CN110507597A - A kind of composition and preparation method thereof, application - Google Patents

A kind of composition and preparation method thereof, application Download PDF

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Publication number
CN110507597A
CN110507597A CN201910771247.6A CN201910771247A CN110507597A CN 110507597 A CN110507597 A CN 110507597A CN 201910771247 A CN201910771247 A CN 201910771247A CN 110507597 A CN110507597 A CN 110507597A
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cell
stem cell
growth factor
tissue
placenta
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杨熙
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Suzhou Nuopu Regenerative Medicine Co Ltd
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Suzhou Nuopu Regenerative Medicine Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A61K38/014—Hydrolysed proteins; Derivatives thereof from animals from connective tissue peptides, e.g. gelatin, collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2053—IL-8
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A61K8/982—Reproductive organs; Embryos, Eggs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/592—Mixtures of compounds complementing their respective functions
    • A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18

Abstract

The present invention discloses a kind of composition, including following raw material: mescenchymal stem cell active factors compound, hydrolysis intacellin, hyaluronic acid and collagen, water, auxiliary reagent；Mescenchymal stem cell active factors compound is prepared by the cell culture supernatant and cell pyrolysis liquid of mescenchymal stem cell by hyperfiltration process；Hydrolysis intacellin is made by placenta tissue by the method for de- cell and enzymic digestion；Active factors content is 0.1-50ug/ml in composition.Invention additionally discloses the preparation method and application of above-mentioned composition.Application stem cell secretion object of the embodiment of the present invention and de- cell placenta tissue reduce the anaphylactoid risk that directly may cause using stem cell, and effectively regulatory factor is all from cell secretion, has higher activity compared to the active factors of recombination.

Description

A kind of composition and preparation method thereof, application
Technical field
The present invention relates to composition and its fabricating technology field more particularly to a kind of compositions of the active factor And preparation method thereof.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSC) is one group of foreign cell group for being derived from matrix, can To organize to obtain from most of human body.They have self-renewal capacity, tissue repair, immunoregulation capability, can be to middle embryo Layer lineage, such as: fat cell, osteocyte, cartilage cell etc., additionally can to and other germ-layer lineage cells point Change, such as breaks up to epidermal cell, vascular endothelial cell.
A large amount of active constituent there are many containing in mescenchymal stem cell cultivating system, such as: stem cell factor (SCF), Fibroblast growth factor (FGF), endothelial growth factor (VEGF), epithelical cell growth factor (EGF), cell colony More than 80 active materials such as stimulating factor, interleukins (IL), collagen, hyaluronic acid.These active materials can answer Reparation and other skin problems for skin wounds, therefore extraction for active material and guarantee foot during the extraction process Enough activity are most important.
Have some active materials and its extraction technique on the market at present, be primarily present following problems:
1, commercially available much to declare to produce the product containing active factors, actually there is no additions, or are added to recombination and live Sex factor but cannot be guaranteed the activity of recombinant factor；
2, a kind of single active factors component is often added in product, but the addition of monofactor can not be sent out well The effect for waving itself, as EGF, FGF, IGF need a variety of factors to need the effect cooperateed with that could effectively play active function；
3, often lack chemical modification from the growth factor that the active factors of genetic engineering obtain, factor active is not It is enough, and it be easy to cause allergic reaction；
4, biologically active prod is easy to inactivate at normal temperature, and the pot-life is short.It can be seen that, it would be highly desirable to it generates a kind of with sufficiently high work The composition of the active factors of property and it can guarantee sufficiently high active preparation method.
Summary of the invention
The purpose of the present invention is to provide a kind of compositions and its preparation method and application of active factor；One side Face, the embodiment of the invention provides a kind of composition, the composition includes following raw material: mescenchymal stem cell active factors are multiple Close object, hydrolysis intacellin, hyaluronic acid and collagen, water, auxiliary reagent；
The mescenchymal stem cell active factors compound is split by the cell culture supernatant of mescenchymal stem cell with cell Solution liquid is prepared by hyperfiltration process；
The hydrolysis intacellin is made by placenta tissue by the method for de- cell and enzymic digestion；
Active factors content is 0.1-50ug/ml in the composition.
As the further improvement of embodiment of the present invention, the mescenchymal stem cell extract from animal, allogeneic or Self, placenta tissue, umbilical cord, marrow, adipose tissue are extracted from or.
As the further improvement of embodiment of the present invention, the hydrolysis intacellin extracts from placenta or the people of animal The placenta that allogeneic placenta, puerpera itself childbirth generate.
As the further improvement of embodiment of the present invention, the mescenchymal stem cell active factors compound includes epidermis Growth factor EGF, fibroblast growth factor FGF, keratinocyte growth factor KGF-2, platelet derived growth factor PDGF, turn Change growth factor TGF-β, vascular endothelial growth factor VEGF, hepatocyte growth factor HGF, insulin-like growth factor I GF, white Cytokine 6, interleukin 8.
As the further improvement of embodiment of the present invention, the auxiliary reagent includes glycerol, 1,3-PD, hydrolysis shell Polysaccharide, mannitol, lanolin, sorbic acid.
On the other hand, the embodiment of the invention also provides the preparation methods of the composition, comprising the following steps:
S1, it is separately cultured mescenchymal stem cell；
S2, mescenchymal stem cell cell is passed on into amplification；
S3, the composition for preparing the active factor, specifically include:
S301, when mescenchymal stem cell MSC degrees of fusion be 70-95% when, stimulate cell, collect supernatant culture solution；
S302, the supernatant medium centrifugal being collected into is removed into residual cells in culture medium, collects supernatant；Use filter membrane The ultrafiltration membrane that aperture is 50K is concentrated, and collects filtered fluid；Filtered fluid is taken to be added in the super filter tube of 3KD, culture solution is concentrated for centrifugation 10 times, sterile water washed once rear ultrafiltration, and collect culture medium concentrate, obtain concentrate A；
S303, mescenchymal stem cell PBS is cleaned 3 times, digestion counts, and takes at least 1 × 10^8, and sterile water is resuspended, ultrasound Smudge cells；
S304, the mixed liquor of smudge cells is centrifuged 10-20min, collects supernatant, removed cell fragment, use filter hole The ultrafiltration membrane of diameter 50K is concentrated, and takes filtered fluid to be added in the super filter tube of 3KD, centrifugal concentrating 10 obtains concentrate B again, will be concentrated Liquid A is 20ml with concentrate B mixing constant volume；Answering for the active factor is prepared to mixed liquor degerming in 0.22um filter Close object；
S4, preparation hydrolysis intacellin, specifically include:
S401, placenta tissue block is prepared；
Red crumbly texture under removing when separating mesenchymal stem cell and remaining placenta tissue are washed with purified water The clot for falling surface, -80 DEG C of freezings after adding DMEM to submerge, then 37 DEG C of water-bath 20min, repeatedly after purified water be added be put into It is shaken on 4 DEG C of shaking table for 24 hours, every 6h changes a deionized water, is carried out with the ammonium hydroxide of 1%Triton x-100 and 0.1% Elution 3 days, deionized water are washed tissue block 1 day；Tissue block is washed 1 day with PBS, and deionized water is washed tissue block 1 day；By tissue Block is weighed after draining and is recorded；
S402, the placenta freeze-drying tissue for preparing different-grain diameter；
Tissue after draining is placed in -80 DEG C of refrigerators for 24 hours, it is for 24 hours by the tissue freeze-drying after freezing, freeze-drying tissue is high Fast pulverizer is crushed；Sub-sieve is carried out with 100um, 70um, 4um cell sieve, separates different-grain diameter；By 100um, 70um partial size Placenta tissue carry out repeat crushing；
S403, preparation hydrolysis intacellin；
With 4um cell sieve sub-sieve, the HCl of every gram of placenta powder addition 9mL 0.1M of 4um partial size will be less than, at room temperature (26 DEG C) concussion 48h is carried out, 1500rpm, 15min are centrifuged 3 times, are taken supernatant deposit that 40ml deionized water is added every time and are mixed It is centrifuged again afterwards, obtains supernatant, with 1M NaOH tune pH to 7.4, be filtered with the filter of 0.22um, hydrolysis tire is made Disk extract.
S5, by mescenchymal stem cell active factors compound, hydrolysis intacellin, hyaluronic acid and collagen, water, Auxiliary reagent is mixed, and the composition that active factor content is 0.1-50ug/ml is prepared.
As the further improvement of embodiment of the present invention, the step S1 is separately cultured the specific step of mescenchymal stem cell Suddenly include:
Healthy donors placenta tissue is taken, is cleaned repeatedly with PBS for several times, removes the amnion on placenta surface.Fritter tissues are taken, are used Brave tooth tweezer wipes the red crumbly texture adhered to above off, retains all blood vessels, and placenta tissue is cut into 1 by physiological saline cleaning ~4mm2Tissue be homogenized block, collagenase digesting, be centrifuged digestive juice 10min, cell precipitation serum-free is without phenol red mesenchyma Stem cell special culture media culture is 37 DEG C in temperature, CO2It is cultivated in the incubator that volume fraction is 5%.
As the further improvement of embodiment of the present invention, the step S2 expands the passage of mescenchymal stem cell cell Specific steps include:
It is passed on when cell confluency rate is up to 70%, 0.05% pancreatin after preheating is added, gently revolving coverage method bottom of bottle, Set 3~5min of digestion in 37 DEG C of incubators；Microscopically observation after digestion 3min；Most cells are rounded, and part cell is Digestion is terminated when through suspending；
It is sucked out after cell suspension and cleans culture bottle with DMEM, after being centrifuged 10min, supernatant is sucked out, it is heavy that cell is resuspended in culture medium It forms sediment, counts, 3000-6000 cell/cm2 density is inoculated in T75 culture bottle, and the is carried out when P1 cell confluency rate is up to 70% Secondary passage operation, 3000-6000 cell/cm2Density is inoculated in T175 culture bottle.
As the further improvement of embodiment of the present invention, the hydrolysis intacellin takes off cell efficiency by placenta tissue Reach 99%-99.9%.
As the further improvement of embodiment of the present invention, the mescenchymal stem cell active factors compound includes epidermis Growth factor EGF, fibroblast growth factor FGF, keratinocyte growth factor KGF-2, platelet derived growth factor PDGF, turn Change growth factor TGF-β, vascular endothelial growth factor VEGF, hepatocyte growth factor HGF, insulin-like growth factor I GF, white Cytokine 6, interleukin 8.
As the further improvement of embodiment of the present invention, the auxiliary reagent includes glycerol, 1,3-PD, hydrolysis shell Polysaccharide, mannitol, lanolin, sorbic acid.
In another aspect, the embodiment of the invention also provides above-mentioned compositions in the drug and/or preparation for preparing wound repair In application.
Compared with prior art, the present invention has following technical effect that
1, the embodiment of the present invention is secreted with mescenchymal stem cell active factors compound, hydrolysis intacellin, transparent Matter acid and collagen are the composition of principle active component, have the function of adjusting skin activity, collocation hydrolysis placenta extracts Object provides nutrition for skin, plays comprehensive coordinative role in the drug that skin restores；
2, the embodiment of the present invention utilizes human stem cell, has powerful regeneration, repairs, differentiation capability, can be obviously promoted and decline The reparative regeneration of old cell, mescenchymal stem cell secretion possess more and content bioactive substance, active factors EGF cooperates the synergistic effect such as FGF and KGF, can support the reparation and proliferation of damaged cell, provide regeneration and cell Proliferation Microenvironment, help the proliferation of dermal fibroblast, promote collage synthesis；
3, stem cell secretion object and de- cell placenta tissue are applied in the embodiment of the present invention, are reduced and are directly used stem cell The anaphylactoid risk that may cause, and effectively regulatory factor is all from cell secretion, for the active factors of recombination With higher activity；
5, the component that the composite reactive factor is used in the embodiment of the present invention is come compared to single or relatively single active factors Synergistic effect can more be played by saying.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is embodiment eluting tissue HE colored graph；
Fig. 2 is 7 days growth curve charts of embodiment fibroblast.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, implement below in conjunction with the present invention Attached drawing in example, technical scheme in the embodiment of the invention is clearly and completely described.Obviously, described embodiment Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field Art personnel all other embodiment obtained without creative labor belongs to the model that the present invention protects It encloses.
Embodiment one
The embodiment of the invention provides a kind of composition, composition includes following raw material: mescenchymal stem cell active factors Compound, hydrolysis intacellin, hyaluronic acid and collagen, water, auxiliary reagent；
Mescenchymal stem cell active factors compound by mescenchymal stem cell cell culture supernatant and cell pyrolysis liquid It is prepared by hyperfiltration process；
Hydrolysis intacellin is made by placenta tissue by the method for de- cell and enzymic digestion；
Active factors content is 0.1-50ug/ml in composition.
Wherein, mescenchymal stem cell extracts from animal, allogeneic or self, or extracts from placenta tissue, umbilical cord, bone Marrow, adipose tissue；Human umbilical cord mesenchymal stem cells, fat mesenchymal stem cell, mesenchymal stem cell, tire can be selected Disk mescenchymal stem cell, menstrual blood mescenchymal stem cell, dental pulp mescenchymal stem cell.
And hydrolyze the tire that intacellin extracts from the placenta of animal or the allogeneic placenta of people, puerpera itself childbirth generate Disk.
Specifically, mescenchymal stem cell active factors compound includes epidermal growth factor EGF, Desmocyte growth factor Sub- FGF, keratinocyte growth factor KGF-2, platelet derived growth factor PDGF, transforming growth factor TGF-β, vascular endothelial growth Factor Ⅴ EGF, hepatocyte growth factor HGF, insulin-like growth factor I GF, interleukin-6, interleukin 8.
In embodiments of the present invention, active factors complex content measurement result in the present embodiment is measured using Elisa method, It is as shown in Table 1:
The various active factors contents of table one
In embodiments of the present invention, auxiliary reagent includes glycerol, 1,3-PD, hydrolysis chitin, mannitol, wool Rouge, sorbic acid.
Embodiment two
The embodiment of the invention also provides the preparation methods of the composition, comprising the following steps:
S1, it is separately cultured mescenchymal stem cell；
S2, mescenchymal stem cell cell is passed on into amplification；
S3, the composition for preparing the active factor, specifically include:
S301, when mescenchymal stem cell MSC degrees of fusion be 70-95% when, stimulate cell, collect supernatant culture solution；
S302, the supernatant medium centrifugal being collected into is removed into residual cells in culture medium, collects supernatant；Use filter membrane The ultrafiltration membrane that aperture is 50K is concentrated, and collects filtered fluid；Filtered fluid is taken to be added in the super filter tube of 3KD, culture solution is concentrated for centrifugation 10 times, sterile water washed once rear ultrafiltration, and collect culture medium concentrate, obtain concentrate A；
S303, mescenchymal stem cell PBS is cleaned 3 times, digestion counts, and takes at least 1 × 10^8, and sterile water is resuspended, ultrasound Smudge cells；
S304, the mixed liquor of smudge cells is centrifuged 20min, collects supernatant, removed cell fragment, use filter sizes The ultrafiltration membrane of 50K is concentrated, and filtered fluid is taken to be added in the super filter tube of 3KD, and centrifugal concentrating 10 obtains to be concentrated for concentrate B again Liquid A and concentrate B mixing constant volume 20ml；The compound of the active factor is prepared to mixed liquor degerming in 0.22um filter Object；
S4, preparation hydrolysis intacellin, specifically include:
S401, placenta tissue block is prepared；
Red crumbly texture under removing when separating mesenchymal stem cell and remaining placenta tissue are washed with purified water The clot for falling surface, -80 DEG C of freezings after adding DMEM to submerge, then 37 DEG C of water-bath 20min, repeatedly after purified water be added be put into It is shaken on 4 DEG C of shaking table for 24 hours, every 6h changes a deionized water, is carried out with the ammonium hydroxide of 1%Triton x-100 and 0.1% Elution 3 days, deionized water are washed tissue block 1 day；Tissue block is washed 1 day with PBS, and deionized water is washed tissue block 1 day；By tissue Block is weighed after draining and is recorded；
S402, the placenta freeze-drying tissue for preparing different-grain diameter；
Tissue after draining is placed in -80 DEG C of refrigerators for 24 hours, it is for 24 hours by the tissue freeze-drying after freezing, freeze-drying tissue is high Fast pulverizer is crushed；Sub-sieve is carried out with 100um, 70um, 4um cell sieve, separates different-grain diameter；By 100um, 70um partial size Placenta tissue carry out repeat crushing；
S403, preparation hydrolysis intacellin；
With 4um cell sieve sub-sieve, the HCl of every gram of placenta powder addition 9mL 0.1M of 4um partial size will be less than, at room temperature (26 DEG C) concussion 48h is carried out, 1500rpm, 15min are centrifuged 3 times, are taken supernatant deposit that 40ml deionized water is added every time and are mixed It is centrifuged again afterwards, obtains supernatant, with 1M NaOH tune pH to 7.4, be filtered with the filter of 0.22um, hydrolysis tire is made Disk extract.
S5, by mescenchymal stem cell active factors compound, hydrolysis intacellin, hyaluronic acid and collagen, water, Auxiliary reagent is mixed, and the composition that active factor content is 0.1-50ug/ml is prepared.
Wherein, step S1 is separately cultured the specific steps of mescenchymal stem cell and includes:
Healthy donors placenta tissue is taken, is cleaned repeatedly with PBS for several times, removes the amnion on placenta surface.Fritter tissues are taken, are used Brave tooth tweezer wipes the red crumbly texture adhered to above off, retains all blood vessels, and placenta tissue is cut into 1 by physiological saline cleaning ~4mm2Tissue be homogenized block, collagenase digesting, be centrifuged digestive juice 10min, cell precipitation serum-free is without phenol red mesenchyma Stem cell special culture media culture is 37 DEG C in temperature, CO2It is cultivated in the incubator that volume fraction is 5%.
Further, the specific steps of mescenchymal stem cell cell passage amplification are included: by step S2
It is passed on when cell confluency rate is up to 70%, 0.05% pancreatin after preheating is added, gently revolving coverage method bottom of bottle, Set 3~5min of digestion in 37 DEG C of incubators；Microscopically observation after digestion 3min；Most cells are rounded, and part cell is Digestion is terminated when through suspending；
It is sucked out after cell suspension and cleans culture bottle with DMEM, after being centrifuged 10min, supernatant is sucked out, it is heavy that cell is resuspended in culture medium It forms sediment, counts, 3000-6000 cell/cm2 density is inoculated in T75 culture bottle, and the is carried out when P1 cell confluency rate is up to 70% Secondary passage operation, 3000-6000 cell/cm2Density is inoculated in T175 culture bottle.
In embodiments of the present invention, hydrolysis intacellin takes off cell efficiency by placenta tissue and reaches 99%-99.9%.
Preferably, mescenchymal stem cell active factors compound includes epidermal growth factor EGF, Desmocyte growth factor Sub- FGF, keratinocyte growth factor KGF-2, platelet derived growth factor PDGF, transforming growth factor TGF-β, vascular endothelial growth Factor Ⅴ EGF, hepatocyte growth factor HGF, insulin-like growth factor I GF, interleukin-6, interleukin 8.
Wherein, auxiliary reagent includes glycerol, 1,3-PD, hydrolysis chitin, mannitol, lanolin, sorbic acid.
After step s4, DNA is extracted with Tiangeng DP304 kit and with trace dna analyzer to measure 100g placenta new DNA content in fresh tissue is about 1789.2ng/mg, the 9.1g freeze-dried powder DNA content average out to 44.6ng/mg after taking off cell, De- cell efficiency is 99.8%；HE stained slice result is carried out to the tissue after de- cell as shown in Figure 1, without obviously may be used in figure The nucleus distribution seen illustrates that de- cell effect reaches requirement, effectively removes anaphylactogen.
Embodiment three
The embodiment of the invention also provides application of the above-mentioned composition in the drug and/or preparation of preparation wound repair.
The influence that composition prepared by the present invention grows dermal fibroblasts is tested, experimental design is as follows:
The hole 1 × 10^4/ of skin fibroblasts is inoculated in six orifice plates, and on six orifice plates, A is arranged: stem cell is living 3 plate of property ingredient compound experimental group；B: 3 plate of culture medium negative control group；It is inoculated with 7 six orifice plates；Stem cell prepared by embodiment 1 Active constituent compound is added in culture medium in 1: 1 ratio, and negative control group adds equivalent DMEM；The culture medium is 90% DMEM+10% fetal calf serum；It takes within second day one piece of six orifice plate by cell dissociation, counts, take three hole average values, it is later same daily One time measured a plate, recorded 7 days growth datas, as a result as shown in Figure 2；
The results show that the fibroblast of addition Stem Cell Activity ingredient compound experimental group culture medium compares negative control group Fibroblast proliferation faster, Stem Cell Activity ingredient compound can promote fibroblastic growth.
Further, the composition prepared by the embodiment of the present invention 2 carries out skin comparative test, and 30 subject's skin are equal There are the impaired problems such as different degrees of small pox, red capillary, are randomly divided into two groups, wherein 15 subjects are A group, apply daily morning and evening The composition finished product is smeared, in addition 15 subjects are B group, and daily smearing is smeared compound not comprising mescenchymal stem cell active factors The composition of the negative control group of object, hydrolysis intacellin, fills in trial report after January, score by resultant effect, and score value is 0-10 points, worse without effect or skin quality is 0-3 points, and unobvious effect is 4-6 points, and significant effect is 7-10 points, as a result such as table It is more satisfied to repairing effect using the subject of the present embodiment composition shown in two；Test result is as shown in Table 2.
The composition and negative control set product of two embodiment 2 of table preparation carry out skin comparative test
Subject's grouping Comprehensive score average value As a result
A group 4.2 Effect is unobvious
B group 7.7 Significant effect
Compared with prior art, the present invention has following technical effect that
1, the embodiment of the present invention is secreted with mescenchymal stem cell active factors compound, hydrolysis intacellin, transparent Matter acid and collagen are the composition of principle active component, have the function of adjusting skin activity, collocation hydrolysis placenta extracts Object provides nutrition for skin, plays comprehensive coordinative role in the drug that skin restores；
2, the embodiment of the present invention utilizes human stem cell, has powerful regeneration, repairs, differentiation capability, can be obviously promoted and decline The reparative regeneration of old cell, mescenchymal stem cell secretion possess more and content bioactive substance, active factors EGF cooperates the synergistic effect such as FGF and KGF, can support the reparation and proliferation of damaged cell, provide regeneration and cell Proliferation Microenvironment, help the proliferation of dermal fibroblast, promote collage synthesis；
3, stem cell secretion object and de- cell placenta tissue are applied in the embodiment of the present invention, are reduced and are directly used stem cell The anaphylactoid risk that may cause, and effectively regulatory factor is all from cell secretion, for the active factors of recombination With higher activity；
5, the component that the composite reactive factor is used in the embodiment of the present invention is come compared to single or relatively single active factors Synergistic effect can more be played by saying.
In the description of above embodiment, particular features, structures, materials, or characteristics can be at any one or more It can be combined in any suitable manner in a embodiment or example.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.

Claims (10)

1. a kind of composition, which is characterized in that the composition includes following raw material: mescenchymal stem cell active factors are compound Object, hydrolysis intacellin, hyaluronic acid and collagen, water, auxiliary reagent；
The mescenchymal stem cell active factors compound by mescenchymal stem cell cell culture supernatant and cell pyrolysis liquid It is prepared by hyperfiltration process；
The hydrolysis intacellin is made by placenta tissue by the method for de- cell and enzymic digestion；
Active factors content is 0.1-50ug/ml in the composition.
2. composition according to claim 1, which is characterized in that the mescenchymal stem cell extracts from animal, of the same race different Body is self, or extracts from placenta tissue, umbilical cord, marrow, adipose tissue.
3. composition according to claim 1, which is characterized in that the hydrolysis intacellin extracts from the placenta of animal Or the placenta that the allogeneic placenta of people, puerpera itself childbirth generate.
4. composition according to claim 1, which is characterized in that the mescenchymal stem cell active factors compound includes Epidermal growth factor EGF, fibroblast growth factor FGF, keratinocyte growth factor KGF-2, platelet derived growth factor PDGF, transforming growth factor TGF-β, vascular endothelial growth factor VEGF, hepatocyte growth factor HGF, insulin-like growth factor Sub- IGF, interleukin-6, interleukin 8.
5. composition according to claim 1, which is characterized in that the auxiliary reagent includes glycerol, 1,3-PD, water Solve chitin, mannitol, lanolin, sorbic acid.
6. a kind of preparation method of composition, which is characterized in that the preparation method comprises the following steps:
S1, it is separately cultured mescenchymal stem cell；
S2, mescenchymal stem cell cell is passed on into amplification；
S3, the composition for preparing the active factor, specifically include:
S301, when mescenchymal stem cell MSC degrees of fusion be 70-95% when, stimulate cell, collect supernatant culture solution；
S302, the supernatant medium centrifugal being collected into is removed into residual cells in culture medium, collects supernatant；Use filter sizes It is concentrated for the ultrafiltration membrane of 50K, collects filtered fluid；Filtered fluid is taken to be added in the super filter tube of 3KD, culture solution is concentrated 10 by centrifugation Times, sterile water washed once rear ultrafiltration, and collect culture medium concentrate, obtain concentrate A；
S303, mescenchymal stem cell is cleaned 3 times with PBS, digestion counts, and takes at least 1 × 10^8, and sterile water is resuspended, and ultrasound is broken Chopping fine born of the same parents；
S304, the mixed liquor of smudge cells is centrifuged 10-20min, removes cell fragment, the ultrafiltration membrane with filter sizes 50K is dense Contracting, takes filtered fluid to be added in the super filter tube of 3KD, 10 times of centrifugal concentrating, obtains concentrate B；Concentrate A and concentrate B are mixed It closes, the compound of the active factor is prepared to mixed liquor degerming in constant volume 20ml, 0.22um filter；
S4, preparation hydrolysis intacellin, specifically include:
S401, placenta tissue block is prepared；
Red crumbly texture under removing when separating mesenchymal stem cell and remaining placenta tissue purified water flushing are removed into table The clot in face, -80 DEG C of freezings after adding DMEM to submerge, then 37 DEG C of water-bath 20min, repeatedly after purified water be added be put into 4 DEG C Shaking table on shake for 24 hours, every 6h changes a deionized water, is eluted with the ammonium hydroxide of 1%Triton x-100 and 0.1% 3 days, deionized water was washed tissue block 1 day；Tissue block is washed 1 day with PBS, and deionized water is washed tissue block 1 day；Tissue block is dripped It weighs and records after dry；
S402, the placenta freeze-drying tissue for preparing different-grain diameter；
Tissue after draining is placed in -80 DEG C of refrigerators for 24 hours, for 24 hours by the tissue freeze-drying after freezing, tissue high speed powder will be lyophilized Broken machine is crushed；Sub-sieve is carried out with 100um, 70um, 4um cell sieve, separates different-grain diameter；By the tire of 100um, 70um partial size Disk tissue carries out repeating crushing；
S403, preparation hydrolysis intacellin；
With 4um cell sieve sub-sieve, the HCl of every gram of placenta powder addition 9mL 0.1M of 4um partial size will be less than, at room temperature (26 DEG C) into Row concussion 48h, 1500rpm, 15min centrifugation 3 times, take supernatant deposit to be added after 40ml deionized water mixes again every time Centrifugation, obtains supernatant, with 1M NaOH tune pH to 7.4, is filtered with the filter of 0.22um, and hydrolysis placenta is made and extracts Object.
S5, by mescenchymal stem cell active factors compound, hydrolysis intacellin, hyaluronic acid and collagen, water, auxiliary Reagent is mixed, and the composition that active factor content is 0.1-50ug/ml is prepared.
7. the preparation method of composition according to claim 6, which is characterized in that the step S1 is separately cultured mesenchyma The specific steps of stem cell include:
Healthy donors placenta tissue is taken, is cleaned repeatedly with PBS for several times, removes the amnion on placenta surface.Fritter tissues are taken, with brave tooth Tweezer wipes the red crumbly texture adhered to above off, retains all blood vessels, physiological saline cleaning, placenta tissue is cut into 1~ 4mm2Tissue be homogenized block, collagenase digesting is centrifuged digestive juice 10min, and cell precipitation serum-free is dry without phenol red mesenchyma Cell special culture media culture is 37 DEG C in temperature, CO2It is cultivated in the incubator that volume fraction is 5%.
8. the preparation method of composition according to claim 6, which is characterized in that the step S2 is by mescenchymal stem cell Cell passes on the specific steps expanded
It is passed on when cell confluency rate is up to 70%, 0.05% pancreatin after preheating is added, gently revolving coverage method bottom of bottle, sets 37 3~5min is digested in DEG C incubator；Microscopically observation after digestion 3min；Most cells are rounded, and part cell has hanged Digestion is terminated when floating；
It is sucked out after cell suspension and cleans culture bottle with DMEM, after being centrifuged 10min, supernatant is sucked out, cell precipitation, meter is resuspended in culture medium Number, 3000-6000 cell/cm2 density is inoculated in T75 culture bottle, carries out for the second time when P1 cell confluency rate is up to 70% Passage operation, 3000-6000 cell/cm2Density is inoculated in T175 culture bottle.
9. the preparation method of composition according to claim 6, which is characterized in that the hydrolysis intacellin is by placenta De- cell efficiency is organized to reach 99%-99.9%；
The mescenchymal stem cell active factors compound include epidermal growth factor EGF, fibroblast growth factor FGF, Keratinocyte growth factor KGF-2, platelet derived growth factor PDGF, transforming growth factor TGF-β, vascular endothelial growth factor VEGF, hepatocyte growth factor HGF, insulin-like growth factor I GF, interleukin-6, interleukin 8；
The auxiliary reagent includes glycerol, 1,3-PD, hydrolysis chitin, mannitol, lanolin, sorbic acid.
10. the answering in the drug and/or preparation of preparation wound repair of composition described in -5 any one according to claim 1 With.
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