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 PDFInfo
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- 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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- 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
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- 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
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- 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]
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- 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]
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- 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
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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- 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
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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
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.
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Cited By (5)
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| 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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Application publication date: 20180918 |