CN110241161A - A kind of yak skin small-molecular peptides preparation method enhancing marrow stromal cell ability - Google Patents
A kind of yak skin small-molecular peptides preparation method enhancing marrow stromal cell ability Download PDFInfo
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The present invention relates to a kind of yak skin small-molecular peptides preparation methods for enhancing marrow stromal cell ability, comprising the following steps: (1) cleans up the yak in Qinghai-tibet skin of fresh band hair, the yak skin through ultraviolet light irradiation, after being irradiated；(2) the yak skin stripping and slicing after irradiating is taken out after extracting in boiling water, pierces through yak skin with thorniness plate；(3) by step, (2) resulting yak skin is put into mass concentration to digest in 0.5% ~ 10% enzymolysis liquid, filters, obtains filtrate；(4) active carbon is added in filtrate, filtering is boiled after being sufficiently stirred, respectively obtain filter residue and for the first time enzymatic hydrolysis solution；(5) filter residue is put into the enzymolysis liquid that mass concentration is 0.5% ~ 8% and digests, and filters, obtains second of enzymatic hydrolysis solution；(6) boiled after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution, separated through differential centrifugation, obtain supernatant；(7) supernatant intercepts the yak skin small-molecular peptides solution that molecular weight is 10 ~ 45kDa through UF membrane, and the yak skin small-molecular peptides solution is freeze-dried to obtain the final product.The method of the present invention is simple, easy to implement.
Description
Technical field
The present invention relates to a kind of preparation process of small-molecular peptides more particularly to a kind of yaks for enhancing marrow stromal cell ability
Ox-hide small-molecular peptides preparation method.
Background technique
Marrow stromal cell originates from the mescenchymal stem cell of embryonic development, is that a kind of multipotency in adult bone marrow is dry thin
Born of the same parents have multi-lineage potential, have differentiation osteoblast, cartilage cell, fat cell and other several phoirocytes
Potential, also can transdifferentiation cardioblast, Skeletal Muscle Cell and the important composition of hematopoieticmicroenviron-ment.Cell cycle
Reflect the variation of ability of cell proliferation.Cell adherence is one of the important way of cell interaction, wide participation cell
Grow the physiology courses such as migration, tissue development, orga- nogenesis.It reflects cell and cultivates in vitro and gradually tend towards stability, into one
The cell Proliferation of step and movement play an important role.
The transfer ability of cell is most important to living cells, is received and moves on the basis of cell adhesion is acted on and being played
The signal of shifting and generate mobile physiological reaction, the enhancing that marrow stromal cell sticks with transfer ability mean bone marrow cell increase
It grows, break up, mature progress faster.
Yak is the peculiar ox kind in Qinghai-Tibet Platean, currently, being always to be carried out with the skin after losing hair or feathers for the research of yak skin, suddenly
The effect of yak hair is omited.
Ox hair is mainly made of keratin, has amino acid long-chain, and cyokeratin is similar to collagen, and there are two types of main eggs
It is white, it is a kind of for the cytoplasm albumen more containing element sulphur, contain cystine；Another kind is the less cellulosic albumen of sulfur-bearing.
And cystine is a kind of sulfur-containing amino acid, is contained in keratin more, is one of amino acid needed by human, it is not bad to human body
Effect.It the effects of pharmaceutically having and promote body cell oxidation and reduction function, increasing white blood cell and pathogen is prevented to develop, also uses
In the acute infectious diseases such as dysentery, typhoid fever, influenza, asthma, neuralgia, eczema and various poisoning illness etc., and there is maintenance albumen
The effect of texture type.
Summary of the invention
A kind of technical problem to be solved by the invention is to provide methods enhancing marrow stromal cell simple, easy to implement
The yak skin small-molecular peptides preparation method of ability.
To solve the above problems, a kind of yak skin small-molecular peptides system for enhancing marrow stromal cell ability of the present invention
Preparation Method, comprising the following steps:
(1) the fresh yak in Qinghai-tibet skin for taking away 1 ~ 2 centimetre of hair of skin is cleaned up, through ultraviolet light irradiation, after being irradiated
Yak skin；
(2) the yak skin after the irradiation is cut into the block of 1 ~ 3cm, takes out after extracting 5 ~ 30 minutes in boiling water, is pierced through with thorniness plate
Yak skin；
(3) by the step, (2) resulting yak skin is put into mass concentration to digest in 0.5% ~ 10% enzymolysis liquid, filters, obtains
Filtrate；The mass ratio of the yak skin and the enzymolysis liquid is 1:5 ~ 1:15；
(4) the active carbon of its quality 3 ~ 10% is added in the filtrate, is boiled after being sufficiently stirred 5 ~ 40 minutes, filters, respectively obtains
Filter residue and for the first time enzymatic hydrolysis solution；
(5) the filter residue is put into the enzymolysis liquid that mass concentration is 0.5% ~ 8% and digests, and filters, obtains second of enzymatic hydrolysis solution；Institute
The mass ratio for stating filter residue and the enzymolysis liquid is 1:8 ~ 1:20；
(6) boiled 5 ~ 40 minutes after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution, through differential centrifugation point
From obtaining supernatant；
(7) the supernatant intercepts the yak skin small-molecular peptides solution that molecular weight is 10 ~ 45kDa through UF membrane, and the yak skin is small
Molecule peptide solution it is freeze-dried to get.
(1) middle ultraviolet lamp irradiation condition refers to that radiation wavelength is 200nm ~ 275nm to the step, and irradiation time is 0.3 ~ 2.0
Hour.
The step enzymolysis liquid that (3) middle mass concentration is 0.5% ~ 10% refers to addition 5 ~ 100g egg in 1L deionized water
White enzyme, solution obtained by being uniformly mixed.
(3) middle enzymatic hydrolysis condition refers to that temperature is 30 ~ 45 DEG C to the step, and the time is 0.5 ~ 8 hour.
The step enzymolysis liquid that (5) middle mass concentration is 0.5% ~ 8% refers to addition 5 ~ 80g albumen in 1L deionized water
Enzyme, solution obtained by being uniformly mixed.
(5) middle enzymatic hydrolysis condition refers to that temperature is 30 ~ 60 DEG C to the step, and the time is 0.5 ~ 12 hour.
The protease refers to bromelain, pepsin, trypsase, cathepsin, papain, withered grass
One of Bacillus protease, thiol protease, alkali protease, compound protease, streptomycete Keratinase, saccharomycete enzyme
Or two kinds or more of compound.
The step (6) in the condition of differential centrifugation separation refer to that first 2000 ~ 3000rpm is centrifuged 5 ~ 30min, then 10000 ~
15000rpm is centrifuged 5 ~ 20min.
(7) middle UF membrane refers to that aperture is 0.01 ~ 0.2 μm of ultrafiltration membrane to the step.
(8) the middle condition being freeze-dried refers to that temperature is -15 DEG C ~ -50 DEG C to the step, and the time is 4 ~ 36 hours.
Compared with the prior art, the present invention has the following advantages:
1, the yak skin small-molecular peptides in the present invention are using peculiar skin of the ox kind yak with hair in Qinghai-Tibet Platean as raw material, after separation
Obtained molecular weight is the yak small-molecular peptides of 10 ~ 45kDa.The exploitation of yak skin small-molecular peptides makes the peculiar money of Qinghai-Tibet Platean
Yak is more preferably utilized in source.
2, the yak skin small-molecular peptides prepared by the present invention are proved through pharmacodynamic experiment, can be by influencing marrow stromal cell
Period, stick with transfer ability have the function that promote Marrow Stromal Cells in Proliferation, marrow stromal cell can be remarkably promoted
Stick and transfer ability, so that more marrow stromal cells enter division stage.It is completed Bone Marrow Cell Cycle and recycles overall process
It is 56.01, bone marrow cell adhesion strength is 55.9%, and marrow basal cell transfer ability reaches 17.07% for 24 hours, reaches within 48 hours
36.68%。
1. influence of the yak skin small-molecular peptides to the marrow stromal cell cell cycle:
It takes 200 mg/ mL yak skin small-molecular peptides to be added in 6 orifice plates to preheat, every group of 3 multiple holes, inoculating cell suspension is connected to interior
In 6 orifice plates containing coverslip；CO2Overnight incubation in incubator, grows cell on cover plate；Taking-up cover plate, PBS rinsing 3 times, often
Secondary 3 min is added fixer (methanol: glacial acetic acid=3:1) and fixes 30 min, and PBS is rinsed 3 times, and 3 min, is added every time
After Giemsa liquid dyes 10 min, cover plate left-hand thread is dried on glass slide, microscopy.
The life process of cell is exactly constantly newborn and death, and significant process is exactly the circulation of cell cycle, between being divided into
Two processes of phase and division stage.Experimental result is as shown in table 1 to be compared with blank group, and yak skin small-molecular peptides are in division stage
Cell is more (P < 0.01), shows that yak skin small-molecular peptides can enhance the proliferative capacity of marrow stromal cell.
1 yak skin small-molecular peptides of table to Bone Marrow Cell Cycle influence (n=6,)
Note: P < 0.01 * P < 0.05 compared to the blank group, * *.
2. influence of the yak skin small-molecular peptides to marrow basal cell's adhesion strength:
It takes the yak skin small-molecular peptides of 200 mg/ mL to be previously added in 6 orifice plates to preheat, by single bone marrow cell with every hole 2
The volume of mL is incubated in the 6 orifice plates of dosing, respectively at the 3rd day half amount of culture changes liquid, the 5th day full dose changes liquid, on day 4
When be sucked out culture solution, PBS gently rinses 2 removing suspension cells, and cell density 5 × 10 is added in each hole5The preparation of/mL normal mouse
Individual cells suspension 1 mL, 37 DEG C, 5% CO2It after 2 h of incubator culture, is gently rinsed with culture solution 2 times, collects and count
Nonadherent bone marrow cell.
Cell adherence refers to that some albumen related with cytoskeleton recombinate in living cells, so that cell occurs to gather
Collection forms the process of cell mass or tissue, reflects cell and cultivates in vitro and gradually tend towards stability, increases to further cell
It grows and movement plays an important role.
Experimental result is as shown in table 2 to be compared with blank control group, and it is thin that yak skin small-molecular peptides can significantly increase bone marrow matrix
The Adhering capacity (P < 0.01) of born of the same parents.
2 yak skin small-molecular peptides of table to bone marrow cell adhesion strength influence (n=6,)
Note: P < 0.01 * P < 0.05 compared to the blank group, * *.
3. influence of the yak skin small-molecular peptides to marrow basal cell's transfer ability:
It takes myelomonocyte suspension to be inoculated in the 6 orifice plates marked with the volume of every 2 mL of hole, is added 200 mg/ mL's
The cell of yak skin small-molecular peptides, culture to 80% is adherent, takes out 6 orifice plates, compares ruler with 10 μ L pipette tips, perpendicular to bottom surface
Horizontal line horizontal line scratch；It is cleaned cell 3 times with PBS buffer solution, washes away the cell fragment of scratch generation；Serum free medium is added,
It is placed in CO2It takes pictures when incubator culture 24,48h, calculates cell migration rate.
The transfer ability of cell is most important to living cells, is bone marrow matrix on the basis of cell adhesion is acted on and being played
The enhancing of cell adhesion and transfer ability mean proliferation of bone marrow cells, differentiation, maturation progress faster.
Experimental result is as shown in Figure 1, blank group is after cultivating 24 h and 48 h, and scratch width has almost no change, cell
Transfer ability is weak, but each administration group, after cultivating 24 h and 48 h, scratch has in varying degrees to narrow, and especially 2 groups, in scratch
The cell of migration increased significantly, 2 groups after cultivating 48 h, scratch healing is obvious.
As shown in table 3, it after the administration of various concentration yak skin small-molecular peptides, is moved after marrow stromal cell culture 24 h and 48 h
Shifting ability compares enhancing with blank control group obviously (P < 0.01), illustrates that yak skin small-molecular peptides can enhance marrow stromal cell
Transfer ability, transfer ability increase significant (P < 0.01).
3 yak skin small-molecular peptides of table to bone marrow cell transfer ability influence (n=6,)
Note: P < 0.01 * P < 0.05 compared to the blank group, * *.
4, the method for the present invention is simple, easy to implement.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is influence of the yak skin small-molecular peptides of the present invention to bone marrow cell transfer ability.
Specific embodiment
A kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability of embodiment 1, including following step
It is rapid:
(1) the fresh yak in Qinghai-tibet skin for taking away 2 centimetres of hairs of skin is cleaned up, shone with the radiation wavelength of 275nm through ultraviolet lamp
It penetrates 2.0 hours, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 3cm, takes out after extracting 30 minutes in boiling water, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 10%, is digested 8 hours in 45 DEG C, mistake
Filter, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 10% refers in 1L deionized water addition 100g protease, be uniformly mixed and
The solution obtained.Protease refers to 30g thiol protease, 20g alkali protease, 5g compound protease, 35g streptomycete keratoprotein
The mixture of enzyme, 10g saccharomycete enzyme.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:5.
(4) the active carbon of its quality 3% is added in filtrate, is boiled after being sufficiently stirred 40 minutes, filter, respectively obtain filter residue and
Enzymatic hydrolysis solution for the first time.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 8%, is digested 12 hours in 60 DEG C, and filtering obtains second of enzymatic hydrolysis
Solution.
Wherein: the enzymolysis liquid that mass concentration is 8% refers to the addition 80g protease in 1L deionized water, is uniformly mixed and obtains
Solution.Protease refers to 3g bromelain, 10g pepsin, 30g trypsase, 10g cathepsin, 7g pawpaw egg
The mixture of white enzyme, 20g subtilopeptidase A.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:8.
(6) boiled 40 minutes after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution, differential centrifugation separation, first
2000rpm is centrifuged 5min, then 10000rpm is centrifuged 5min, obtains supernatant.
(7) the Ultra filtration membrane that supernatant via hole diameter is 0.01 μm, the yak skin small molecule that interception molecular weight is 10 ~ 20kDa
Peptide solution, the yak skin small-molecular peptides solution in -15 DEG C be freeze-dried 4 hours to get.
A kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability of embodiment 2, including following step
It is rapid:
(1) the fresh yak in Qinghai-tibet skin for taking away 1 centimetre of hair of skin is cleaned up, shone with the radiation wavelength of 200nm through ultraviolet lamp
It penetrates 0.3 hour, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 1cm, extracts in boiling water and takes out after five minutes, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 1%, is digested 0.5 hour in 30 DEG C, mistake
Filter, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 1% refers to the addition 10g protease in 1L deionized water, is uniformly mixed and obtains
Solution.Protease refers to 1g bromelain, 0.5g pepsin, 2g trypsase, 2g cathepsin, 0.5g pawpaw egg
The mixture of white enzyme, 4g subtilopeptidase A.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:15.
(4) the active carbon of its quality 10% is added in filtrate, is boiled after being sufficiently stirred 5 minutes, filter, respectively obtain filter residue and
Enzymatic hydrolysis solution for the first time.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 0.5%, is digested 0.5 hour in 30 DEG C, filtering, is obtained second
Digest solution.
Wherein: the enzymolysis liquid that mass concentration is 0.5% refers in 1L deionized water addition 5g protease, be uniformly mixed and
The solution obtained.Protease refers to 0.4g thiol protease, 0.1g alkali protease, 4g compound protease, 0.3g streptomycete cutin
The mixture of protease, 0.2g saccharomycete enzyme.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:20.
(6) boiled 5 minutes after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution, differential centrifugation separation, first
3000rpm is centrifuged 30min, then 15000rpm is centrifuged 20min, obtains supernatant.
(7) the Ultra filtration membrane that supernatant via hole diameter is 0.2 μm, the yak skin small molecule that interception molecular weight is 25 ~ 45kDa
Peptide solution, the yak skin small-molecular peptides solution in -25 DEG C be freeze-dried 36 hours to get.
A kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability of embodiment 3, including following step
It is rapid:
(1) the fresh yak in Qinghai-tibet skin for taking away 1.5 centimetres of hairs of skin is cleaned up, with the radiation wavelength of 223nm through ultraviolet lamp
Irradiation 1.0 hours, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 2cm, takes out after extracting 15 minutes in boiling water, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 5%, is digested 4 hours in 40 DEG C, mistake
Filter, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 5% refers to the addition 50g protease in 1L deionized water, is uniformly mixed and obtains
Solution.Protease refers to the mixture of 15g subtilopeptidase A, 25g thiol protease, 10g alkali protease.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:10.
(4) the active carbon of its quality 5% is added in filtrate, is boiled after being sufficiently stirred 20 minutes, filter, respectively obtain filter residue and
Enzymatic hydrolysis solution for the first time.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 6%, is digested 6 hours in 50 DEG C, and filtering obtains second of enzymatic hydrolysis
Solution.
Wherein: the enzymolysis liquid that mass concentration is 6% refers to the addition 60g compound protease in 1L deionized water, is uniformly mixed
Obtained by solution.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:10.
(6) boiled 30 minutes after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution, differential centrifugation separation, first
2500rpm is centrifuged 15min, then 13000rpm is centrifuged 18min, obtains supernatant.
(7) the Ultra filtration membrane that supernatant via hole diameter is 0.05 μm, the yak skin small molecule that interception molecular weight is 10 ~ 45kDa
Peptide solution, the yak skin small-molecular peptides solution in -50 DEG C be freeze-dried 24 hours to get.
A kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability of embodiment 4, including following step
It is rapid:
(1) the fresh yak in Qinghai-tibet skin for taking away 2 centimetres of hairs of skin is cleaned up, shone with the radiation wavelength of 245nm through ultraviolet lamp
It penetrates 0.5 hour, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 3cm, extracts in boiling water and takes out after ten minutes, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 4%, is digested 7 hours in 45 DEG C, mistake
Filter, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 4% refers to the addition 40g thiol protease in 1L deionized water, is uniformly mixed
Obtained by solution.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:8.
(4) the active carbon of its quality 8% is added in filtrate, is boiled after being sufficiently stirred 10 minutes, filter, respectively obtain filter residue and
Enzymatic hydrolysis solution for the first time.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 7%, is digested 8 hours in 60 DEG C, and filtering obtains second of enzymatic hydrolysis
Solution.
Wherein: the enzymolysis liquid that mass concentration is 7% refers to the addition 70g bromelain in 1L deionized water, is uniformly mixed
Obtained by solution.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:15.
(6) boiled 15 minutes after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution, differential centrifugation separation, first
2500rpm is centrifuged 20min, then 15000rpm is centrifuged 5min, obtains supernatant.
(7) the Ultra filtration membrane that supernatant via hole diameter is 0.1 μm, the yak skin small molecule that interception molecular weight is 20 ~ 40kDa
Peptide solution, the yak skin small-molecular peptides solution in -20 DEG C be freeze-dried 18 hours to get.
A kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability of embodiment 5, including following step
It is rapid:
(1) the fresh yak in Qinghai-tibet skin for taking away 2 centimetres of hairs of skin is cleaned up, shone with the radiation wavelength of 225nm through ultraviolet lamp
It penetrates 1.5 hours, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 1cm, takes out after extracting 25 minutes in boiling water, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 5.5%, is digested 0.5 hour in 40 DEG C,
Filtering, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 5.5% refers in 1L deionized water addition 55g protease, be uniformly mixed and
The solution obtained.Protease refer to 5g bromelain, 0.6g streptomycete Keratinase, 25g saccharomycete enzyme, 1g pepsin,
3g trypsase, 0.4g cathepsin, 10g papain, 10g subtilopeptidase A mixture.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:13.
(4) the active carbon of its quality 7.5% is added in filtrate, is boiled after being sufficiently stirred 10 minutes, filters, respectively obtains filter residue
Solution is digested with first time.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 4%, is digested 12 hours in 55 DEG C, and filtering obtains second of enzymatic hydrolysis
Solution.
Wherein: the enzymolysis liquid that mass concentration is 4% refers to the addition 40g protease in 1L deionized water, is uniformly mixed and obtains
Solution.Protease refers to the mixture of 12g thiol protease, 22g alkali protease, 6g compound protease.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:12.
(6) boiled 20 minutes after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution, differential centrifugation separation, first
3000rpm is centrifuged 5min, then 10000rpm is centrifuged 20min, obtains supernatant.
(7) the Ultra filtration membrane that supernatant via hole diameter is 0.01 μm, the yak skin small molecule that interception molecular weight is 10 ~ 40kDa
Peptide solution, the yak skin small-molecular peptides solution in -35 DEG C be freeze-dried 30 hours to get.
In above-described embodiment 1 ~ 5, protease can also be bromelain, pepsin, trypsase, histone
Enzyme, papain, subtilopeptidase A, thiol protease, alkali protease, compound protease, streptomycete keratoprotein
The compound of one or both of enzyme, saccharomycete enzyme or more.
Claims (10)
1. a kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability, comprising the following steps:
(1) the fresh yak in Qinghai-tibet skin for taking away 1 ~ 2 centimetre of hair of skin is cleaned up, through ultraviolet light irradiation, after being irradiated
Yak skin；
(2) the yak skin after the irradiation is cut into the block of 1 ~ 3cm, takes out after extracting 5 ~ 30 minutes in boiling water, is pierced through with thorniness plate
Yak skin；
(3) by the step, (2) resulting yak skin is put into mass concentration to digest in 0.5% ~ 10% enzymolysis liquid, filters, obtains
Filtrate；The mass ratio of the yak skin and the enzymolysis liquid is 1:5 ~ 1:15；
(4) the active carbon of its quality 3 ~ 10% is added in the filtrate, is boiled after being sufficiently stirred 5 ~ 40 minutes, filters, respectively obtains
Filter residue and for the first time enzymatic hydrolysis solution；
(5) the filter residue is put into the enzymolysis liquid that mass concentration is 0.5% ~ 8% and digests, and filters, obtains second of enzymatic hydrolysis solution；Institute
The mass ratio for stating filter residue and the enzymolysis liquid is 1:8 ~ 1:20；
(6) boiled 5 ~ 40 minutes after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution, through differential centrifugation point
From obtaining supernatant；
(7) the supernatant intercepts the yak skin small-molecular peptides solution that molecular weight is 10 ~ 45kDa through UF membrane, and the yak skin is small
Molecule peptide solution it is freeze-dried to get.
2. a kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability as described in claim 1, special
Sign is: (1) middle ultraviolet lamp irradiation condition refers to that radiation wavelength is 200nm ~ 275nm to the step, and irradiation time is 0.3 ~ 2.0
Hour.
3. a kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability as described in claim 1, special
Sign is: the step enzymolysis liquid that (3) middle mass concentration is 0.5% ~ 10% refers to addition 5 ~ 100g albumen in 1L deionized water
Enzyme, solution obtained by being uniformly mixed.
4. a kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability as described in claim 1, special
Sign is: (3) middle enzymatic hydrolysis condition refers to that temperature is 30 ~ 45 DEG C to the step, and the time is 0.5 ~ 8 hour.
5. a kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability as described in claim 1, special
Sign is: the step enzymolysis liquid that (5) middle mass concentration is 0.5% ~ 8% refers to addition 5 ~ 80g albumen in 1L deionized water
Enzyme, solution obtained by being uniformly mixed.
6. a kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability as described in claim 1, special
Sign is: (5) middle enzymatic hydrolysis condition refers to that temperature is 30 ~ 60 DEG C to the step, and the time is 0.5 ~ 12 hour.
7. a kind of yak skin small-molecular peptides preparation method of enhancing marrow stromal cell ability as claimed in claim 3 or 5,
Be characterized in that: the protease refers to bromelain, pepsin, trypsase, cathepsin, papain, withered
Straw mycoproteinase, thiol protease, alkali protease, compound protease, streptomycete Keratinase, one in saccharomycete enzyme
Kind or two kinds or more of compound.
8. a kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability as described in claim 1, special
Sign is: the step (6) in the condition of differential centrifugation separation refer to that first 2000 ~ 3000rpm is centrifuged 5 ~ 30min, then 10000 ~
15000rpm is centrifuged 5 ~ 20min.
9. a kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability as described in claim 1, special
Sign is: (7) middle UF membrane refers to that aperture is 0.01 ~ 0.2 μm of ultrafiltration membrane to the step.
10. a kind of yak skin small-molecular peptides preparation method for enhancing marrow stromal cell ability as described in claim 1, special
Sign is: (8) the middle condition being freeze-dried refers to that temperature is -15 DEG C ~ -50 DEG C to the step, and the time is 4 ~ 36 hours.
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