Fig. 3 is that the nerve fibre that chick embryonic dorsal root ganglion (DRG) is grown in the case where NGF modifies the effect of hair sill is (micro-
Mirror photo).
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Nerve growth factor (Nerve growth factor, NGF) is the neurotrophic factor being found earliest, in 20
The fifties in century is confirmed by discovery for the first time.The pentamer albumen matter compound that NGF is made of 3 subunits (α, β and γ), molecule
Quality is 130-140kDa.β subunit is the functional activity unit of NGF, the dimer-β subunit (β NGF) being made of double-strand, point
Son amount is 26kDa.The general entitled injection mouse nerve growth factor (Mouse of nerve growth factor in following embodiments
Nerve Growth Fact for Injection), for trade name grace through multiple, manufacturer is the road biology work of Xiamen Beijing University
Journey Co., Ltd, be purchased from Sinopharm Chemical Reagent Co., Ltd., specification be 18ug (>=9000AU)/.
Embodiment 1, preparation peptide growth factor modify hair sill
One, preparation method
Peptide growth factor modification hair sill is prepared in accordance with the following steps:
(1) preparation of the hair sill through HCl treatment
Taking length is several of the hair (stereoscan photograph is as shown in Figure 1A) of 1cm, is placed in the water-soluble of 75wt% alcohol
60min is impregnated in liquid, soaking temperature is 37 DEG C, and distilled water cleans drying, and drying temperature is 37 DEG C, drying time 15min.So
8mg hair is placed in the 10mL aqueous hydrochloric acid solution that mass concentration is 10% afterwards, impregnates 10min at 37 DEG C；It steams after completion of the reaction
Distilled water is rinsed, and is freeze-dried 36h, is obtained hair sill (stereoscan photograph is as shown in Figure 1B).
(2) NGF modifies the preparation of the hair sill through HCl treatment
Take the hair sill 8mg being prepared in above-mentioned steps (1), nerve growth factor 5ng, N- hydroxysuccinimidyl acyl sub-
Amine and the equal 0.5mg of 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride are added sequentially in 10mL distilled water, and 4 DEG C
Lower stirring 48h (speed of agitator 400rpm) obtains the hair sill of NGF modification (stereoscan photograph is as shown in Figure 1 C).
The hair scale piece and no significant difference of hair surface it can be seen from the stereoscan photograph in Fig. 1.This show due to
Hydrochloric acid is only modified hair material surface, and the time is short and mild condition, therefore does not influence the overall structure of hair.
Two, infrared spectrum measurement
Measuring method: the hair base for taking the original sample of hair used in above-mentioned preparation method one, step (1) to be prepared respectively
Material, the NGF being prepared modify each several of three groups of materials of hair sill.By sample head and the tail both ends with taping on, protect
The length of the intermediate tested part of card is 2cm, diameter 2mm.Sample room leaves no gaps as far as possible, the sample bundled is inserted into red
The sample placing position of external spectrum instrument.Then parameter is set: resolution ratio 4cm-1, scanning times are 30 times, scanning range 4000-
400cm-1, the scanning of traveling wave of going forward side by side number, be absorbed spectroscopic data.Finally, obtained data are analyzed and processed, obtain infrared
Spectrogram.As shown in Figure 2.
In infrared spectrum (Fig. 2), 2924.39cm-1With 2853.25cm-1Place is-OH hydroxyl stretching vibration characteristic peak,
1648.69cm-1For the stretching vibration characteristic peak of-C=O, 1235.32cm-1For the stretching vibration characteristic peak of-C-O, several groups of features
The characterized carboxylic group in peak.Fatty acid, the keratin on the surface (Fig. 2A) etc. are securely connected hair by disulfide bond as former state, and
Hair surface forms special complicated protection layer, therefore infrared spectroscopy shows that it has no obvious characteristic peak.Hair is carried out as former state
Surface treatment, makes hair surface expose carboxyl, to be grafted, therefore hair sill (Fig. 2 B) exposes characteristic peak.
NGF modifies fatty acid, the keratin of the amino of NGF and hair sill surface in the hair sill (Fig. 2 C) through HCl treatment
Carboxyl be combined into amido bond, therefore peak acuity is weaker than hair sill.ATR-FTIR is the result shows that NGF is successfully grafted
To hair sill surface.
Three, bioactivity
Still there is bioactivity in order to verify NGF in the NGF prepared in above-mentioned preparation method one modification hair sill, it is existing
The method co-cultured using NGF modification hair sill and chick embryonic dorsal root ganglion, verifies the activity of NGF.The specific method is as follows:
Nerve growth factor modification hair sill is co-cultured with the chick embryonic dorsal root ganglion extracted in instar chicken embryo from 8 days.Wherein train
The system of supporting is 10 200 μ L and 100 μ L of μ L, DMEM of chicken plasma.After co-culturing for 24 hours, optical microphotograph microscopic observation dorsal root ganglion mind
Growing state through aixs cylinder.
As a result as shown in Figure 3: chick embryonic dorsal root ganglion is modifying one lateral process of hair sill close to nerve growth factor
It is longer, and protrusion can be grown along material direction.Prove that encapsulating nerve growth factor modification hair sill can discharge
Nerve growth factor out, and nerve growth factor has bioactivity.
Claims (10)
1. a kind of preparation method of peptide growth factor modification hair sill, includes the following steps:
(1) hair is placed in the aqueous solution of alcohol and is impregnated, taken out hair and cleaned and dried；Hair after drying is placed in
It is impregnated in the aqueous solution of acid, takes out hair and cleaned and dried, obtain hair sill；
(2) under the action of initiator, step (1) the hair sill and peptide growth factor react in water, obtain
Hair sill is modified to the peptide growth factor.
2. preparation method according to claim 1, it is characterised in that: in step (1), the length of the hair is 0.5~
1cm。
3. preparation method according to claim 1 or 2, it is characterised in that: in step (1), in the aqueous solution of the alcohol,
The mass concentration of alcohol is 75%；The time impregnated in the aqueous solution of alcohol is 30~90min.
4. preparation method according to claim 1 or 2, it is characterised in that: in step (1), the drying is drying, described
The temperature of drying is 20~37 DEG C, and the time is 10~20min.
5. preparation method according to claim 1 or 2, it is characterised in that: in step (1), in the aqueous solution of the acid, acid
Mass concentration be 1%~15%；The mass volume ratio of the aqueous solution of hair and the acid after the drying is (1~10)
Mg:10mL；And/or the time that the hair after the drying impregnates in the aqueous solution of acid is 5~15min, the temperature of immersion is
20~37 DEG C.
6. preparation method according to claim 1 or 2, it is characterised in that: in step (1), the drying is freeze-drying, described
The time of freeze-drying is 24~48h.
7. preparation method according to claim 1 or 2, it is characterised in that: in step (2), the initiator, the hair
The ratio of sill, the peptide growth factor and the water is (1~100) mg:(10~200) ng:(1~10) ng:1mL.
8. preparation method according to claim 1 or 2, it is characterised in that: the peptide growth factor is nerve growth factor
Son, brain-derived neurotrophic factor, neurenergen 3, Neurotrophin-4, neurenergen -5, neurotrophin-6, mind
Through nutrient -7, basic fibroblast growth factor, acid fibroblast growth factor, epidermal growth factor, red blood cell
Generate element, spongiocyte strain derived neurotrophic factor, ciliary neurotrophic factor, motor neuron nutrition factor -1, movement
Neurotrophic factor -2, Bone Morphogenetic Protein, the antibody of Nogo or chondroitin sulfate antibody；And/or
The initiator is that Geniposide, glutaraldehyde or mass ratio are 1:(1~10) n-hydroxysuccinimide and 1- (3- diformazan
Aminopropyl) -3- ethyl-carbodiimide hydrochloride.
9. preparation method according to claim 1 or 2, it is characterised in that: the reaction carries out under stirring conditions, institute
The revolving speed for stating stirring is 200~500rpm；The temperature of the reaction is 0~4 DEG C, and the time is 24~48h.
10. the peptide growth factor that preparation method of any of claims 1-9 is prepared modifies hair sill.
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| WO2014147459A1 (en) * | 2013-03-20 | 2014-09-25 | N.O.M. Coatings Sia | Composition of artificial hair and production method thereof |
| CN105288749A (en) * | 2015-05-20 | 2016-02-03 | 北京航空航天大学 | Preparation method of slow-release polypeptide growth factor biological material scaffold |
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| WO2014147459A1 (en) * | 2013-03-20 | 2014-09-25 | N.O.M. Coatings Sia | Composition of artificial hair and production method thereof |
| CN105288749A (en) * | 2015-05-20 | 2016-02-03 | 北京航空航天大学 | Preparation method of slow-release polypeptide growth factor biological material scaffold |
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