CN108752419A - 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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CN108752419A
CN108752419A CN201810624685.5A CN201810624685A CN108752419A CN 108752419 A CN108752419 A CN 108752419A CN 201810624685 A CN201810624685 A CN 201810624685A CN 108752419 A CN108752419 A CN 108752419A
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polypeptide
freeze
stem cells
mesenchymal stem
umbilical cord
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谢崇林
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 will be less than aqueous solution concentration, freeze-drying outside the bag filter of 7000Da containing molecular weight, much peptide freeze-dried powder；2, purified to polypeptide freeze-dried powder initial gross separation with DA201-C types macroreticular resin, successively with 40%, 50%, 60%, 70% ethanol gradient elution, each gradient elution 3BV collects each gradient eluent, concentrates, 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 technology
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 Include the potential of the differentiation and developments such as tendon, ligament, liver cell, cardiac muscle cell and marrow stromal cell.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 of mesenchymal stem cell substitutes source.HUCMSCs, which is one kind as mesenchymal stem cell, has self more New and multi-lineage potential adult stem cell.Theoretically, under certain condition, hUCMSCs can be directed differentiation in body Various functions cell, form any kind of tissue and organ, that is, have " plasticity ".Also, it is dry thin with medulla mesenchyma Born of the same parents compare, and hUCMSCs has 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, and this cell is utilized so as to more efficiently obtain.
Invention content
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 enzymolysis and is gone out through alkali protease Alcalase2.4L+ trypsin digestions Enzymatic treatment obtains pot marigold Seed Storage Protein enzymolysis solution.Enzymolysis liquid is fitted into the bag filter that molecular cut off is 7000Da and is dialysed, Bag filter external water solution decompression containing molecular weight less than 7000Da is concentrated, it is then that the polypeptide solution of concentration is dry in freezing Freeze-drying is to get pot marigold seed polypeptide freeze-dried powder in dry machine.
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 types macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with absolute ethyl alcohol, then use deionization Water is cleaned standby and is used；The polypeptide of a concentration of 50mg/mL freeze-drying amidin is flowed through into chromatography with the flow velocity of 0.5BV/h under room temperature 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 velocitys 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 that is 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 are freeze-dried to obtain F40 crude products to no alcohol taste.
Step 3, gel filtration chromatography purifying
F40 crude products are made into the sample solution of a concentration of 50mg/mL, use column dimension for the Sephadex of 1.6 × 100cm G-15 gel filtration chromatographies are 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 feverfew pot marigold, cleans and dries, is ground into 80 mesh fine powders.
Preferably, alkali protease Alcalase2.4L enzyme activities are 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 proportioning 3:1, pH 7.4, temperature 60 C, substrate mass concentration 40g/L, enzyme concentration 6%, enzymolysis time 6h condition enzymes Solution.
Preferably, 90 DEG C of holding 20min enzyme deactivations are warming up to after enzymolysis.
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 enzymolysis and is gone out through alkali protease Alcalase2.4L+ trypsin digestions Enzymatic treatment obtains pot marigold Seed Storage Protein enzymolysis solution.Enzymolysis liquid is fitted into the bag filter that molecular cut off is 7000Da and is dialysed, Bag filter external water solution decompression containing molecular weight less than 7000Da is concentrated, it is then that the polypeptide solution of concentration is dry in freezing Freeze-drying is to get pot marigold seed polypeptide freeze-dried powder in dry machine.
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 types macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with absolute ethyl alcohol, then use deionization Water is cleaned standby and is used；The polypeptide of a concentration of 50mg/mL freeze-drying amidin is flowed through into chromatography with the flow velocity of 0.5BV/h under room temperature 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 use 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 products.
Step 3, gel filtration chromatography purifying
F50 crude products are made into the sample solution of a concentration of 50mg/mL, use column dimension for the Sephadex of 1.6 × 100cm G-15 gel filtration chromatographies are 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 feverfew pot marigold, cleans and dries, is ground into 80 mesh fine powders.
Preferably, alkali protease Alcalase2.4L enzyme activities are 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 proportioning 3:1, pH 7.4, temperature 60 C, substrate mass concentration 40g/L, enzyme concentration 6%, enzymolysis time 6h condition enzymes Solution.
Preferably, 90 DEG C of holding 20min enzyme deactivations are warming up to after enzymolysis.
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 enzymolysis and is gone out through alkali protease Alcalase2.4L+ trypsin digestions Enzymatic treatment obtains pot marigold Seed Storage Protein enzymolysis solution.Enzymolysis liquid is fitted into the bag filter that molecular cut off is 7000Da and is dialysed, Bag filter external water solution decompression containing molecular weight less than 7000Da is concentrated, it is then that the polypeptide solution of concentration is dry in freezing Freeze-drying is to get pot marigold seed polypeptide freeze-dried powder in dry machine.
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 types macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with absolute ethyl alcohol, then use deionization Water is cleaned standby and is used；The polypeptide of a concentration of 50mg/mL freeze-drying amidin is flowed through into chromatography with the flow velocity of 0.5BV/h under room temperature 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 use 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 products to no alcohol taste.
Step 3, gel filtration chromatography purifying
F70 crude products are made into the sample solution of a concentration of 50mg/mL, use column dimension for the Sephadex of 1.6 × 100cm G-15 gel filtration chromatographies are 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 feverfew pot marigold, cleans and dries, is ground into 80 mesh fine powders.
Preferably, alkali protease Alcalase2.4L enzyme activities are 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 proportioning 3:1, pH 7.4, temperature 60 C, substrate mass concentration 40g/L, enzyme concentration 6%, enzymolysis time 6h condition enzymes Solution.
Preferably, 90 DEG C of holding 20min enzyme deactivations are warming up to after enzymolysis.
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.
Advantageous 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.
Description of the drawings
Fig. 1 is the Sephadex G-15 gel filtration chromatography figures of F40 crude products；
Fig. 2 is the Sephadex G-15 gel filtration chromatography figures of F50 crude products；
Fig. 3 is the Sephadex G-15 gel filtration chromatography figures of F60 crude products；
Fig. 4 is the Sephadex G-15 gel filtration chromatography figures of F70 crude products；
Fig. 5 is the influence that polypeptide F40, F50, F60, F70 are proliferated umbilical cord mesenchymal stem cells.
Specific implementation mode
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but the guarantor of the present 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 feverfew 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 proportioning 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 enzymolysis 20min carries out destroy the enzyme treatment and obtains pot marigold Seed Storage Protein enzymolysis solution.It is the saturating of 7000Da that enzymolysis liquid, which is packed into molecular cut off, It dialyses, aqueous solution outside the bag filter containing molecular weight less than 7000Da is concentrated under reduced pressure 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 types macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with absolute ethyl alcohol, then use deionization Water is cleaned standby and is used；The polypeptide of a concentration of 50mg/mL freeze-drying amidin is flowed through into chromatography with the flow velocity of 0.5BV/h under room temperature 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 use 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 of a concentration of 50mg/mL, use column dimension for 1.6 × The Sephadex G-15 gel filtration chromatographies of 100cm are 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, it is added containing the DMEM/F12 culture mediums that volume fraction is 10% fetal calf serum, 100U/mL penicillin, 100mg/L streptomysins, It is placed on 37 DEG C, volume fraction 5%CO2It is cultivated in incubator.The later half amounts of 3d change liquid, liquid are changed after 1 week again, hereafter every 3d Change 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 as stated above, obtains second of tissue block adherent training Foster cell.The umbilical cord mesenchymal stem cells that second of tissue cultures obtains are taken to 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 are 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 mediums of final concentration of 10 μ g/mL, control group use the 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 values A (size of absorbance and the quantity of living cells are proportional).Cell is calculated according to surveyed OD values Proliferation rate, cell proliferation rate=(experimental group OD values-blank group OD values)/(control group OD values-blank group OD values) × 100%.With Control group proliferation rate is 100%.
Two, experimental result
1, gel filtration chromatography purification result
The sample solution of a concentration of 50mg/mL is made into F40, F50, F60, F70 crude product, use column dimension for 1.6 × The Sephadex G-15 gel filtration chromatographies of 100cm are 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 groups umbilical cord mesenchymal stem cells are proliferated Rate is significantly higher (P < 0.05), polypeptide F60 group umbilical cord mesenchymal stem cells proliferation rates and control group difference unobvious (P > 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 the essentiality content of the present 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 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 through alkali protease Alcalase2.4L+ trypsin digestions, after enzymolysis heating carry out at enzyme deactivation Manage to obtain pot marigold Seed Storage Protein enzymolysis solution.Enzymolysis liquid is fitted into the bag filter that molecular cut off is 7000Da and is dialysed, will be contained There is bag filter external water solution decompression of the molecular weight less than 7000Da to concentrate, 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 types macroreticular resin：
(1) first by resin with soaked in absolute ethyl alcohol for 24 hours, be then washed till 220nm without absorption with absolute ethyl alcohol, then be washed with deionized water It is spare after net；The polypeptide of a concentration of 50mg/mL freeze-drying amidin is flowed through into chromatographic column with the flow velocity of 0.5BV/h under room temperature, is 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 1BV/h flow velocitys with deionized water, is in charge of collection eluent, measures the conductance of water Rate, when conductivity is down to it is suitable with deionized water when, the polypeptide that is adsorbed on macroporous absorbent resin is lyophilized using 40% ethyl alcohol Powder is eluted, and 3BV is eluted, and collects eluent, and 45 DEG C of vacuum concentrated by rotary evaporations are freeze-dried to obtain F40 crude products to no alcohol taste.
Step 3, gel filtration chromatography purifying
F40 crude products are made into the sample solution of a concentration of 50mg/mL, use 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 F40.
2. polypeptide according to claim 1, it is characterised in that：The pot marigold seed is the kind of feverfew 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 activities are 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 proportioning 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 enzymolysis.
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.
CN201810624685.5A 2018-06-16 2018-06-16 A kind of polypeptide and the biological use for improving umbilical cord mesenchymal stem cells amplification efficiency Withdrawn CN108752419A (en)

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Application publication date: 20181106