CN108823271A - A kind of polypeptide and the biological use for improving umbilical cord mesenchymal stem cells amplification efficiency - Google Patents

A kind of polypeptide and the biological use for improving umbilical cord mesenchymal stem cells amplification efficiency Download PDF

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CN108823271A
CN108823271A CN201810624684.0A CN201810624684A CN108823271A CN 108823271 A CN108823271 A CN 108823271A CN 201810624684 A CN201810624684 A CN 201810624684A CN 108823271 A CN108823271 A CN 108823271A
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谢崇林
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N5/0668—Mesenchymal stem cells from other natural sources
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

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Abstract

A kind of biological use the invention discloses polypeptide and for improving umbilical cord mesenchymal stem cells amplification efficiency, polypeptide production methods are：1, pot marigold seed digests, and enzymolysis liquid is packed into the bag filter that molecular cut off is 7000Da and dialyses, and aqueous solution concentration, freeze-drying outside the bag filter of 7000Da will be less than containing molecular weight, much peptide freeze-dried powder；2, purified with DA201-C type macroreticular resin to polypeptide freeze-dried powder initial gross separation, successively with 40%, 50%, 60%, 70% ethanol gradient elution, each gradient elution 3BV collects each gradient eluent, is concentrated, is freeze-dried to obtain F40, F50, F70 crude product；3, gel filtration chromatography purifies：The corresponding fraction of the highest chromatographic peak of absorbance in sample solution is collected according to chromatogram, freeze-drying obtains polypeptide F40, F50, F70 respectively.Polypeptide of the present invention is remarkably improved umbilical cord mesenchymal stem cells proliferation rate, can be used for umbilical cord mesenchymal stem cells rapid amplifying.

Description

A kind of polypeptide and the biological use for improving umbilical cord mesenchymal stem cells amplification efficiency
Technical field
The invention belongs to field of medicaments, are related to a kind of polypeptide and the life for improving umbilical cord mesenchymal stem cells amplification efficiency Object purposes.
Background technique
Mescenchymal stem cell was a kind of stem cell with self-renewing, proliferation and multi-lineage potential, in head in 1966 First found from marrow by Friedenstein etc., numerous studies find mescenchymal stem cell have inwardly, in, outer 3 germinal layers Potential including differentiation and developments such as tendon, ligament, liver cell, cardiac muscle cell and marrow stromal cells.And it is filled between Adult Human Bone Marrow source Matter stem cell population and Proliferation, Differentiation potential decline with the increase at age, and the acquisition of donor mescenchymal stem cell must row marrow It punctures, due to disease, patient often has the factors such as infection, constitution be weaker to also limit autologous bone marrow mesenchymal stem cells Using.Therefore the hot spot that new source for mesenchymal stem cells is stem-cell research both at home and abroad at present is found, human umbilical cord mesenchymal is dry Cell (human umbilical cordmesenchymal stem cells, hUCMSCs) have abundance, to donor without Influence, be easy to acquire and transport, without allosome rejection, avoid many advantages, such as dispute of ethic, therefore hUCMSCs is expected to become The ideal substitution source of mesenchymal stem cell.HUCMSCs is that one kind has self more as mesenchymal stem cell New and multi-lineage potential adult stem cell.Theoretically, under certain condition, hUCMSCs can be directed differentiation in body Various functioning cells, form any kind of tissue and organ, that is, have " plasticity ".Also, it is dry thin with medulla mesenchyma Cell phase ratio, hUCMSCs have many advantages, such as and become the research hotspot of medical field in recent years.
Because hUCMSCs all has tremendous potential in terms of basic research and clinical application, its culture is expanded There is still a need for further researchs, utilize this cell so as to more efficiently obtain.
Summary of the invention
It is done it is an object of the invention to overcome the deficiencies of the prior art and provide a kind of polypeptide and for improving umbilical cord mesenchyma The biological use of cell amplification efficiency.
The present invention is achieved through the following technical solutions：
Technical solution one：
A kind of polypeptide promoting umbilical cord mesenchymal stem cells proliferation, is made by the steps：
The preparation of step 1, pot marigold seed polypeptide freeze-dried powder
Pot marigold seed is heated up after enzymatic hydrolysis and is gone out through alkali protease Alcalase2.4L+ trypsin digestion Enzymatic treatment obtains pot marigold Seed Storage Protein enzymatic hydrolysis solution.Enzymolysis liquid is fitted into the bag filter that molecular cut off is 7000Da and is dialysed, It will be concentrated under reduced pressure containing aqueous solution outside bag filter of the molecular weight less than 7000Da, it is then that the polypeptide solution of concentration is dry in freezing Freeze-drying is in dry machine to get pot marigold seed polypeptide freeze-dried powder.
Step 2, macroreticular resin initial gross separation purifying
Initial gross separation purifying is carried out by hydrophobicity to polypeptide freeze-dried powder using DA201-C type macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with dehydrated alcohol, then use deionization Water is cleaned standby and is used；Amidin is lyophilized in the polypeptide that concentration is 50mg/mL under room temperature, chromatography is flowed through with the flow velocity of 0.5BV/h Column detects the light absorption value A220 of efflux with UV detector, using A220=0.05 as breakthrough point；
(2) after end of the sample, chromatographic column is rinsed with 1BV/h flow velocity with deionized water, is in charge of collection eluent, measures water Conductivity, when conductivity is down to it is suitable with deionized water when, using 40% ethyl alcohol to the polypeptide being adsorbed on macroporous absorbent resin Freeze-dried powder is eluted, and 3BV is eluted, and collects eluent, and 45 DEG C of vacuum concentrated by rotary evaporations to no alcohol taste are freeze-dried to obtain F40 crude product.
Step 3, gel filtration chromatography purifying
F40 crude product is made into the sample solution that concentration is 50mg/mL, uses column dimension for the Sephadex of 1.6 × 100cm G-15 gel filtration chromatography is further isolated and purified again, applied sample amount 1mL, and eluent is deionized water, flow velocity 15mL/h, Detection wavelength 220nm collects the corresponding elution fraction of the highest chromatographic peak of absorbance in sample solution according to chromatogram, and freezing is dry It is dry, obtain polypeptide F40.
Preferably, the pot marigold seed is the seed of compositae plant pot marigold, cleans and dries, is ground into 80 mesh fine powders.
Preferably, alkali protease Alcalase2.4L enzyme activity is 2.4AU/g.
Preferably, the enzyme activity of trypsase is 2500U/g.
Preferably, pot marigold seed is through alkali protease Alcalase2.4L (2.4AU/g)+trypsase (2500U/g) In compound proportion 3:1, pH 7.4, temperature 60 C, substrate mass concentration 40g/L, enzyme concentration 6%, enzymolysis time 6h condition enzyme Solution.
Preferably, 90 DEG C of holding 20min enzyme deactivations are warming up to after enzymatic hydrolysis.
It preferably, will be dense in 55 DEG C of rotary evaporation decompressions less than aqueous solution outside the bag filter of 7000Da containing molecular weight Contracting.
Application of the aforementioned polypeptides in terms of promoting umbilical cord mesenchymal stem cells proliferation.
Technical solution two：
A kind of polypeptide promoting umbilical cord mesenchymal stem cells proliferation, is made by the steps：
The preparation of step 1, pot marigold seed polypeptide freeze-dried powder
Pot marigold seed is heated up after enzymatic hydrolysis and is gone out through alkali protease Alcalase2.4L+ trypsin digestion Enzymatic treatment obtains pot marigold Seed Storage Protein enzymatic hydrolysis solution.Enzymolysis liquid is fitted into the bag filter that molecular cut off is 7000Da and is dialysed, It will be concentrated under reduced pressure containing aqueous solution outside bag filter of the molecular weight less than 7000Da, it is then that the polypeptide solution of concentration is dry in freezing Freeze-drying is in dry machine to get pot marigold seed polypeptide freeze-dried powder.
Step 2, macroreticular resin initial gross separation purifying
Initial gross separation purifying is carried out by hydrophobicity to polypeptide freeze-dried powder using DA201-C type macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with dehydrated alcohol, then use deionization Water is cleaned standby and is used；Amidin is lyophilized in the polypeptide that concentration is 50mg/mL under room temperature, chromatography is flowed through with the flow velocity of 0.5BV/h Column detects the light absorption value A220 of efflux with UV detector, using A220=0.05 as breakthrough point；
(2) after end of the sample, chromatographic column is rinsed with the flow velocity of 1BV/h with deionized water, is in charge of collection eluent, measures water Conductivity, when conductivity is down to it is suitable with deionized water when, successively using 40%, 50% ethyl alcohol to being adsorbed on macroporous absorption Polypeptide freeze-dried powder on resin carries out gradient elution, and each gradient elution 3BV collects 50% ethanol eluate, 45 DEG C of vacuum rotations Inspissation is reduced to no alcohol taste, is freeze-dried to obtain F50 crude product.
Step 3, gel filtration chromatography purifying
F50 crude product is made into the sample solution that concentration is 50mg/mL, uses column dimension for the Sephadex of 1.6 × 100cm G-15 gel filtration chromatography is further isolated and purified again, applied sample amount 1mL, and eluent is deionized water, flow velocity 15mL/h, Detection wavelength 220nm collects the corresponding elution fraction of the highest chromatographic peak of absorbance in sample solution according to chromatogram, and freezing is dry It is dry, obtain polypeptide F50.
Preferably, the pot marigold seed is the seed of compositae plant pot marigold, cleans and dries, is ground into 80 mesh fine powders.
Preferably, alkali protease Alcalase2.4L enzyme activity is 2.4AU/g.
Preferably, the enzyme activity of trypsase is 2500U/g.
Preferably, pot marigold seed is through alkali protease Alcalase2.4L (2.4AU/g)+trypsase (2500U/g) In compound proportion 3:1, pH 7.4, temperature 60 C, substrate mass concentration 40g/L, enzyme concentration 6%, enzymolysis time 6h condition enzyme Solution.
Preferably, 90 DEG C of holding 20min enzyme deactivations are warming up to after enzymatic hydrolysis.
It preferably, will be dense in 55 DEG C of rotary evaporation decompressions less than aqueous solution outside the bag filter of 7000Da containing molecular weight Contracting.
Application of the aforementioned polypeptides in terms of promoting umbilical cord mesenchymal stem cells proliferation.
Technical solution three：
A kind of polypeptide promoting umbilical cord mesenchymal stem cells proliferation, is made by the steps：
The preparation of step 1, pot marigold seed polypeptide freeze-dried powder
Pot marigold seed is heated up after enzymatic hydrolysis and is gone out through alkali protease Alcalase2.4L+ trypsin digestion Enzymatic treatment obtains pot marigold Seed Storage Protein enzymatic hydrolysis solution.Enzymolysis liquid is fitted into the bag filter that molecular cut off is 7000Da and is dialysed, It will be concentrated under reduced pressure containing aqueous solution outside bag filter of the molecular weight less than 7000Da, it is then that the polypeptide solution of concentration is dry in freezing Freeze-drying is in dry machine to get pot marigold seed polypeptide freeze-dried powder.
Step 2, macroreticular resin initial gross separation purifying
Initial gross separation purifying is carried out by hydrophobicity to polypeptide freeze-dried powder using DA201-C type macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with dehydrated alcohol, then use deionization Water is cleaned standby and is used；Amidin is lyophilized in the polypeptide that concentration is 50mg/mL under room temperature, chromatography is flowed through with the flow velocity of 0.5BV/h Column detects the light absorption value A220 of efflux with UV detector, using A220=0.05 as breakthrough point；
(2) after end of the sample, chromatographic column is rinsed with the flow velocity of 1BV/h with deionized water, is in charge of collection eluent, measures water Conductivity, when conductivity is down to it is suitable with deionized water when, successively using 40%, 50%, 60%, 70% ethyl alcohol to absorption Polypeptide freeze-dried powder on macroporous absorbent resin carries out gradient elution, and each gradient elution 3BV collects 70% ethanol eluate, 45 DEG C of vacuum concentrated by rotary evaporations are freeze-dried to obtain F70 crude product to no alcohol taste.
Step 3, gel filtration chromatography purifying
F70 crude product is made into the sample solution that concentration is 50mg/mL, uses column dimension for the Sephadex of 1.6 × 100cm G-15 gel filtration chromatography is further isolated and purified again, applied sample amount 1mL, and eluent is deionized water, flow velocity 15mL/h, Detection wavelength 220nm collects the corresponding elution fraction of the highest chromatographic peak of absorbance in sample solution according to chromatogram, and freezing is dry It is dry, obtain polypeptide F70.
Preferably, the pot marigold seed is the seed of compositae plant pot marigold, cleans and dries, is ground into 80 mesh fine powders.
Preferably, alkali protease Alcalase2.4L enzyme activity is 2.4AU/g.
Preferably, the enzyme activity of trypsase is 2500U/g.
Preferably, pot marigold seed is through alkali protease Alcalase2.4L (2.4AU/g)+trypsase (2500U/g) In compound proportion 3:1, pH 7.4, temperature 60 C, substrate mass concentration 40g/L, enzyme concentration 6%, enzymolysis time 6h condition enzyme Solution.
Preferably, 90 DEG C of holding 20min enzyme deactivations are warming up to after enzymatic hydrolysis.
It preferably, will be dense in 55 DEG C of rotary evaporation decompressions less than aqueous solution outside the bag filter of 7000Da containing molecular weight Contracting.
Application of the aforementioned polypeptides in terms of promoting umbilical cord mesenchymal stem cells proliferation.
Beneficial effect：
Polypeptide provided by the invention can significantly improve the proliferation rate of umbilical cord mesenchymal stem cells, can be used between umbilical cord filling The external rapid amplifying of matter stem cell enriches the cell source of umbilical cord mesenchymal stem cells.
Detailed description of the invention
Fig. 1 is the Sephadex G-15 gel filtration chromatography figure of F40 crude product；
Fig. 2 is the Sephadex G-15 gel filtration chromatography figure of F50 crude product；
Fig. 3 is the Sephadex G-15 gel filtration chromatography figure of F60 crude product；
Fig. 4 is the Sephadex G-15 gel filtration chromatography figure of F70 crude product；
Fig. 5 is the influence that polypeptide F40, F50, F60, F70 are proliferated umbilical cord mesenchymal stem cells.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this Protect range.
One, experimental method
1, the preparation of pot marigold seed polypeptide freeze-dried powder
Pot marigold seed (seed of compositae plant pot marigold is cleaned and dried, is ground into 80 mesh fine powders) believes alkalinity through Novi Protease A lcalase2.4L (2.4AU/g)+trypsase (2500U/g) is in compound proportion 3:1, pH 7.4, temperature 60 C, It is digested under conditions of substrate mass concentration 40g/L, enzyme concentration 6%, enzymolysis time 6h, 90 DEG C of holdings is warming up to after enzymatic hydrolysis 20min carries out destroy the enzyme treatment and obtains pot marigold Seed Storage Protein enzymatic hydrolysis solution.It is the saturating of 7000Da that enzymolysis liquid, which is packed into molecular cut off, It dialyses, will be concentrated under reduced pressure containing aqueous solution outside bag filter of the molecular weight less than 7000Da in 55 DEG C of rotary evaporations, so in analysis bag The polypeptide solution of concentration is lyophilized in freeze drier afterwards to get pot marigold seed polypeptide freeze-dried powder.
2, macroreticular resin initial gross separation purifies
Initial gross separation purifying is carried out by hydrophobicity to polypeptide freeze-dried powder using DA201-C type macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with dehydrated alcohol, then use deionization Water is cleaned standby and is used；Amidin is lyophilized in the polypeptide that concentration is 50mg/mL under room temperature, chromatography is flowed through with the flow velocity of 0.5BV/h Column detects the light absorption value A220 of efflux with UV detector, using A220=0.05 as breakthrough point；
(2) after end of the sample, chromatographic column is rinsed with the flow velocity of 1BV/h with deionized water, is in charge of collection eluent, measures water Conductivity, when conductivity is down to it is suitable with deionized water when, successively using 40%, 50%, 60%, 70% ethyl alcohol to absorption Polypeptide freeze-dried powder on macroporous absorbent resin carries out gradient elution, each gradient elution 3BV, collects each gradient eluent, and 45 DEG C vacuum concentrated by rotary evaporation is freeze-dried to obtain F40, F50, F60, F70 crude product to no alcohol taste.
3, gel filtration chromatography purifies
F40, F50, F60, F70 crude product are made into the sample solution that concentration is 50mg/mL, use column dimension for 1.6 × The Sephadex G-15 gel filtration chromatography of 100cm is further isolated and purified again, and applied sample amount 1mL, eluent is deionization Water, flow velocity 15mL/h, Detection wavelength 220nm collect the highest chromatographic peak pair of absorbance in each sample solution according to chromatogram The elution fraction answered, freeze-drying, respectively obtains polypeptide F40, F50, F60, F70.
4, to the Effect of promoting growth of umbilical cord mesenchymal stem cells
Separation, the culture of umbilical cord mesenchymal stem cells：By the full-term pregnancy obtained under aseptic condition childbirth fetal cord, use PBS is washed 3 times, removes remaining haemocyte.Cutting navel cord at the segment of 2.0cm or so and reject blood vessel (1 umbilical vein, 2 Arteria umbilicalis), gel tissue therein is taken out, size about 1mm is cut into3Tissue block, be subsequently placed in culture bottle.6h Afterwards, be added containing volume fraction be 10% fetal calf serum, 100U/mL penicillin, 100mg/L streptomysin DMEM/F12 culture medium, It is placed on 37 DEG C, volume fraction 5%CO2It is cultivated in incubator.The later half amount of 3d changes liquid, liquid is changed after 1 week again, hereafter every 3d It changes liquid 1 time.Cell growth status around observation adherent tissue is digested when cell reaches 80%-90% degrees of fusion with pancreatin Passage, and tissue is transferred in a new culture bottle, continue to cultivate according to the above method, obtains second of tissue block adherent training Feeding cell.The umbilical cord mesenchymal stem cells for taking second of tissue cultures to obtain carry out following experiments.
The influence of polypeptide F40, F50, F60, F70 to umbilical cord mesenchymal stem cells proliferation activity：By second of tissue cultures After obtained umbilical cord mesenchymal stem cells culture to logarithmic phase, adjust to 2 × 106A/mL, in 96 holes, flat tissue culture plate is every 50 μ L of cell suspension is added in hole, if experimental group and control group, experimental group is divided into 4 groups, is separately added into polypeptide F40, F50, F60, F70 Each 50 μ L of DMEM/F12 culture medium of final concentration of 10 μ g/mL, control group use the culture medium culture without polypeptide, and another setting is only Blank group containing culture medium is placed in cell incubator and handles cell 72h, 10 μ L CCK-8 solution of addition, with enzyme-linked after 4h Immune detector detects OD450 value A (size of absorbance and the quantity of living cells are proportional).Cell is calculated according to surveyed OD value Proliferation rate, cell proliferation rate=(experimental group OD value-blank group OD value)/(control group OD value-blank group OD value) × 100%.With Control group proliferation rate is 100%.
Two, experimental result
1, gel filtration chromatography purification result
The sample solution that concentration is 50mg/mL is made into F40, F50, F60, F70 crude product, use column dimension for 1.6 × The Sephadex G-15 gel filtration chromatography of 100cm is further isolated and purified again, and applied sample amount 1mL, eluent is deionization Water, flow velocity 15mL/h, Detection wavelength 220nm collect the highest chromatography of absorbance in each sample solution according to chromatogram 1~4 The corresponding elution fraction in peak, freeze-drying, respectively obtains polypeptide F40, F50, F60, F70.
2, to the Effect of promoting growth of umbilical cord mesenchymal stem cells
As a result as shown in table 1 and Fig. 5.Compared with the control group, polypeptide F40, F50, F70 group umbilical cord mesenchymal stem cells are proliferated Rate is significantly higher (P < 0.05), polypeptide F60 group umbilical cord mesenchymal stem cells proliferation rate and unobvious (the P > of control group difference 0.05)。
The influence that table 1 polypeptide F40, F50, F60, F70 are proliferated umbilical cord mesenchymal stem cells
Polypeptide provided by the invention can significantly improve the proliferation rate of umbilical cord mesenchymal stem cells, can be used between umbilical cord filling The external rapid amplifying of matter stem cell enriches the cell source of umbilical cord mesenchymal stem cells.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know Protection scope of the present invention should not be confined to the specific embodiment by road.

Claims (8)

1. a kind of polypeptide for promoting umbilical cord mesenchymal stem cells proliferation, which is characterized in that be made by the steps：
The preparation of step 1, pot marigold seed polypeptide freeze-dried powder
Pot marigold seed is through alkali protease Alcalase2.4L+ trypsin digestion, and heating carries out at enzyme deactivation after enzymatic hydrolysis Manage to obtain pot marigold Seed Storage Protein enzymatic hydrolysis solution.Enzymolysis liquid is fitted into the bag filter that molecular cut off is 7000Da and is dialysed, will be contained There is aqueous solution outside bag filter of the molecular weight less than 7000Da to be concentrated under reduced pressure, then by the polypeptide solution of concentration in freeze drier Middle freeze-drying is to get pot marigold seed polypeptide freeze-dried powder.
Step 2, macroreticular resin initial gross separation purifying
Initial gross separation purifying is carried out by hydrophobicity to polypeptide freeze-dried powder using DA201-C type macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with dehydrated alcohol, then be washed with deionized water It is spare after net；Amidin is lyophilized in the polypeptide that concentration is 50mg/mL under room temperature, chromatographic column is flowed through with the flow velocity of 0.5BV/h, used UV detector detects the light absorption value A220 of efflux, using A220=0.05 as breakthrough point；
(2) after end of the sample, chromatographic column is rinsed with the flow velocity of 1BV/h with deionized water, is in charge of collection eluent, measures the electricity of water Conductance, when conductivity is down to it is suitable with deionized water when, it is successively big to being adsorbed on using 40%, 50%, 60%, 70% ethyl alcohol Polypeptide freeze-dried powder progress gradient elution on macroporous adsorbent resin, each gradient elution 3BV, 70% ethanol eluate of collection, 45 DEG C Vacuum concentrated by rotary evaporation is freeze-dried to obtain F70 crude product to no alcohol taste.
Step 3, gel filtration chromatography purifying
F70 crude product is made into the sample solution that concentration is 50mg/mL, uses column dimension for the Sephadex G- of 1.6 × 100cm 15 gel filtration chromatographies are further isolated and purified again, applied sample amount 1mL, and eluent is deionized water, flow velocity 15mL/h, inspection Wavelength 220nm is surveyed, the corresponding elution fraction of the highest chromatographic peak of absorbance in sample solution is collected according to chromatogram, freezing is dry It is dry, obtain polypeptide F70.
2. polypeptide according to claim 1, it is characterised in that：The pot marigold seed is the kind of compositae plant pot marigold Son is cleaned and is dried, is ground into 80 mesh fine powders.
3. polypeptide according to claim 1, it is characterised in that：Alkali protease Alcalase2.4L enzyme activity is 2.4AU/ g。
4. polypeptide according to claim 1, it is characterised in that：The enzyme activity of trypsase is 2500U/g.
5. polypeptide according to claim 1, it is characterised in that：Pot marigold seed is through alkali protease Alcalase2.4L (2.4AU/g)+trypsase (2500U/g) is in compound proportion 3:1, pH 7.4, temperature 60 C, substrate mass concentration 40g/L, It is digested under conditions of enzyme concentration 6%, enzymolysis time 6h.
6. polypeptide according to claim 1, it is characterised in that：90 DEG C of holding 20min enzyme deactivations are warming up to after enzymatic hydrolysis.
7. polypeptide according to claim 1, it is characterised in that：The bag filter external water of 7000Da will be less than containing molecular weight Solution is concentrated under reduced pressure in 55 DEG C of rotary evaporations.
8. application of the polypeptide described in claim 1 in terms of promoting umbilical cord mesenchymal stem cells proliferation.
CN201810624684.0A 2018-06-16 2018-06-16 A kind of polypeptide and the biological use for improving umbilical cord mesenchymal stem cells amplification efficiency Withdrawn CN108823271A (en)

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