CN108543064A - A kind of quick reparation liquid and preparation method thereof for burn and scald - Google Patents

A kind of quick reparation liquid and preparation method thereof for burn and scald Download PDF

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Publication number
CN108543064A
CN108543064A CN201810439269.8A CN201810439269A CN108543064A CN 108543064 A CN108543064 A CN 108543064A CN 201810439269 A CN201810439269 A CN 201810439269A CN 108543064 A CN108543064 A CN 108543064A
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Prior art keywords
scald
burn
stem cell
cell
growth factor
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CN201810439269.8A
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Chinese (zh)
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吴金芸
叶华衍
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Bio Tech Development (yancheng) Co Ltd
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Bio Tech Development (yancheng) Co Ltd
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Priority to CN201810439269.8A priority Critical patent/CN108543064A/en
Publication of CN108543064A publication Critical patent/CN108543064A/en
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • A61K38/1808Epidermal growth factor [EGF] urogastrone
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • A61K38/1825Fibroblast growth factor [FGF]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • A61K38/1858Platelet-derived growth factor [PDGF]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0014Skin, i.e. galenical aspects of topical compositions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • A61P17/02Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

Abstract

The quick reparation liquid and preparation method thereof that the present invention relates to a kind of for burn and scald.The stem cell obtained using conventional separation, through molecule clone technology " overexpression " people's fibronectin(Fibronectin, FN), using the stem cell culture supernatant of cell factor and overexpression FN rich in various autocrines, it is applied to the repairing and treating of burned patient and obstinate ulcer wound surface.The inventive method is simple and easy to do, is suitable for the large-scale production of the stem cell culture supernatant of separate sources.And by such products application in the repairing and treating of burned patient and obstinate ulcer wound surface.It is applied to medicine conversion cause for stem cell and one new path is provided.

Description

A kind of quick reparation liquid and preparation method thereof for burn and scald
Technical field
The invention belongs to biotechnologies, are related to a kind of quick reparation liquid and preparation method thereof for burn and scald.
Background technology
Studies have reported that after severe trauma, large-area burns, orthopedic the surface of a wound covering have become it is one very heavy The problem of wanting, other than autologous patient is made skin graft, people using allograft of skin and other associated restoration materials cover The surface of a wound, but no matter the transplanting of self or heterogenous skin is limited by donor source and immunological rejection.Urgently find one Kind is preferably without rejection, the natural drug for promoting skin injury reparation, promoting skin regeneration.
Stem cell has unique differentiation potential, can cure the wound that tissue is difficult to self-healing, can treat and clinically be difficult to control More disease, stem cell are a kind of early stage neoblasts with self-replacation and more differentiation potentials, and medical field is referred to as For " general-purpose cell ".Stem cell has various biological characteristics, most importantly has self-renewing and Multidirectional Differentiation Potential and unlimited multiplication capacity.Stem cell also has the ability of secretion cytokine profiles, human epidermal growth factor (EGF) , human horny cell growth factor-2(KGF), human fibroblastic growth factor(FGF), human blood platelets sample derivative growth factor (PDGF), fibronectin(FN)Deng.
Mescenchymal stem cell (mesenchymal stem cells, MSC) is one group of foreign cell group for being derived from matrix, It can organize to obtain from most of human body.Lot of experiments shows that mescenchymal stem cell has epidermal cell differentiation potential, can Promote the healing of skin of being wound.The hidden danger that bone marrow derived mescenchymal stem cell is polluted due to there is virus, and with the donor age Increase, cell quantity and amplification, differentiation capability occur being decreased obviously trend, are not suitable for batch and prepare.And epidermal stem cells are It is present in a kind of skin progenitor cell in the gap between basal layer of epidermis and hair follicle root, the specificity as skin histology is dry thin Born of the same parents can not only maintain the chief functional cells of skin metabolism, also be closely related with the reparation of the surface of a wound, in surface of a wound environment It is divided into keratinocyte, is transferred to skin wound and repairing skin wound surfaces.
Invention content
To solve the above problems, the present invention provides a kind of quick reparation liquid and preparation method thereof for burn and scald, this is fast Speed repairs liquid energy and enough promotes epidermal growth, shortens the Wound healing time.The stem cell obtained using conventional separation, through molecule Clone technology " overexpression " people's fibronectin(Fibronectin, FN).Using rich in various autocrines cell factor and It is overexpressed the stem cell culture supernatant of FN, is applied to the repairing and treating of burned patient and obstinate ulcer wound surface.
In order to achieve the above objects and other related objects, the present invention provides a kind of quick reparation liquid for burn and scald, packet It includes and derives containing epithelical cell growth factor, human horny cell growth factor-2, human fibroblastic growth factor, human blood platelets sample The culture supernatant of growth factor and fibronectin stem cell.
The a concentration of of culture supernatant of the present invention is greater than or equal to 1 × 1010A/ml.
The present invention provides a kind of quick liquid of repairing for burn and scald in burned patient and obstinate ulcer wound surface Application in repairing and treating.
The present invention provide it is a kind of for burn and scald it is quick repair liquid in traumatic surface, prevent answering in the formation of scar With.
The present invention provides a kind of quick preparation method for repairing liquid for burn and scald, includes the following steps:
(1), the separation of conventional mescenchymal stem cell, obtains mescenchymal stem cell;
(2), using molecule clone technology, (1) mescenchymal stem cell that fibronectin channel genes step obtains was carried out Expression;Empirically (1) the middle umbilical cord mesenchymal stem cells obtained are transfected room conventional steps;Next day renews and fresh contains 800 μ g/ The opti-MEM of mlG418 changes liquid, continues culture one week;
(3) cells and supernatant, is collected, and detects the expression of fibronectin in its culture supernatant;Including following step Suddenly:
1. after cell amplification was passaged to for about 20 generations, cell quantity >=1010
2. changing fresh serum-free stem cell media into, continue culture 3 days;
3. collecting cells and supernatant, mixing;
4. sample is taken to measure the expression of FN in supernatant for ELISA detection kit;
5. according to testing result, connecting protein 10 μ g/ every for fibre-bearing by remaining sample is aseptic subpackaged, and it is dry to carry out freezing It is dry, as finished product.
In said program, related content is explained as follows:
Fibronectin(FN):A large amount of studies and clinical application is it was demonstrated that FN plays ten in the reparation and healing of wound Divide important role, is the key substance for promoting wound healing;Foreign countries have been carried out human plasma FN burn, wound, septicemia, The application study of infection etc..Nishida is proved by zoopery:By FN be coated onto rat skin wound surface its healing Speed is obviously accelerated.To the patient with auxotype ulcer of the cornea, patient is treated in the form of medicament for the eyes, after medication 2 days, table Skin just starts a large amount of hyperplasia.After three weeks, ulcer completely disappears, and does not generate scar and side effect.FN, Ke Yiyu have been applied to ulcer Wound collagen combines and forms FN protective layers, not only improves migration and the proliferation of epithelial cell to repair wound, and prevent germ Infection.The result of study of the units such as Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences also turns out:FN and some media are coordinated As external-applied ointment treatment by mechanical trauma and burn, wound healing time reduces 1/3-1/2 than control group, no to infect, without scar It is trace, without any side effects.
Epithelical cell growth factor(EGF):The study found that EGF can promote epithelial cell, fibroblastic proliferation;Increase The vigor of strong epidermal cell;The aging for delaying epidermal cell makes each composition of skin keep best physiological status;Stimulate cell The synthesis of some outer macromoleculars (such as hyaluronic acid and collagen are white) and secretion, skin care, are to determine skin vigor and health Source.
Human blood platelets sample derivative growth factor(PDGF):Platelet derived growth factor PDGF is the rising star of removing wrinkle and resisting aging, The fibroblast method for acting on skin corium reaches fine anti-ageing wrinkle improvement effect.The impaired epithelial cell of induction and endothelium are thin Born of the same parents' division growth;The formation and regeneration for promoting blood vessel, guarantee is provided for wound repair.There are obvious curative effects for anti-aging;Energy Stimulated vascular smooth muscle cell, fibroblast, spongiocyte division and proliferation;Myofibroblast can be promoted to generate glue Original, especially I types and III Collagen Type VI;The growth and update for promoting Skin Cell, promoted the physiological activity of skin histology with it is newly old Metaboilic level enhances the immunity and self-repairing capability of Skin Cell.
Human fibroblastic growth factor(FGF):For the fresh surface of a wound caused by daily friction, scratch, scald, tumble injury etc. Quickly repair;Rubefaction caused by solar radiation and abuse low value cosmetics and environmental pollution, take off skin, be thinning, is sensitive etc. it is fast Speed is repaired;Skin early ageing, sallow, wrinkle, relaxation etc. are quickly repaired;Pachylosis, climate change make dry skin, skin ash The reparations such as yellow, dark and gloomy unglazed, furfur.
Human horny cell growth factor-2(KGF):The physiology courses such as the metabolism of epithelial cell can be specifically stimulated, including thin Regeneration, differentiation and migration of born of the same parents etc.;Tissue specificity is strong, stablizes, and safety is good.KGF acts only on epithelial cell, passes through its spy Specific receptor can start epithelial cell and be fed back to the coordination signal of subcutaneous interstitial tissue while stimulating epithelial cell growth, Promote neoblastic formation, safety is good, without potential side effect;There is stimulating growth point to newborn or aging epithelial cell Split effect;Have the function of significantly removing scar and Antiradiation injury.
The stem cell that the present invention is obtained using conventional separation, through molecule clone technology " overexpression " people's fibronectin (FN).Using the stem cell culture supernatant of cell factor and overexpression FN rich in various autocrines, it is applied to burn and scald and suffers from The repairing and treating of person and obstinate ulcer wound surface.It is related to a kind of quick reparation liquid can be used for burn and scald.The invention is easy easily Row is suitable for the large-scale production of the stem cell culture supernatant of separate sources, prepares " the quick reparative factor of burn and scald ".And it should Class products application is in the repairing and treating of burned patient and obstinate ulcer wound surface.It is applied to medicine for stem cell and converts cause One new path is provided.
Specific implementation mode
The present invention is further described below by way of specific implementation mode, but the present invention is not limited only to following embodiment.
Embodiment:
A kind of quick reparation liquid for burn and scald, including contain epithelical cell growth factor, human horny cell growth factor-2, people The culture supernatant of fibroblast growth factor, human blood platelets sample derivative growth factor and fibronectin stem cell.
The a concentration of of the culture supernatant is greater than or equal to 1 × 1010A/ml.
A kind of quick reparation liquid for burn and scald is in the repairing and treating of burned patient and obstinate ulcer wound surface Application.
It is a kind of for burn and scald it is quick repair liquid in traumatic surface, prevent application in the formation of scar.
A kind of quick preparation method for repairing liquid for burn and scald includes the following steps:
(1), the separation of conventional mescenchymal stem cell, obtains mescenchymal stem cell;
By taking human umbilical mesenchymal cell detaches as an example, human umbilical cord mesenchymal stem cells are prepared using improvement tissue block adherent method.
(2), using molecule clone technology, (1) mescenchymal stem cell that fibronectin channel genes step is obtained into Row is overexpressed;Commission structure hFN-PUC57 eukaryon expression plasmids;Structure is wrapped up by liposome Lipofectamine 20000 The hFN-PUC57 eukaryon expression plasmids built up, empirically with step, (1) the middle umbilical cord mesenchymal stem cells obtained carry out room routine Transfection;The control liposome of plasmid-free is set simultaneously;Next day renews the fresh opti-MEM containing 800 μ g/ml G418 and changes liquid, continues Culture one week;Find control liposome hole complete cell death under the microscope after a week, and transfecting hole cell has more Cheng Ke The cell mass of grand shape growth;Large amplification is routinely carried out with the culture solution without G418.
(3) cells and supernatant, is collected, and detects the expression of fibronectin in its culture supernatant;Including with Lower step:
1. after cell amplification was passaged to for about 20 generations, cell quantity >=1010
2. changing fresh serum-free stem cell media into, continue culture 3 days;
3. collecting cells and supernatant, mixing;
4. sample is taken to measure the expression of FN in supernatant for ELISA detection kit;
5. according to testing result, connecting protein 10 μ g/ every for fibre-bearing by remaining sample is aseptic subpackaged, and it is dry to carry out freezing It is dry, as finished product.
Product inspection method:
The first step:The Kunming mouse 10 for selecting 10-12 week old, scalding model foundation is carried out with electric iron in its stomach wall both sides.It answers Pay attention to striving allowing both sides scald degree consistent with area;Next day, the side culture medium of 100 μ l cultures(It is fresh, not Exposing cell)It smears, as a control group;The sterile water for injection dissolving freeze-drying finished product of 100 μ l of the other side, and smeared, As experimental group.
As a result:Experimental group was in the 4th day and started to form a scab, and started within the 7th day decrustation, fully recovered within ten days 100%;Control group the 4th Its its surface of a wound still has a little diffusate, still has within 15 days 3/10 wounds in mice still to fail to heal;
Conclusion:Can be used for " the quick reparative factor of burn and scald " the present invention relates to one kind and have effects that quickly to repair burn and scald.
Second step:Using in the first step, three mouse for failing healing, reuse the sterile note with 100 μ l after detection The freeze-drying finished product with water dissolution is penetrated, and is smeared;
As a result:Find that the surface of a wound is formed a scab, and starts decrustation, and heal after seven days after three days;
Conclusion:Can be used for " the quick reparative factor of burn and scald " the present invention relates to one kind and have effects that quickly to repair burn and scald, and And still there is repairing and treating effect to cureless ulcer wound surface.
It is described herein and claimed invention is not limited to the ranges of particular aspects disclosed here, because of these sides Face is intended as illustrations of several aspects of the invention.It is expected that any equivalent aspect is all in the scope of the present invention.In fact, removing Except those of shown here and description, different modifications of the invention are for those of ordinary skills from foregoing description It will be apparent.Such modification, which is also intended to, to be fallen within the scope of the appended claims.In case of conflict, to include fixed Subject to the present disclosure of justice.

Claims (5)

1. a kind of quick reparation liquid for burn and scald, it is characterised in that:It is thin including containing epithelical cell growth factor, people's cutin The intracellular growth factor, human fibroblastic growth factor, human blood platelets sample derivative growth factor and fibronectin stem cell Culture supernatant.
2. the quick reparation liquid according to claim 1 for burn and scald, it is characterised in that:The culture supernatant it is dense Degree is more than or equal to 1 × 1010A/ml.
3. a kind of quick reparation liquid as described in claim 1 for burn and scald is created in burned patient and obstinate ulcer Application in the repairing and treating in face.
4. it is a kind of it is as described in claim 1 for burn and scald it is quick repair liquid in traumatic surface, prevent in the formation of scar Application.
5. a kind of quick preparation method for repairing liquid for burn and scald, it is characterised in that:Include the following steps:
(1), the separation of conventional mescenchymal stem cell, obtains mescenchymal stem cell;
(2), using molecule clone technology, (1) mescenchymal stem cell that fibronectin channel genes step obtains was carried out Expression;Empirically (1) the middle umbilical cord mesenchymal stem cells obtained are transfected room conventional steps;Next day renews and fresh contains 800 μ g/ The opti-MEM of mlG418 changes liquid, continues culture one week;
(3) cells and supernatant, is collected, and detects the expression of fibronectin in its culture supernatant;Including following step Suddenly:
1. after cell amplification was passaged to for about 20 generations, cell quantity >=1010
2. changing fresh serum-free stem cell media into, continue culture 3 days;
3. collecting cells and supernatant, mixing;
4. sample is taken to measure the expression of FN in supernatant for ELISA detection kit;
5. according to testing result, connecting protein 10 μ g/ every for fibre-bearing by remaining sample is aseptic subpackaged, and it is dry to carry out freezing It is dry, as finished product.
CN201810439269.8A 2018-05-09 2018-05-09 A kind of quick reparation liquid and preparation method thereof for burn and scald Pending CN108543064A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110755453A (en) * 2019-12-05 2020-02-07 伯仕利生物科技发展(盐城)有限公司 Preparation method of freeze-dried powder rich in hEGF and used for wound skin repair
CN110878287A (en) * 2019-12-05 2020-03-13 伯仕利生物科技发展(盐城)有限公司 Preparation method and application of stem cell supernatant over-expressing TAT-KGF
CN110904049A (en) * 2019-12-05 2020-03-24 伯仕利生物科技发展(盐城)有限公司 Preparation method and application of stem cell supernatant rich in VEGF and FGF
CN111358749A (en) * 2020-05-09 2020-07-03 山东兴瑞生物科技有限公司 Composition for promoting skin wound healing and preparation method thereof
JP2020137518A (en) * 2019-02-25 2020-09-03 株式会社細胞応用技術研究所 Additive

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CN105708860A (en) * 2016-01-26 2016-06-29 深圳爱生再生医学科技有限公司 Stem cell preparation for repairing skin ulcer
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020137518A (en) * 2019-02-25 2020-09-03 株式会社細胞応用技術研究所 Additive
CN110755453A (en) * 2019-12-05 2020-02-07 伯仕利生物科技发展(盐城)有限公司 Preparation method of freeze-dried powder rich in hEGF and used for wound skin repair
CN110878287A (en) * 2019-12-05 2020-03-13 伯仕利生物科技发展(盐城)有限公司 Preparation method and application of stem cell supernatant over-expressing TAT-KGF
CN110904049A (en) * 2019-12-05 2020-03-24 伯仕利生物科技发展(盐城)有限公司 Preparation method and application of stem cell supernatant rich in VEGF and FGF
CN111358749A (en) * 2020-05-09 2020-07-03 山东兴瑞生物科技有限公司 Composition for promoting skin wound healing and preparation method thereof
CN111358749B (en) * 2020-05-09 2023-01-03 山东兴瑞生物科技有限公司 Composition for promoting skin wound healing and preparation method thereof

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Application publication date: 20180918