CN106540244A - A kind of dog mesenchymal stem cell injection and its preparation method and application - Google Patents
A kind of dog mesenchymal stem cell injection and its preparation method and application Download PDFInfo
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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
- 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
- A61K38/38—Albumins
- A61K38/385—Serum albumin
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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
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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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
- A61K9/10—Dispersions; Emulsions
Abstract
A kind of dog mesenchymal stem cell injection of the present invention and its preparation method and application, including：Dog mesenchymal stem cells MSCs, calciparine, dog blood albumin and electrolyte solution, cleverly with the mescenchymal stem cell of dog derived from bone marrow, and which is combined with other compositions be prepared into parenteral solution form, that is dog mesenchymal stem cells MSCs parenteral solution, the traditional therapy of dog dermal tissue insult is broken, bring stem cell therapy into Medical pet field, its stability is high, good effect, use safety, it is suitable to the preservation and transport of long period, it is without any side effects, it is that clinical large-scale use is laid a good foundation.
Description
Technical field
The invention belongs to biomedicine field, more particularly to a kind of dog mesenchymal stem cell injection and preparation method thereof and
Using.
Background technology
With the improvement of people's living standards, the acceleration of urbanization process, the diminution of household size and aging population
Aggravation, pet dog by become people life important companion, raise quantity it is more and more.The dermopathic incidence of disease of current dog
It is higher, wherein also include some it is unexpected scald burn, wound, the dog widespread skin wound that surgical operation etc. is caused.At present by
It is poor in therapeutic effect, significantly impact viewing and admiring and economic worth for dog.
It is anti-by the various factors skin histology destruction for causing and the whole body that may occur that skin trauma generally refers to skin
Should.Although skin trauma wound type and injured degree are different, the mechanism of its healing is basically identical, main to include local
The processes such as inflammatory reaction, cell propagation, revascularization, connective tissue formation, contraction of wounds and wound reconstruction.Stem-cell therapy is made
For a kind of emerging tissue damage repairing and treating means, more and more apply in human clinical medicine.Mescenchymal stem cell
The features such as (Mesenchymal Stem Cell, MSC) is because with easy, low immunogenicity, Multidirectional Differentiation and strong paracrine is separated
And be widely used.MSC to the repair of skin trauma through wound healing whole process.By fluorescence localization etc.
Method proves that MSC can be divided into various Skin Cells in the surface of a wound, directly acts on wound site, accelerating wound healing.MSC exists
Also there is to surrounding environment after transplanting obvious immunoregulation effect, and support that the various rush survivals of NK cell secretion and rush are moved
The cell factor and growth factor of shifting.MSC suppresses host T cell by the activity of IL-10 and TGF-β in impact host T cell
Propagation, reduces wound inflammation reaction.2008, year Krasnodembskaya finds：MSC can secrete one kind and resist micro- life with wide spectrum
The LL-37 peptides of thing effect, this active peptide is by destroying the cell membrane of bacterium and raising direct restricting bacterial macrophage activity
Chemokine receptors, so as to reduce the generation of wound inflammation, acceleration of wound reparation.In addition, MSC can adjust host's TNF-α
Generation with the inflammatory effect of mediate excessive, and inflammatory phase reduce NK cell functions, reduce IFN-γ activity.Conversely,
The later stage of inflammation, TGF- α can stimulate the MSC of implantation to produce various agglutinant growth factors and cell factor, stimulate the surface of a wound
Angiogenesiss in bed.Although people have gained some understanding to the mechanism of MSC wound healings at present, but still have many problems simultaneously
It is unclear, such as MSC promote skin ultrastructure action pathway how manyWhich approach is most important
MSC has been used for tissue injury reparation on Human clinical, achieves preferable therapeutic effect.But at present with regard to dog
Stem-cell research is also few, and application of the stem cell in dog disease treatment is also less.In order to preferably improve the medical treatment of pet dog
Level, increases the research of the stem cell of dog, it has also become the task of top priority.The research of dog stem cell biology not only can make people more
The biological character and physical signs of deep understanding dog, it helps strengthen the research of the disease treatment to dog.MSC may be into
For dog skin trauma or a new direction of other histoorgan repairing and treatings.
The content of the invention
Instant invention overcomes shortcoming of the prior art, there is provided a kind of dog mesenchymal stem cell injection and its preparation side
Method, which has the advantages such as stability height, good effect, the clinical large-scale use of safe, adaptation, has a extensive future, can be
The treatment of the dermal tissue insult of pet dog opens new road.
In order to solve above-mentioned technical problem, the present invention is achieved by the following technical solutions：
A kind of dog mesenchymal stem cell injection, including：Dog mesenchymal stem cells MSCs, calciparine, dog blood albumin and
Electrolyte solution.
Further, the concentration of the calciparine is 0.5%, is that one kind contains polysaccharide compound, and has and prevent cell
Assemble the preparation of agglomerating function.
Further, the concentration of the dog blood albumin stoste is 5-10%；It is dry containing dog medulla mesenchyma in 1ml parenteral solutions
Cell 1 × 105It is individual.
A kind of preparation method of dog mesenchymal stem cell injection, there is provided dog mesenchymal stem cells MSCs preservation liquid, precooling,
Standby, mesenchyme stem cell preserving fluid includes calciparine, dog blood albumin and electrolyte solution；There is provided dog medulla mesenchyma dry thin
Born of the same parents, dog mesenchymal stem cells MSCs is added in mesenchyme stem cell preserving fluid；By resuspended mode, between adjustment is added to
It is 1 × 10 that mesenchymal stem cells preserve the dog mesenchymal stem cells MSCs quantity in liquid5Individual cell/1ml.
Further, wherein the preparation of the dog mesenchymal stem cells MSCs is comprised the following steps：
(1) dog marrow is taken：Dog routinely loses hair or feathers after anaesthetizing, sterilizes, drape, uses in left side or right side ilium under sterile working
No. 16 bone marrow aspiration pin punctures, with preprepared containing anti-coagulants, anti-coagulants is 3000U heparin, bone marrow extraction liquid 3ml in
In centrifuge tube, 3ml PBS are added, 6ml marrow dilutions are made；
(2) separation of dog bone marrow nucleated cell：The Ficoll liquid of 3ml 1.077g/ml is taken, centrifuge tube is added, then is slowly added
Enter 6ml marrow dilutions, 2500r/min centrifugation 20min carefully draw the white cloud and mist layer of karyocyte formation in centrifuge tube,
The DMEM containing hyclone 10%, 1000r/min centrifugation 5min are added, upper liquid is carefully siphoned away after centrifugation, ox containing tire is added
The DMEM of serum 10%, uniformly makes cell suspension with liquid-transfering gun piping and druming；
(3) original cuiture of dog mesenchymal stem cells MSCs：By cell suspension inoculation in blake bottle, 37 DEG C of 5%CO are placed in2
After 24h is cultivated in incubator, liquid observation of cell are changed；Then liquid, period daily inverted phase contrast microscope were once changed per 3 days
The Morphology of observation of cell；
(4) Secondary Culture of dog mesenchymal stem cells MSCs：When cell is paved with Tissue Culture Plate, careful suction abandons culture dish
Interior nutrient solution, is rinsed 2 times with PBS, is added 0.25% appropriate trypsin solution, is slightly shaken up, and is put
Born of the same parents, terminate digestion with the complete medium containing 10% hyclone, blow and beat repeatedly the bottle wall of cell attachment growth, until bottle wall is thin
Born of the same parents all come off, and make cell suspension, and continuing gently piping and druming cell suspension makes cell scatter；
(5) identification of dog mesenchymal stem cells MSCs；
(6) skeletonization of dog mesenchymal stem cells MSCs and into fat break up：Treat that growth of mesenchymal stem cells is converged to 80%-90%
When right, remove culture medium, washed with PBS 3 times, add skeletonization and lipoblast induction liquid Fiber differentiation, one is changed per 2-3d
The metamorphosis of secondary nutrient solution, daily observation of cell growth conditions and cell, carries out cell induction identification in good time, after induction into
Osteocyte, lipoblast carry out alizarin red, oil red O stain identification.
Further, the identification of the dog mesenchymal stem cells MSCs detects BMMSCs by immunofluorescence dyeing method
The expression of CD34 and CD44 albumen, for using resist for rabbit-anti CD34 and the anti-CD44 of mouse, and two resist the goat anti-rabbit igg for FITC marks
With the sheep anti-mouse igg of FITC marks, comprise the following steps：
(1) mesenchymal stem cells MSCs in the 3rd generation is taken, is seeded on the culture plate that gelatin was coated with, make cell be affixed on training
Foster plate, after cell attachment 24h, suctions out nutrient solution, 2 times is washed with PBS；
(2) add under 4% paraformaldehyde room temperature and fix 20min, remove fixer, PBS is washed 3 times, each 5min；
(3) saturating agent is added, and saturating agent is 0.1%TritonX-100, PBS, agent is sucked after incubation at room temperature 15min；
PBS is washed 3 times, each 5min；
(4) confining liquid is added, and confining liquid is 10%FBS, PBS, is incubated at room temperature 30min, removes confining liquid, 3 are washed with PBS
It is secondary, each 5min；
(5) be separately added into it is one anti-(the anti-CD44 of mouse, 1:200 and rabbit-anti CD34,1：100), 4 DEG C of lucifuge overnight incubations, PBS are washed
Wash 3 times, each 5min；
(6) be separately added into FITC marks it is two anti-(sheep anti-mouse igg and goat anti-rabbit igg, 1：500), room temperature lucifuge incubation
30min, PBS are washed 3 times, each 5min；
(7) 1 μ g/mL DAPI lucifuges are added to redye 5min, PBS is washed 3 times, each 5min；
(8) anti-fluorescence quenching is added, coloration result is observed under inverted fluorescence microscope and is taken pictures.
Further, application of the dog mesenchymal stem cell injection in treatment dog dermal tissue insult.
Compared with prior art, the invention has the beneficial effects as follows：
A kind of dog mesenchymal stem cell injection of the present invention and its preparation method and application, cleverly with dog marrow
The mescenchymal stem cell in source, and which is combined the parenteral solution form that is prepared into, i.e. dog mesenchymal stem cells MSCs with other compositions
Parenteral solution, has broken the traditional therapy of dog dermal tissue insult, brings stem cell therapy into Medical pet field, and which is stable
Property high, good effect, using safety, be suitable to the preservation and transport of long period, it is without any side effects, be clinical large-scale use
Lay a good foundation.
Description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, together with embodiments of the present invention for explaining the present invention,
It is not construed as limiting the invention, in the accompanying drawings：
Fig. 1 is the skeletonization adipogenic induction differentiation of dog marrow MSC；
Fig. 2 is dog marrow MSC identified by immunofluorescence；
Fig. 3 is the area for treating 8d and 12d wound models；
Fig. 4 is the comparison of dog skin trauma model wound healing rate after 8 days；
Fig. 5 is the observation of the dog skin trauma surface of a wound.
Specific embodiment
The preferred embodiments of the present invention are illustrated below in conjunction with accompanying drawing, it will be appreciated that preferred reality described herein
Apply example and be merely to illustrate and explain the present invention, be not intended to limit the present invention.
First, the preparation of dog derived from bone marrow mescenchymal stem cell：
(1) marrow is taken
Dog anesthesia after routinely depilation, sterilization, drape, under sterile working under sterile working respectively at left side or right side ilium
With No. 16 bone marrow aspiration pin punctures, with it is preprepared containing anti-coagulants (3000U heparin) bone marrow extraction liquid 3ml in 15ml from
In heart pipe.Bone marrow fluid 3ml is taken, is added 3ml to contain dual anti-PBS (two-fold dilution), is made 6ml marrow dilutions.
(2) Ficoll liquid density-gradient centrifugation method is separated
3ml 1.077g/ml Ficoll liquid is taken, the centrifuge tube of 15ml is added, is slow added into 6ml marrow dilutions,
2500r/min refrigerated centrifuges are centrifuged 20min.After centrifugation, it is careful draw that karyocyte formed it is white nebulous be layered in
The centrifuge tube of 15ml, adds the DMEM containing hyclone 10%, 1000r/min centrifugation 5min.It is careful after centrifugation to draw upper liquid,
The DMEM containing hyclone 10% is added, and cell suspension is uniformly made with liquid-transfering gun piping and druming.
(3) original cuiture of dog mesenchymal stem cells MSCs
By cell suspension inoculation in 60mm2Culture dish, is placed in 37 DEG C of 5%CO2After 24h is cultivated in incubator, change liquid and observe
Cell；Then liquid, the Morphology of period daily inverted phase contrast microscope observation of cell were once changed per 3 days.
(4) Secondary Culture of dog mesenchymal stem cells MSCs
When cell is paved with 80% region of Tissue Culture Plate, the nutrient solution abandoned in 1.3.3 culture dishes is carefully inhaled, is rushed with PBS
Wash 2 times, add 0.25% appropriate trypsin solution, slightly shake up, put vitellophag at room temperature.With containing 10% hyclone
Complete medium terminate digestion, blow and beat repeatedly the bottle wall of cell attachment growth, until bottle wall cell all comes off, make cell
Suspension, continuing gently piping and druming cell suspension makes cell scatter.
2nd, the identification of dog mesenchymal stem cells MSCs
Fig. 2 is dog BM-MSCs identified by immunofluorescence, by immunofluorescence dyeing method detect dog bone marrow MSCs CD34 and
The expression of CD44 albumen.One for using resists for rabbit-anti CD34 and the anti-CD44 of mouse, and two resist the goat anti-rabbit igg and FITC for FITC marks
The sheep anti-mouse igg of mark.
(1) mesenchymal stem cells MSCs in the 3rd generation is taken, is seeded on the culture plate that gelatin was coated with, make cell be affixed on training
Foster plate, after cell attachment 24h, suctions out nutrient solution, 2 times is washed with PBS；
(2) add under 4% paraformaldehyde room temperature and fix 20min, remove fixer, PBS is washed 3 times, each 5min；
(3) saturating agent (0.1%TritonX-100, PBS) is added, after incubation at room temperature 15min, agent is sucked；PBS is washed
3 times, each 5min；
(4) confining liquid (10%FBS, PBS) incubation at room temperature 30min is added, is removed confining liquid, is washed 3 times with PBS, every time
5min；
(5) be separately added into it is one anti-(the anti-CD44 of mouse, 1:200 and rabbit-anti CD34,1：100), 4 DEG C of lucifuge overnight incubations.PBS is washed
Wash 3 times, each 5min；
(6) be separately added into FITC marks it is two anti-(sheep anti-mouse igg and goat anti-rabbit igg, 1：500), room temperature lucifuge incubation
30min.PBS is washed 3 times, each 5min；
(7) 1 μ g/mL DAPI lucifuges are added to redye 5min, PBS is washed 3 times, each 5min；
(8) anti-fluorescence quenching is added, coloration result is observed under inverted fluorescence microscope and is taken pictures.
3rd, the Multidirectional Differentiation of dog mesenchymal stem cells MSCs
(1) Osteoblast Differentiation identification
Take 60mm2The P3 of culture dish culture is for dog mesenchymal stem cells MSCs, to be generated when growing to 80%-90% degree of converging,
Remove culture medium, 3 times are washed with PBS.Gegenbaur's cell induction liquid is added (to refer to mescenchymal stem cell Osteoblast Differentiation culture medium
(ODM) specification is prepared) Fiber differentiation, the conventional complete culture solution of control group addition, nutrient solution of replacing per 2-3d.It is daily to see
The metamorphosis of cell growth state and cell is examined, cell induction identification is carried out in good time, the Gegenbaur's cell after induction carries out alizarin
Red colouring is identified.
As a result as shown in Figure 1A, B.Calcium tubercle after the 21st day Alizarin red staining of dog mesenchymal stem cells MSCs osteogenic induction
Take on a red color, calcium tubercle does not then occur in control group.
(2) identify into fat differentiation
Take 60mm2The P3 of culture dish culture is for dog mesenchymal stem cells MSCs, to be generated when growing to 80%-90% degree of converging,
Remove culture medium, 3 times are washed with PBS.Lipoblast induction liquid is added (with reference to mescenchymal stem cell into fat differential medium
(ADM) specification is prepared) Fiber differentiation, the conventional complete culture solution of control group addition, nutrient solution of replacing per 2-3d.It is daily to see
The metamorphosis of cell growth state and cell is examined, cell induction identification is carried out in good time, the lipoblast after induction carries out oil red O
Dyeing identification.
As a result as shown in C, D of Fig. 1.Fat drips after the 15th day oil red O stain of dog mesenchymal stem cells MSCs adipogenic induction
Take on a red color, fat drips does not then occur in control group.
4th, the preparation of dog mesenchymal stem cell injection
A kind of dog mesenchymal stem cell injection, it includes following components：
The quantity of dog mesenchymal stem cells MSCs is 1 × 105Individual/ml；
Mass volume ratio is 5-10% dog blood albumin；
Mass volume ratio is 0.5% calciparine；
Balance of electrolyte solution.
100ml stem cell injection liquid is prepared such as, the parenteral solution is by dog blood albumin stoste 25ml (mass volume ratio final concentration
For 5%), (final concentration of 0.5%) of mass volume ratio, electrolyte solution are 94.5ml and dog medulla mesenchyma to calciparine 0.5ml
Stem cell constitutes.In addition to mescenchymal stem cell, parenteral solution remaining composition need to be prepared in advance, 4 DEG C of pre- cold standbies, dog medulla mesenchyma
Stem cell is finally resuspended to make single cell suspension in this solution, the number of dog mesenchymal stem cells MSCs in every milliliter of parenteral solution
Measure as 1 × 105It is individual.The ratio of the equal representation quality (g) of mass volume ratio of the present invention and volume (ml).
In 2-15 DEG C of environment temperature, inner cell is maintained as single cell suspension shape to dog mesenchymal stem cells MSCs within 48 hours
State, cell viability are maintained at more than 85%.
Wherein, calciparine is that one kind contains polysaccharide compound, with preventing the agglomerating function of cell aggregation；Blood albumin is equal
For clinical injection liquid composition, can be cells with nutrient, beneficial to the metabolism of cell.
5th, dog mesenchymal stem cell injection is to dog dermal tissue insult therapeutic test
(1) foundation of dog skin trauma model
Dog routinely loses hair or feathers after anaesthetizing, sterilizes, drape, and under sterile working, scapular region, buttocks both sides, are just being 3 × 3cm respectively
Square skin wounds, used as test group, left side is as a control group on right side.
(2) the local injection treatment of dog mesenchymal stem cells MSCs
Injecting pathway：Injecting away from local subcutaneous at wound 5cm for first day and the 3rd day after wound model foundation.
Right side test group wound surrounding subcutaneous injection 1ml contains 1 × 105The culture completely of individual/ml mesenchymal stem cells MSCs
Liquid, left side control group same method injection electrolyte solutions of the 1ml without mesenchymal stem cells MSCs, routinely wound is processed wound
Method is processed.
(3) assessment of wound
After injection of bone marrow mescenchymal stem cell, daily wound healing situation, surface of a wound area are recorded, record the surface of a wound
Healing time.
As a result show, its injection site observed after local injection mesenchymal stem cells MSCs, injection site all without
The bad reactions such as red and swollen heat pain, dog only itself are also reacted without significantly uncomfortable, and food and drink etc. are normal；By comparative experiments group and right
Area according to the wound surface of group is it is found that during wound healing 1-2 all, the area of the experimental group surface of a wound is most of than control group
Surface of a wound area little (Fig. 3)；Can be found by comparing the healing rate in wound healing one week, the speed ratio of experimental group wound healing
Control group is fast (Fig. 4), and Fig. 5 is the observation of the dog skin trauma surface of a wound.
Finally it should be noted that：The preferred embodiments of the present invention are these are only, the present invention is not limited to, although
The present invention is described in detail with reference to embodiment, for a person skilled in the art, which still can be to aforementioned
Technical scheme described in each embodiment is modified, or equivalent is carried out to which part technical characteristic, but it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements made etc. should be included in the protection of the present invention
Within the scope of.
Claims (7)
1. a kind of dog mesenchymal stem cell injection, it is characterised in that include：Dog mesenchymal stem cells MSCs, calciparine, dog blood
Albumin and electrolyte solution.
2. a kind of dog mesenchymal stem cell injection according to claim 1, it is characterised in that the concentration of the calciparine is
0.5%, it is that one kind contains polysaccharide compound, and with preventing the preparation of the agglomerating function of cell aggregation.
3. a kind of dog mesenchymal stem cell injection according to claim 1, it is characterised in that the dog blood albumin stoste
Concentration be 5-10%；Contain dog mesenchymal stem cells MSCs 1 × 10 in 1ml parenteral solutions5It is individual.
4. a kind of preparation method of dog mesenchymal stem cell injection according to claim 1, it is characterised in that dog bone is provided
Bone marrow-drived mesenchymal stem preserves liquid, and precooling, standby, mesenchyme stem cell preserving fluid includes calciparine, dog blood albumin and electrolysis
Matter solution；Dog mesenchymal stem cells MSCs is provided, dog mesenchymal stem cells MSCs is added in mesenchyme stem cell preserving fluid；
By resuspended mode, it is 1 × 10 that adjustment is added to the dog mesenchymal stem cells MSCs quantity in mesenchyme stem cell preserving fluid5
Individual cell/1ml.
5. a kind of preparation method of dog mesenchymal stem cell injection according to claim 4, it is characterised in that wherein described
The preparation of dog mesenchymal stem cells MSCs is comprised the following steps：
(1) dog marrow is taken：Dog routinely loses hair or feathers after anaesthetizing, sterilizes, drape, in left side or right side ilium with No. 16 under sterile working
Bone marrow aspiration pin puncture, with preprepared containing anti-coagulants, anti-coagulants is 3000U heparin, and bone marrow extraction liquid 3ml is in centrifugation
In pipe, 3ml PBS are added, 6ml marrow dilutions are made；
(2) separation of dog bone marrow nucleated cell：The Ficoll liquid of 3ml 1.077g/ml is taken, centrifuge tube is added, is slow added into
6ml marrow dilutions, 2500r/min centrifugation 20min carefully draw the white cloud and mist layer of karyocyte formation in centrifuge tube, plus
Enter the DMEM containing hyclone 10%, 1000r/min centrifugation 5min, upper liquid is carefully siphoned away after centrifugation, ox blood containing tire is added
Clear 10% DMEM, uniformly makes cell suspension with liquid-transfering gun piping and druming；
(3) original cuiture of dog mesenchymal stem cells MSCs：By cell suspension inoculation in blake bottle, 37 DEG C of 5%CO are placed in2Culture
After 24h is cultivated in case, liquid observation of cell are changed；Then liquid, period daily inverted phase contrast microscope observation were once changed per 3 days
The Morphology of cell；
(4) Secondary Culture of dog mesenchymal stem cells MSCs：When cell is paved with Tissue Culture Plate, careful suction is abandoned in culture dish
Nutrient solution, is rinsed 2 times with PBS, is added 0.25% appropriate trypsin solution, is slightly shaken up, put vitellophag at room temperature, is used
Complete medium containing 10% hyclone terminates digestion, blows and beats repeatedly the bottle wall of cell attachment growth, until bottle wall cell is complete
Portion comes off, and makes cell suspension, and continuing gently piping and druming cell suspension makes cell scatter；
(5) identification of dog mesenchymal stem cells MSCs；
(6) skeletonization of dog mesenchymal stem cells MSCs and into fat break up：Treat growth of mesenchymal stem cells to 80%-90% degree of converging
When, remove culture medium, washed with PBS 3 times, add skeletonization and lipoblast induction liquid Fiber differentiation, change per 2-3d and once train
The metamorphosis of nutrient solution, daily observation of cell growth conditions and cell, carries out cell induction identification in good time, and the skeletonization after induction is thin
Born of the same parents, lipoblast carry out alizarin red, oil red O stain identification.
6. a kind of preparation method of dog mesenchymal stem cell injection according to claim 5, it is characterised in that the dog bone
The identification of bone marrow-drived mesenchymal stem detects the expression of BMMSCs CD34 and CD44 albumen by immunofluorescence dyeing method, uses
One resist for rabbit-anti CD34 and the anti-CD44 of mouse, two resist the sheep anti-mouse igg of the goat anti-rabbit igg and FITC marks for FITC marks, bag
Include following steps：
(1) mesenchymal stem cells MSCs in the 3rd generation is taken, is seeded on the culture plate that gelatin was coated with, make cell be affixed on culture plate,
After cell attachment 24h, nutrient solution is suctioned out, 2 times are washed with PBS；
(2) add under 4% paraformaldehyde room temperature and fix 20min, remove fixer, PBS is washed 3 times, each 5min；
(3) saturating agent is added, and saturating agent is 0.1%TritonX-100, PBS, agent is sucked after incubation at room temperature 15min；PBS
Washing 3 times, each 5min；
(4) confining liquid is added, confining liquid is 10%FBS, PBS, is incubated at room temperature 30min, removes confining liquid, washed with PBS 3 times,
Each 5min；
(5) be separately added into it is one anti-(the anti-CD44 of mouse, 1:200 and rabbit-anti CD34,1：100), 4 DEG C of lucifuge overnight incubations, PBS washings 3
It is secondary, each 5min；
(6) be separately added into FITC marks it is two anti-(sheep anti-mouse igg and goat anti-rabbit igg, 1：500), room temperature lucifuge incubation 30min,
PBS is washed 3 times, each 5min；
(7) 1 μ g/mL DAPI lucifuges are added to redye 5min, PBS is washed 3 times, each 5min；
(8) anti-fluorescence quenching is added, coloration result is observed under inverted fluorescence microscope and is taken pictures.
7. a kind of dog mesenchymal stem cell injection according to claim 1, it is characterised in that the dog mescenchymal stem cell
Application of the parenteral solution in treatment dog dermal tissue insult.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107912424A (en) * | 2017-12-01 | 2018-04-17 | 佛山科学技术学院 | A kind of special room-temperature extender of dog mescenchymal stem cell and application |
| CN116808076A (en) * | 2023-08-25 | 2023-09-29 | 北京臻宠生物科技有限公司 | Application method of mesenchymal stem cell repair factor in canine wound treatment |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102920735A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection, preparation method and application thereof in preparing medicine for treating diabetes |
| CN105596372A (en) * | 2015-12-15 | 2016-05-25 | 天津卫硕生物科技有限公司 | Method for treating various severe skin damages by using autogeneic bone marrow-derived mesenchymal stem cells (BMSCs) |
-
2016
- 2016-12-06 CN CN201611108623.6A patent/CN106540244A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102920735A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection, preparation method and application thereof in preparing medicine for treating diabetes |
| CN105596372A (en) * | 2015-12-15 | 2016-05-25 | 天津卫硕生物科技有限公司 | Method for treating various severe skin damages by using autogeneic bone marrow-derived mesenchymal stem cells (BMSCs) |
Non-Patent Citations (3)
| Title |
|---|
| 付小兵等: "骨髓间充质干细胞自体移植提高猪皮肤创面修复质量的初步研究", 《中华医学杂志》 * |
| 张贝莹等: "犬骨髓间充质干细胞的分离培养与鉴定", 《第25届广东省科技进步活动月畜牧兽医学术与科技创新发展大会论文集》 * |
| 方佳等: "犬骨髓间充质干细胞的分离培养与鉴定", 《黑龙江畜牧兽医》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107912424A (en) * | 2017-12-01 | 2018-04-17 | 佛山科学技术学院 | A kind of special room-temperature extender of dog mescenchymal stem cell and application |
| CN116808076A (en) * | 2023-08-25 | 2023-09-29 | 北京臻宠生物科技有限公司 | Application method of mesenchymal stem cell repair factor in canine wound treatment |
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