CN108542915A - A kind of drug and preparation method thereof promoting wound healing - Google Patents
A kind of drug and preparation method thereof promoting wound healing Download PDFInfo
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- CN108542915A CN108542915A CN201810715021.XA CN201810715021A CN108542915A CN 108542915 A CN108542915 A CN 108542915A CN 201810715021 A CN201810715021 A CN 201810715021A CN 108542915 A CN108542915 A CN 108542915A
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- 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
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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
- A61K31/716—Glucans
- A61K31/722—Chitin, chitosan
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- 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
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
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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
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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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
Abstract
The invention discloses a kind of drugs promoting wound healing, and the drug includes fat mesenchymal stem cell and chitosan fluid film.And the weight ratio of the fat mesenchymal stem cell and chitosan fluid film is 60~70:30~40.And the preparation method for the drug for promoting wound healing is also disclosed, described method includes following steps：Fat mesenchymal stem cell and chitosan fluid film are prepared respectively；Fat mesenchymal stem cell chitosan fluid film is wrapped up, is stored in liquid nitrogen cryogenics tank.Fat mesenchymal stem cell and chitosan fluid film are used in combination the present invention, can be effectively promoted the healing of wound, more preferable than fat mesenchymal stem cell effect is used alone.Drug Platelet-rich plasm in the present invention and growth factor, each component compatibility is reasonable, synergistic, collectively promotes the healing of wound；The production stage of drug is simple, process optimization, it is easy to accomplish large-scale production.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of drug and preparation method thereof promoting wound healing.
Background technology
The patient of large-area burns or diabetes seriously affects limb function and form, very often due to wound repair difficulty
To leading to death.Therefore, how efficiently quickly wound healing becomes research hotspot in recent years.Cell replacement therapy
As effective therapy for the treatment of severe trauma.Mescenchymal stem cell (mesenchymal stem cells, MSCS) comes
Derived from the mesoblastic a kind of multipotential stem cell of mesoderm growing early stage, multi-lineage potential, can secrete soluble factor and transdifferentiation promotees
Into wound healing, still there is multi-lineage potential after continuous passage culture and freezen protective.MSCS can be used as ideal cell and use
In cell replacement therapy.The main source of MSCS is Adult Human Bone Marrow at present.Due to Adult Human Bone Marrow source MSCS (bone marrow
BM-derived MSC, BMMSC) cell quantity and Proliferation, Differentiation potential declines with the increase at age, and viral infection rate is high, and
The acquisition palpus row bone marrow puncture of donor MSCS, source is restricted.Therefore find a kind of new sources MSCS, it has also become cell replaces
For the hot spot of Therapy study.
Fat stem cell (adipose-derived stem cells, ADSCs) is to be detached from adipose tissue in recent years
A kind of obtained stem cell with multi-lineage potential.Research finds that ADSCs cells can stablize proliferation and become feeble and die in vitro
Rate is low, while there are materials to be easy for it, a small amount of tissue can obtain a large amount of stem cells, suitable for large-scale culture, to body injury
The advantages that small, and it is derived from a wealth of sources, and cylinder storage amount is big, suitable for autotransplantation.
Abilities of the ADSCs with secrete cytokines, matrix, can promote the revascularization of transplant fat particle, enhance
The stabilization of the fatty granule cell of transplanting, while ADSCs carries out Proliferation, Differentiation by template of fatty granule cell.ADSCs has
The ability for breaking up lipoblast, can build up around it, is divided into be implanted into internal fatty granule cell as holder
Adipocyte and autologous fat mescenchymal stem cell and vascular endothelial cell.
Certain repair can be played by being repaired to wound face using fat mesenchymal stem cell (ADSCs), but
It is there are the situation that cell is easy to run off, to influence its repairing effect.
Invention content
For present situation, the present invention provides a kind of drug promoting wound healing, and the drug includes that fat mesenchymal is dry thin
Born of the same parents and chitosan fluid film, can reduce the loss of cell, give full play to the work that fat mesenchymal stem cell promotes wound healing
With.
The present invention also provides a kind of preparation methods of the drug of promotion wound healing, by fat mesenchymal stem cell shell
Glycan fluid film wraps up, and can effectively reduce the loss of cell.
In order to solve the above technical problem, the present invention provides following technical solutions：
A kind of drug promoting wound healing, the drug further include chitosan fluid film and isometric by being rich in blood
The solution of platelet-poor plasma and skin factor composition；Wherein, the weight of the fat mesenchymal stem cell and chitosan fluid film
Than being 60~70:30~40.
Preferably, in the solution, the volume by volume concentration of the Platelet-rich plasm is 3-6%；The epidermal growth
The working concentration of the factor is 3-8ng/ml.
Preferably, the weight ratio of the fat mesenchymal stem cell and chitosan fluid film is 60~70:30~40.
The invention also discloses the preparation methods for the drug for promoting wound healing, and described method includes following steps：
(1) fat mesenchymal stem cell and chitosan fluid film are prepared respectively；
(2) fat mesenchymal stem cell chitosan fluid film is wrapped up, is stored in liquid nitrogen cryogenics tank, the liquid nitrogen is low
The temperature of warm tank is -196 DEG C.
Wherein, the preparation method of the fat mesenchymal stem cell includes the following steps：
(1) fat lump is obtained, fat lump is cleaned up, the big blood vessel of connective tissue and finding of naked eye is cut off, then uses PBS
After buffer solution (phosphate buffer) cleans repeatedly, adipose tissue is cut into 1mm3Size is collected in 15ml centrifuge tubes, is added
A concentration of 0.1~0.5% isometric clostridiopetidase A, 37 DEG C of shaker water baths digest 60min；
(2) it is then added in isometric α-MEM culture mediums containing 10% serum, 200 mesh filter screens, which filter out, not to be digested
Tissue block, 1000rpm centrifuges 10 minutes, abandons supernatant, PBS buffer solution is rinsed 3 times, and precipitation is resuspended in containing 10% newborn bovine serum
α-MEM in, be inoculated in 3 100mm culture dishes, in a concentration of 5%CO2Incubator in, cultivated 2 days under the conditions of 37 DEG C,
Cell not adherent in culture solution is discarded, the α-MEM culture solutions renewed continue to cultivate, and replace a subculture within every 2 days；
(3) when stem cell growth to 80% fusion between fat, with a concentration of 0.25% pancreatin and a concentration of 0.02%
EDTA solution digestions pass on, and choose the 3rd generation cell and carry out phenotypic evaluation；
(4) after phenotypic evaluation, fat stem cell suspension is mixed with 30ml fat and is done carefully to get fat mesenchymal
Born of the same parents.
Preferably, the quantity of step (5) described fat stem cell is 1 × 106/ml。
Preferably, the clostridiopetidase A is collagenase type I.
Wherein, in the preparation method of above-mentioned fat mesenchymal stem cell, the method that the cell carries out phenotypic evaluation is：
(1) the 3rd fat subsitutes stem cell is collected, with containing 0.5%BSA and 0.1%NaN3PBS buffer solution washing lotion wash 2 times；
(2) plus 2% paraformaldehyde fixes 15 minutes under the conditions of 4 DEG C, is washed 2 times with PBS buffer solution washing lotion；
(3) add primary antibody, be incubated 30 minutes under the conditions of 4 DEG C；
(4) it is washed again 2 times with PBS buffer solution washing lotion, finally often pipe adds machine liquid on 0.5ml to be resuspended, then is removed with membrane filtration
Cell mass, upper machine, facs analysis.
Preferably, the primary antibody include FITC mark anti-human CD10, CD13, CD29, CD105, CD54, CD166, CD45,
CD34 monoclonal antibodies do Isotype control with species with IgG subclass FITC labelled antibodies.
The preparation method of the chitosan fluid film is：Chitosan is dissolved in 0.2% spirit of vinegar, gelatinous stream is prepared
Body film is used in combination the NaOH of 1N (1N=1 gram equivalents/liter) to neutralize, after filtering to obtain the final product.
The drug first punctures drug when specifically used, fat mesenchymal stem cell is first coated in wound, so
Chitosan fluid film is covered on the wound for having smeared fat mesenchymal stem cell afterwards, it can shape after the coating of chitosan fluid film
At flexible, transparent membrane, can be lost in avoid the cell of smearing；And the film oxygen permeability is good, and there is hemostasis, the reduction surface of a wound to ooze
The double effects for going out and avoiding the surface of a wound to dry.Simultaneously research shows that chitosan has broad spectrum antibiotic activity, people's epidermis can be promoted thin
Intracellular growth and local immunoregulation effect, using rear wound surface convergence, clothing is not glued in the drying of scab face, finishing.
In the present invention, Platelet-rich plasm and epidermal growth factor can be used well-known to those skilled in the art normal
Prepared by regulation Preparation Method, further include solvent in the present invention, and solvent uses stem cell factor, main component behaviour soma
Porcine HGF, deionized water, butanediol, hexylene glycol, propylene glycol, compound amino acid, glucan, extract solution from aloe, poly- paddy
Propylhomoserin and vitamin E etc..
The beneficial effects of the invention are as follows：
Fat mesenchymal stem cell and chitosan fluid film are used in combination the present invention, can be effectively promoted being cured for wound
It closes, it is more preferable than fat mesenchymal stem cell effect is used alone.Drug Platelet-rich plasm in the present invention and growth factor,
Each component compatibility is reasonable, synergistic, collectively promotes the healing of wound；The production stage of drug is simple, and process optimization is easy to
It accomplishes scale production.
Description of the drawings
Fig. 1 is influence of the different disposal to healing speed；
Fig. 2 is that the 14th, 18,21 day different disposal healing speed compares；
Fig. 3 is different disposal Wound Histology and Pathological Physiology situation of change；
The case where Fig. 4 is the variation of different disposal epithelial tissue；
Fig. 5 is influences of the CSH to the survival rate of the ADSCs surface of a wound；
Fig. 6 is influence of the different disposal to wounds in mice GFP protein expressions；
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
It is described in more detail below specific embodiments of the present invention.It should be appreciated that may be realized in various forms the present invention
Without should be limited by embodiments set forth here.It is to be able to be best understood from this hair on the contrary, providing these embodiments
It is bright, and the scope of the present invention can be completely communicated to those skilled in the art.
"comprising" or " comprising " as mentioned in working as in specification in the whole text and claim are an open language, therefore are answered
It is construed to " including but not limited to ".Specification subsequent descriptions are to implement the better embodiment of the present invention, and so description is
For the purpose of the rule of specification, it is not limited to the scope of the present invention.Protection scope of the present invention is when regarding appended power
Profit requires subject to institute's defender.
Embodiment 1
A kind of drug promoting wound healing, the drug include that weight ratio is 60:40 fat mesenchymal stem cell and
Chitosan fluid film, in isometric solution, Platelet-rich plasm volumetric concentration is 3%, the working concentration of epidermal growth factor
For the solution of 3ng/ml, the solvent stem cell factor of solution, main component is human stem cell growth factor, deionization
Water, butanediol, hexylene glycol, propylene glycol, compound amino acid, glucan, extract solution from aloe, polyglutamic acid and vitamin E etc..
The preparation method of the drug of above-mentioned promotion wound healing, described method includes following steps：
(1) fat mesenchymal stem cell and chitosan fluid film are prepared respectively；
(2) fat mesenchymal stem cell, Platelet-rich plasm and epidermal growth factor chitosan fluid film are wrapped up,
It is stored in liquid nitrogen cryogenics tank, the temperature of the liquid nitrogen cryogenics tank is -196 DEG C.
Wherein, the preparation method of the fat mesenchymal stem cell includes the following steps：
(1) fat lump (umbilical cord) is obtained, fat lump is cleaned up, the big blood vessel of connective tissue and finding of naked eye is cut off,
After being cleaned repeatedly with PBS buffer solution again, adipose tissue is cut into 1mm3Size is collected in 15ml centrifuge tubes, is added a concentration of
0.1% isometric collagenase type I, 37 DEG C of shaker water baths digest 60min；
(2) it is then added in isometric α-MEM culture mediums containing 10% serum, 200 mesh filter screens, which filter out, not to be digested
Tissue block, 1000rpm centrifuges 10 minutes, abandons supernatant, PBS buffer solution is rinsed 3 times, and precipitation is resuspended in containing 10% newborn bovine serum
α-MEM in, be inoculated in 3 100mm culture dishes, in a concentration of 5%CO2Incubator in, cultivated 2 days under the conditions of 37 DEG C,
Cell not adherent in culture solution is discarded, the α-MEM culture solutions renewed continue to cultivate, and replace a subculture within every 2 days；
(3) when stem cell growth to 80% fusion between fat, with a concentration of 0.25% pancreatin and a concentration of 0.02%
EDTA solution digestions pass on, and choose the 3rd generation cell and carry out phenotypic evaluation；
(4) it is 1 × 10 by quantity after phenotypic evaluation6The fat stem cell suspension of/ml is mixed with 30ml fat,
Up to fat mesenchymal stem cell.
Wherein, in the preparation method of above-mentioned fat mesenchymal stem cell, the method that the cell carries out phenotypic evaluation is：
(1) the 3rd fat subsitutes stem cell is collected, with containing 0.5%BSA and 0.1%NaN3PBS buffer solution washing lotion wash 2 times；
(2) plus 2% paraformaldehyde fixes 15 minutes under the conditions of 4 DEG C, is washed 2 times with PBS buffer solution washing lotion；
(3) plus primary antibody (including FITC marks anti-human CD10, CD13, CD29, CD105, CD54, CD166, CD45, CD34 mono-
It is anti-, with species Isotype control is done with IgG subclass FITC labelled antibodies), it is incubated 30 minutes under the conditions of 4 DEG C；
(4) it is washed again 2 times with PBS buffer solution washing lotion, finally often pipe adds machine liquid on 0.5ml to be resuspended, then is removed with membrane filtration
Cell mass, upper machine, facs analysis.
The preparation method of the chitosan fluid film is：Chitosan is dissolved in 0.2% spirit of vinegar, gelatinous stream is prepared
Body film is used in combination the NaOH of 1N (1N=1 gram equivalents/liter) to neutralize, after filtering to obtain the final product.
Embodiment 2
A kind of drug promoting wound healing, the drug include that weight ratio is 65:35 fat mesenchymal stem cell and
Chitosan fluid film, in isometric solution, Platelet-rich plasm volumetric concentration is 4%, and the work of epidermal growth factor is dense
Degree is 5ng/ml solution, and the solvent stem cell factor of solution, main component is human stem cell growth factor, deionization
Water, butanediol, hexylene glycol, propylene glycol, compound amino acid, glucan, extract solution from aloe, polyglutamic acid and vitamin E etc..
The preparation method of the drug of above-mentioned promotion wound healing, described method includes following steps：
(1) fat mesenchymal stem cell and chitosan fluid film are prepared respectively；
(2) fat mesenchymal stem cell, Platelet-rich plasm and epidermal growth factor chitosan fluid film are wrapped up,
It is stored in liquid nitrogen cryogenics tank, the temperature of the liquid nitrogen cryogenics tank is -196 DEG C.
Wherein, the preparation method of the fat mesenchymal stem cell includes the following steps：
(1) fat lump is obtained, fat lump is cleaned up, the big blood vessel of connective tissue and finding of naked eye is cut off, then uses PBS
After buffer solution cleans repeatedly, adipose tissue is cut into 1mm3Size is collected in 15ml centrifuge tubes, is added a concentration of 0.3%
Isometric collagenase type I, 37 DEG C of shaker water baths digest 60min；
(2) it is then added in isometric α-MEM culture mediums containing 10% serum, 200 mesh filter screens, which filter out, not to be digested
Tissue block, 1000rpm centrifuges 10 minutes, abandons supernatant, PBS buffer solution is rinsed 3 times, and precipitation is resuspended in containing 10% newborn bovine serum
α-MEM in, be inoculated in 3 100mm culture dishes, in a concentration of 5%CO2Incubator in, cultivated 2 days under the conditions of 37 DEG C,
Cell not adherent in culture solution is discarded, the α-MEM culture solutions renewed continue to cultivate, and replace a subculture within every 2 days；
(3) when stem cell growth to 80% fusion between fat, with a concentration of 0.25% pancreatin and a concentration of 0.02%
EDTA solution digestions pass on, and choose the 3rd generation cell and carry out phenotypic evaluation；
(4) it is 1 × 10 by quantity after phenotypic evaluation6The fat stem cell suspension of a/ml is mixed with 30ml fat
It closes to get fat mesenchymal stem cell.
Wherein, in the preparation method of above-mentioned fat mesenchymal stem cell, the method that the cell carries out phenotypic evaluation is：
(1) the 3rd fat subsitutes stem cell is collected, with containing 0.5%BSA and 0.1%NaN3PBS buffer solution washing lotion wash 2 times；
(2) plus 2% paraformaldehyde fixes 15 minutes under the conditions of 4 DEG C, is washed 2 times with PBS buffer solution washing lotion；
(3) plus primary antibody (including FITC marks anti-human CD10, CD13, CD29, CD105, CD54, CD166, CD45, CD34 mono-
It is anti-, with species Isotype control is done with IgG subclass FITC labelled antibodies), it is incubated 30 minutes under the conditions of 4 DEG C；
(4) it is washed again 2 times with PBS buffer solution washing lotion, finally often pipe adds machine liquid on 0.5ml to be resuspended, then is removed with membrane filtration
Cell mass, upper machine, facs analysis.
The preparation method of the chitosan fluid film is：Chitosan is dissolved in 0.2% spirit of vinegar, gelatinous stream is prepared
Body film is used in combination the NaOH of 1N to neutralize, after filtering to obtain the final product.
Embodiment 3
A kind of drug promoting wound healing, the drug include that weight ratio is 70:30 fat mesenchymal stem cell and
Chitosan fluid film, in isometric solution, Platelet-rich plasm volumetric concentration is 6%, and the work of epidermal growth factor is dense
Degree is 8ng/ml solution, and the solvent stem cell factor of solution, main component is human stem cell growth factor, deionization
Water, butanediol, hexylene glycol, propylene glycol, compound amino acid, glucan, extract solution from aloe, polyglutamic acid and vitamin E etc..
The preparation method of the drug of above-mentioned promotion wound healing, described method includes following steps：
(1) fat mesenchymal stem cell and chitosan fluid film are prepared respectively；
(2) fat mesenchymal stem cell, Platelet-rich plasm and epidermal growth factor chitosan fluid film are wrapped up,
It is stored in liquid nitrogen cryogenics tank, the temperature of the liquid nitrogen cryogenics tank is -196 DEG C.
Wherein, the preparation method of the fat mesenchymal stem cell includes the following steps：
(1) fat lump is obtained, fat lump is cleaned up, the big blood vessel of connective tissue and finding of naked eye is cut off, then uses PBS
After buffer solution cleans repeatedly, adipose tissue is cut into 1mm3Size is collected in 15ml centrifuge tubes, is added a concentration of 0.5%
Isometric collagenase type I, 37 DEG C of shaker water baths digest 60min；
(2) it is then added in isometric α-MEM culture mediums containing 10% serum, 200 mesh filter screens, which filter out, not to be digested
Tissue block, 1000rpm centrifuges 10 minutes, abandons supernatant, PBS buffer solution is rinsed 3 times, and precipitation is resuspended in containing 10% newborn bovine serum
α-MEM in, be inoculated in 3 100mm culture dishes, in a concentration of 5%CO2Incubator in, cultivated 2 days under the conditions of 37 DEG C,
Cell not adherent in culture solution is discarded, the α-MEM culture solutions renewed continue to cultivate, and replace a subculture within every 2 days；
(3) when stem cell growth to 80% fusion between fat, with a concentration of 0.25% pancreatin and a concentration of 0.02%
EDTA solution digestions pass on, and choose the 3rd generation cell and carry out phenotypic evaluation；
(4) it is 1 × 10 by quantity after identifying6The fat stem cell suspension of/ml mixed with 30ml fat to get
Fat mesenchymal stem cell.
Wherein, in the preparation method of above-mentioned fat mesenchymal stem cell, the method that the cell carries out phenotypic evaluation is：
(1) the 3rd fat subsitutes stem cell is collected, with containing 0.5%BSA and 0.1%NaN3PBS buffer solution washing lotion wash 2 times；
(2) plus 2% paraformaldehyde fixes 15 minutes under the conditions of 4 DEG C, is washed 2 times with PBS buffer solution washing lotion；
(3) plus primary antibody (including FITC marks anti-human CD10, CD13, CD29, CD105, CD54, CD166, CD45, CD34 mono-
It is anti-, with species Isotype control is done with IgG subclass FITC labelled antibodies), it is incubated 30 minutes under the conditions of 4 DEG C；
(4) it is washed again 2 times with PBS buffer solution washing lotion, finally often pipe adds machine liquid on 0.5ml to be resuspended, then is removed with membrane filtration
Cell mass, upper machine, facs analysis.
The preparation method of the chitosan fluid film is：Chitosan is dissolved in 0.2% spirit of vinegar, gelatinous stream is prepared
Body film is used in combination the NaOH of 1N to neutralize, after filtering to obtain the final product.
In order to prove that effectiveness of the invention, the present invention have carried out following experiment：
One, the foundation of mouse skin full-thickness defects model
SPF grades of inbred strais C57/B6 mouse (8 weeks, female, 20-23g) 40 are taken, mouse is placed on anesthesia induction respectively
(3% isoflurane, 100% oxygen flow IL/min) is anaesthetized in box.Mouse is taken out to be placed on electric pad and keeps body temperature, connects anesthesia
Machine (l% isofluranes) goes the hair of most mouse back skin, exposed skin to use after Animal Anesthesia satisfaction with electric hair cutter and depilatory agent
75% alcohol takes off iodine after twice of iodophor disinfection.Mouse back with endless knife be two a diameter of 6mm round full thickness skin lack
Damage, with 5-0 silk suture viscous silica gel rings around defect, and covers transparent dressing film.Respectively at postoperative 0d, 3d, 7d, 10d,
14d, 18d, 21d, digital camera is taken pictures wounds in mice and marks surface of a wound size, each surface of a wound to repeat three with sterile transparent squared paper
It is secondary.1.60 softwares of National Institute of Health (NIH) Image calculate surface of a wound area, calculate Wound healing rate
=100 × (- n days surface of a wound areas of 0 day surface of a wound area)/0 day surface of a wound area.
Two, defect of skin treatment of animals model is grouped：
It is divided into according to requirement of experiment and establishes mouse skin full-thickness defects model and be randomly divided into 4 groups：
A group animals：Simple physiological saline smears the surface of a wound；
B group animals：Simple CSH is applied to the surface of a wound；
C group animals：1million ADSCs multiple spots are applied to the surface of a wound and surface of a wound surrounding subcutaneous；
D group animals：Covering CSH (uses this hair after the ADSCs multiple spots of 1million are applied to the surface of a wound and surface of a wound surrounding subcutaneous
Bright drug).
Three, mouse skin is drawn materials：
Mouse materials are put to death when distinguishing 3d, 7d, 10d, 14d, 21d after surgery.It notes in 10% abdominal cavities chloraldurate 4ml/kg
Anesthesia is penetrated, fixed, disinfection, paving is single, and 1cm starts to cut off skin with sharp weapon around the surface of a wound, and dissociate subcutaneous tissue, removes bilateral wound
After covering weave, be divided into four parts (on ice operate), be soaked in respectively 10% neutral formalin solution (for surface of a wound HE detections),
It is placed in cryopreservation tube and freezes in liquid nitrogen container (test serum protein expression), be placed in liquid nitrogen Rapid-Freezing Method half an hour time in cryopreservation tube
It deposits in -80 DEG C of refrigerators, another is embedded using OCT embedding mediums at once, is placed on freezing microtome and is sliced, and it is aobvious to be inverted fluorescence
Micro mirror observes luciferase expression.
Four, skin histology paraffin section
(1) step：
A, it draws materials：It is put into 4% paraformaldehyde by the hair on removal skin after above-mentioned steps taking-up skin histology fixed
More than for 24 hours；
B, the finishing after fixing；Tissue is taken out from fixer, respectively with blade by the longitudinal sectional two halves of skin；
Dehydration：Gradient alcohol dehydration is carried out in the following order：70% alcohol (1h), 80% (1h), 95% alcohol (2 times,
1h/ times), 100% alcohol (3 times, 0.5h/ times, 0.5h/ times, 1h/ times)；
C, transparent：Xylene soak (2 times, 1h/ times)；
D, waxdip：65 DEG C of paraffin impregnate 3-4h；
E, it embeds：Skin histology block is positioned in embedded box fixed；
F, paraffin section：After routine paraffin wax embedding, 3-4 μm of slice, mount is in load fragment；
G, it dewaxes：Conventional xylene dewaxes, and graded ethanol is cleaned to aquation；
H, hematoxylin dyes：Disseminate 10-15mings, washing；
I, break up, return indigo plant：1% hydrochloride alcohol breaks up the several seconds, washing, and the unsaturated carbonate lithium oil blackeite several seconds, flowing water rinses 15-
30min；
J, red colouring：Disseminate 5-15s in 1% alcohol-soluble Yihong；
K, rule dehydration, transparent, mounting；
L, micro- microscopic observation and each multiple morphological change of skin biopsy is taken.
Five, mice skin tissue frozen section
(1) it draws materials
Take skin histology to enter in ice-cold PBS buffer solution, clean residual blood, with double-pole method by skin it is longitudinal sectional be several groups
Knit block.
(2) fixed
Skin histology is put into 4% neutral paraformaldehyde solution, 4 DEG C are fixed 24 hours or more.
(3) it rinses
PBS buffer solution repeatedly rinses skin histology.
(4) it is dehydrated
Gradient sucrose dehydration is carried out in the following order：Pass through 10% sucrose, 20% sucrose, 30% 4 DEG C of sucrose solution successively
It impregnates, it is degree to be sink to bottom of bottle with skin histology.
(5) it embeds
Skin histology block is embedded with frost embedding medium OCT within 1-2 hours before slice.
(6) it is sliced
Tissue block is modified, and embedding medium excessive around tissue is cut, and there are about 1-2mm's around tissue under normal circumstances
Embedding medium side, both sides are required parallel.Fixing organization blocks first repair tissue block, and skin layers institutional framework is contained generally, is then adjusted
Whole freezing microtome slice thickness is 5 μm and is sliced that cutting temperature is advisable with -20 DEG C~-23 DEG C.
(7) patch
Direct patch in cryogenic freezing slicer, patch are sealed and use as early as possible for -20 DEG C after room temperature drying.
Six, Pan-ck immunohistochemical stainings
(1) 3%H2O2Deionized water is incubated 10 minutes deactivating endogenous peroxydases, and PBS buffer solution is washed, and 2 minutes × 3；
(2) 37 DEG C of primary antibody is added dropwise to be incubated 1 hour, PBS buffer solution is washed, and 2 minutes × 3；
(3) 37 DEG C of secondary antibody is added dropwise to be incubated 30 minutes, PBS buffer solution is washed, and 2 minutes × 3；
(4) DAB working solutions are applied to develop the color；
(5) distilled water fully rinses；
(6) it is dehydrated：50% ethyl alcohol, 3 minutes → 70% ethyl alcohol 3 minutes
(7) haematoxylin is redyed
3 minutes (core is blue) → originally washing 1 minute → hydrochloride alcohol differentiation 10 seconds (chromatin is clear in nucleus) → from
1 minute → distillation washing 10 seconds is originally washed to wash → returning 30 seconds blue → 1 minute
(8) it is dehydrated
50% ethyl alcohol, 3 minutes → 75% ethyl alcohol, 3 minutes → 80% ethyl alcohol, 3 minutes → 95% ethyl alcohol 3 minutes (twice) →
100% ethyl alcohol 3 minutes (twice)
(9) transparent
3 minutes → TOII of TOI, 3 minutes → TO III 3 minutes.
(10) mounting
It takes appropriate neutral gum to instill the tissue of glass slide, coverslip is taken gradually to cover from one end, avoid that gas occurs
Bubble is inverted ordinary light observed under electron microscope and takes pictures.
Seven, green cells quantitative analysis
In light 4 groups of mice skin tissues slice under the microscope, every group of 3 animals, every animal takes two tissue blocks, every piece
4-5 pieces are cut, every piece chooses the 2-3 visual field, and therefore, every group is chosen about 60 visuals field altogether, using stereoscopy grid system meter
The green fluorescence of number unit area is expressed as positive cell number, takes each cell mean for statistical analysis.
Eight, Western Blot Protein Detections
(1) extraction of histone
By skin histology take it is a certain amount of be placed in the sterile no enzyme 2ml EP pipes of import, be added quality (mg) with lysate volume
(ul) it than the 2%SDS lysates for 1 to 20, being placed in electric homogenizer, " B " shelves rotating speed is used to be homogenized 5-10s on ice, eye is seen,
Shaking loses naked eyes visible tissue particle, is subsequently placed in Ultrasonic Cell Disruptor and is crushed (5s/ times, 5 times).It is subsequently placed at
It heats 10 minutes in boiling water, then 14000rpm/min, centrifuges 10min, draw supernatant, then with 5x sample-loading buffers and 2-ME
Mixed liquor (volume ratio is 95 to 5) is configured to can be used for the albumen sample of Western Blot, boils 5min in boiling water again, immediately
Using or packing be stored in -40 DEG C, it is spare, sample detection after boiling 5min again is also placed in boiling water before taking out every time.
(2) Western blot methods
Glue：Prepare 12% separation gel and 5% spacer gel.Separation gel is first perfused, after room temperature 30min after its polymerization is good
Reperfu- sion spacer gel is inserted into sample-adding comb, sample-adding comb is carefully extracted after polymerizeing completely, to prevent the destruction of well.
Sample-adding：It is mixed in proportion with 5 × SDS-PAGE sample-loading buffers with sample (80 μ g albumen), 100 DEG C are boiled 5min,
It is immediately passed to cooled on ice, centrifuges 12000rpm/min, 1min.It is hole-specifically loaded, 1 × SDS-PAGE loading buffers are added in remaining hole
Liquid, injection volume are 20 μ l.
Electrophoresis：With Bio-Rad vertical electrophoresis apparatus, 8V/cm voltages are added on gel, after dye front enters separation gel
Voltage is increased to 15V/cm and continues electrophoresis, until bromophenol blue reaches the bottom of separation gel, stops electrophoresis.
Transferring film：Gel is removed after electrophoresis, cuts off extra Blank gel, and 8 What- are cut by its size after trimming
Man 3M filter paper and a NC film, NC films impregnate two hours in deionized water in advance, by 4 filter paper-gel-NC films -4
The sequence of filter paper stack neatly be placed on it is wet turn on negative plate, remove the bubble in gap with the light pressure of clean glass rod, with
The voltage transferring film 60min of 100V.After transferring film, film is taken out, the upper left corner is cut and makes marks, visible pre-dyed after transferring film success
The band of the albumen of Marker is clearly transferred on film, contaminates NC films with the beginning of spring red S, it is seen that there is clearly protein band on film,
Gel is dyed through Coomassie brilliant blue, decoloration verification is without western blot.
The detection of destination protein:
A, after the beginning of spring red S dyes film, film is washed by film rinsed clean, then with TBS three times, each 5min with deionized water.
B, 5% skimmed milk power room temperature closing NC films 1h.
C, TBS/T washes film 5 times, each 10min.
D, 5% skimmed milk power is with rabbit anti-mouse VEGF monoclonal antibodies 1：800, goat-anti rabbit GFP monoclonal antibodies and rabbit-anti
Mouse G, APDH polyclonal antibody 1：1000 dilution proportion primary antibodies, 4 DEG C of overnight incubations.
E, TBS/T washes film three times, each 5min.
F, it is transferred to 1：1000 HRP donkey anti-rabbits IgG or 1：In 1000 HRP goat anti-rabbit iggs.It is incubated at room temperature 2h.
G, TBST washes film 5 times, each 10min.
H, film is taken out, enhances in chemiluminescence luminescence reagent and reacts 30s.
I, exposure image scans and shoots photo, with ias analysis result.
Nine, test result：
(1) ADSCs promotes wound healing
After mouse full thickness dermal model is made, according to different processing methods around the surface of a wound multi-point injection physiology
The processing such as brine, ADSCs or coating CSH, and recorded the surface of a wound areal calculation surface of a wound in 0d, 3d, 7d, 10d, 14d, 18d, 21d days
Healing rate.The experimental results showed that 14d, 18d, 21d, award CSH, wounds in mice healing speed after ADSCs, CSH+ADSCs treatment
Degree is more apparent than Normal group to be speeded (P ＜ 0.05).14d, 18d CSH+ADSCs treatment group wounds in mice healing rate ratio
CSH group mouse obviously speed.Wounds in mice healing rate ratio ADSCs treatment groups of 18d CSH+ADSCs treatment groups mouse is apparent
Accelerate.(P ＜ 0.05) (as depicted in figs. 1 and 2).
(2) Wound Histology and Pathological Physiology variation
When mouse wound healing 14d, 18d, 21d, wound circumference diameter 1cm area full thickness skin tissues is taken to carry out respectively
Frozen section is dyed through HE, and experimental result is shown, as shown in figure 3, granulation tissue progressive additive in wounds in mice skin histology,
The gradual thickening of collagenous fibres, while simultaneously flap coverage finally makes the surface of a wound heal completely to epithelial cell constantly proliferation.Through CSH,
After ADSCs, CSH+ADSCs treatment, in 14d, 18d, granulation tissue thickness, re-epithelialization etc. are bright in 21d surface of a wound skin histologies
Aobvious better than Normal group.
(3) ADSCs and CSH promotes wound tissue epithelialization
When mouse wound healing 14d, periwound skin tissue freezing section is taken, with Pan-CK antibody test surface of a wound groups
Epithelialization is knitted, as shown in figure 4, result is shown：Normal group wounds in mice epithelial tissue hyperplasia is slow, gives CSH, ADSCs,
CSH+ADSCs treats epithelium posterius hyperblastosis speed and accelerates, and epithelial tissue thickness increases.
(4) CSH improves ADSCs survivals and promotes wounds in mice healing
After mouse wound healing 14d Shi Qu periwound skins tissue freezing section, directly under fluorescence microscope into
Row observation display, the visible dotted green fluorescence (as shown in Figure 5) of ADSCs and CSH+ADSCs treatment groups, and CSH+ADSCs groups are glimmering
Luminous intensity and quantity are higher than ADSCs groups (P ＜ 0.05), it is seen that CSH can increase ADSCs the surface of a wound survival rate, without injecting
A groups, the B groups of ADSCs also shows faint fluorescence, may be the fluorescence that autologous tissue sends out.
(5) ADSCs and CSH makes the up-regulated expression of VEGF
After the surface of a wound full thickness dermal model surgery when 14d, each group wound tissue is taken to extract row Western after albumen
Blot methods detect, and as a result show：ADSCs groups and the apparent up-regulation of CSH+ADSCs group wounds in mice GFP protein expressions and CSH+
ADSCs group wounds in mice GFP protein expressions are apparently higher than ADSCs groups (P ＜ 0.05) (as shown in Figure 6), CSH groups, ADSCs groups,
The apparent up-regulation (P ＜ 0.05) of CSH+ADSCs group wounds in mice vegf proteins expression, and CSH+ADSCs group ratio ADSCs group mouse are created
Face vegf protein expression obviously increases (P<0.05), it is seen that CSH can improve the survival of ADSCs.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention belong to the scope of protection of present invention.
Claims (10)
1. a kind of drug promoting wound healing, which is characterized in that the drug includes fat mesenchymal stem cell and chitosan
Fluid film.
2. the drug according to claim 1 for promoting wound healing, which is characterized in that the drug further includes chitosan stream
Body film and isometric solution being made of Platelet-rich plasm and skin factor；
Wherein, the weight ratio of the fat mesenchymal stem cell and chitosan fluid film is 60~70:30~40.
3. the drug according to claim 1 for promoting wound healing, which is characterized in that
In the solution, the volume by volume concentration of Platelet-rich plasm is 3-6%, and the working concentration of the epidermal growth factor is
3-8ng/ml。
4. a kind of preparation method of the drug of promotion wound healing as described in claims 1 to 3, which is characterized in that the side
Method includes the following steps：
(1) fat mesenchymal stem cell and chitosan fluid film are prepared respectively；
(2) fat mesenchymal stem cell chitosan fluid film is wrapped up, is stored in liquid nitrogen cryogenics tank.
5. the preparation method of the drug of promotion wound healing according to claim 4, which is characterized in that filled between the fat
The preparation method of matter stem cell includes the following steps：
(1) fat lump is obtained, fat lump is cleaned up, the big blood vessel of connective tissue and finding of naked eye is cut off, then is buffered with PBS
After liquid cleans repeatedly, adipose tissue is cut into 1mm3Size is collected in 15ml centrifuge tubes, is added a concentration of 0.1~0.5%
Isometric clostridiopetidase A, 37 DEG C of shaker water baths digest 60min；
(2) it is then added in isometric α-MEM culture mediums containing 10% serum, 200 mesh filter screens filter out the group not digested
Block is knitted, 1000rpm is centrifuged 10 minutes, abandons supernatant, and PBS buffer solution is rinsed 3 times, and precipitation is resuspended in the α-containing 10% newborn bovine serum
In MEM, it is inoculated in 3 100mm culture dishes, in a concentration of 5%CO2Incubator in, cultivate 2 days, discard under the conditions of 37 DEG C
Not adherent cell in culture solution, the α-MEM culture solutions renewed continue to cultivate, and replace a subculture within every 2 days；
(3) molten with a concentration of 0.25% pancreatin and a concentration of 0.02%EDTA when stem cell growth to 80% fusion between fat
Liquid had digestive transfer culture chooses the 3rd generation cell and carries out phenotypic evaluation；
(4) after phenotypic evaluation, fat stem cell suspension is mixed with 30ml fat to get fat mesenchymal stem cell.
6. the preparation method of fat mesenchymal stem cell according to claim 5, which is characterized in that step (4) described fat
The quantity of fat stem cell is 1 × 106A/ml.
7. the preparation method of fat mesenchymal stem cell according to claim 5, which is characterized in that the clostridiopetidase A is I
Collagenase Type.
8. the preparation method of fat mesenchymal stem cell according to claim 5, which is characterized in that the cell carries out table
The method that type is identified is：
(1) the 3rd fat subsitutes stem cell is collected, with containing 0.5%BSA and 0.1%NaN3PBS buffer solution washing lotion wash 2 times；
(2) plus 2% paraformaldehyde fixes 15 minutes under the conditions of 4 DEG C, is washed 2 times with PBS buffer solution washing lotion；
(3) add primary antibody, be incubated 30 minutes under the conditions of 4 DEG C；
(4) it is washed again 2 times with PBS buffer solution washing lotion, finally often pipe adds machine liquid on 0.5ml to be resuspended, then removes cell with membrane filtration
Group, upper machine, facs analysis.
9. the preparation method of fat mesenchymal stem cell according to claim 8, which is characterized in that the primary antibody includes
FITC marks anti-human CD10, CD13, CD29, CD105, CD54, CD166, CD45 and CD34 monoclonal antibody, with species with IgG subclass
FITC labelled antibodies do Isotype control.
10. the preparation method of the drug of promotion wound healing according to claim 4, which is characterized in that the chitosan
The preparation method of fluid film is：Chitosan is dissolved in 0.2% spirit of vinegar, gelatinous fluid film is prepared, the NaOH of 1N is used in combination
It neutralizes, after filtering to obtain the final product.
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| CN112175902A (en) * | 2020-10-19 | 2021-01-05 | 上海交通大学医学院附属第九人民医院 | Method for separating adipose-derived stem cells |
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| CN107213455A (en) * | 2017-06-13 | 2017-09-29 | 广州赛莱拉干细胞科技股份有限公司 | A kind of stem cell medicine and its preparation method and application |
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| CN101361990A (en) * | 2008-09-03 | 2009-02-11 | 陕西瑞盛生物科技有限公司 | Double layer artificial skin and preparation method thereof |
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