CN106563161A - Novel drug combination for cosmetic purposes - Google Patents
Novel drug combination for cosmetic purposes Download PDFInfo
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- CN106563161A CN106563161A CN201610983646.5A CN201610983646A CN106563161A CN 106563161 A CN106563161 A CN 106563161A CN 201610983646 A CN201610983646 A CN 201610983646A CN 106563161 A CN106563161 A CN 106563161A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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Abstract
The invention provides a drug combination and uses thereof. The drug combination includes mesenchymal stem cells and soft tissue filling agent. The soft tissue filling agent includes at least one selected from collagen, hyaluronic acid, calcium hydroxyapatite (microcrystalline porcelain), polylactic acid, fat, and platelet-rich plasma. The drug combination is used to enhance the vitality of cells, such as fibroblasts. Compared with the prior art, the drug combination has the advantages of low immunogenicity, quick effect, naturalness, longer-lasting effect, and low risk. The drug combination can be effectively used to remove wrinkles, resist ageing, repair scars and wounds, and the like.
Description
Technical field
The present invention relates to biomedicine field, specifically, the present invention relates to medical composition and its use.
Background technology
From ancient times to the present, U.S.A is an eternal topic.But Time fleets past, no matter how you maintain and cherish, and can not hinder
Only skin relaxes naturally and wrinkle is generated.This make us particularly numerous Mies that like to be beautiful appear aging expression, self-confident and personal evil spirit
Power weakens, and produces negative feeling, have impact on the quality of work and life.Equally, because the face that causes of the factor such as acne, wound
Depressed scar has also brought unlimited worried and pain.
From the point of view of the structure of skin, the thickness of dermal tissue is in close relations with the number of its fibrous tissue and substrate, and with
The compactness of skin, plumpness relaxes closely related with wrinkling, in recent years by increasing Skin cosmesis scholar
Concern.Along with the aging of life rationality and extraneous various stimulations, the speed that collagen protein, hyaluronic acid are lost in skin corium
Begin to accelerate, supply is not as good as consume；Simultaneously the structure of collagen protein and hyaluronic acid is also progressively destroyed, and is lost original
Elastic force；And fibroblastic quantity that collagen protein and hyaluronic acid can be secreted in skin histology is reduced, synthesis capability
Also gradually weaken, this causes skin to start to become lax, wrinkle occurs.Depressed scar is destroyed mainly due to skin corium,
Skin corium is thinning, local dent, and fibrocyte is reduced, and secretion collagen protein and elastic fiberss reduce caused.
However, how effective delay skin aging, beautifies the problem that appearance is still that people constantly seek and solve.
The content of the invention
It is contemplated that at least solving one of technical problem in correlation technique to a certain extent.For this purpose, the present invention is carried
A kind of pharmaceutical composition that can be used for and prevent skin from relaxing with wrinkle generation is naturally gone out, the pharmaceutical composition has immunogen
Property low, instant effect, naturality, the advantage that effect is lasting, risk is low.
In a first aspect of the present invention, the present invention proposes a kind of pharmaceutical composition, and described pharmaceutical composition is used to improve
Cytoactive.Embodiments in accordance with the present invention, the pharmaceutical composition includes：Mescenchymal stem cell；And soft tissue filler, its
In, the soft tissue filler is included selected from collagen protein, hyaluronic acid, calcium hydroxyapatite (crystallite porcelain), polylactic acid, fat
At least one of with platelet rich plasma.Improve cytoactive, eliminate wrinkle using pharmaceutical composition according to embodiments of the present invention
Stricture of vagina, beauty, compared to prior art, overcome the unstable defect of autologous fibroblasts transplanting slowly effect, effect, overcome
The adipose cell survival rate at a specified future date of autologous fat transplantation is unstable, and is easily affected by gravity and sagging, and fat absorption is uneven,
The not good defect of therapeutic effect, overcomes the unfavorable factors such as fat stem cell transplant operation risk is high, range of application is limited.This
Outward, using pharmaceutical composition according to embodiments of the present invention, soft tissue filler is used with mesenchyma stem cell combined, soft tissue
Filler can maintain mescenchymal stem cell in certain space and volume range, not only can play protection and lubricate thin
The effect of born of the same parents, additionally it is possible to improve the activity of efficiency factor in the range of this, is more efficiently promoting the increase of target cell activity.
The pharmaceutical composition of the embodiment of the present invention has low immunogenicity, instant effect, naturality, the advantage that effect is lasting, risk is low.
Embodiments in accordance with the present invention, aforementioned pharmaceutical compositions can further include following additional technical feature at least
One of：
Embodiments in accordance with the present invention, the mescenchymal stem cell is umbilical cord mesenchymal stem cells.Umbilical cord mesenchyma is dry thin
Born of the same parents' wide material sources, immunogenicity is lower, and medication is safer, and umbilical cord mesenchymal stem cells itself can secrete collagen protein simultaneously
The factor including including SCF, bFGF, VEGF, IGF etc. is further secreted, stimulates fibroblast survival and propagation in skin corium
And collagen protein, hyaluronic acid and elastic fiber are further secreted, moreover umbilical cord mesenchymal stem cells can also be further
Promote the generation of vascularization, play a part of nutrition corium and subcutaneous relevant cell, the pharmaceutical composition of the embodiment of the present invention
Drug effect is more persistently, effectively.
Embodiments in accordance with the present invention, the mescenchymal stem cell is P1～P6 for mescenchymal stem cell.Inventor passes through
Experiment finds that being not so good as the cell of early generation, the cytokine amount of secretion more than the mescenchymal stem cell cell state in P6 generations also has
Reduced, P1～P6 lives for mescenchymal stem cell itself is in good condition, cytoactive and cytokine secretion are stronger for improving cell
Property is better.
Embodiments in accordance with the present invention, the mescenchymal stem cell is being suspended in culture medium, be suspended in normal saline or patch
Wall is incubated at what the form in culture medium was provided.Embodiments in accordance with the present invention, the mescenchymal stem cell is with above-mentioned three kinds of shapes
The one of which of formula is provided, and the good cell state of stem cell is filled between can keeping.
Embodiments in accordance with the present invention, the mescenchymal stem cell is being suspended in culture medium or be suspended in normal saline
Form is provided, and the density of the mescenchymal stem cell is 0.1*106-10*106Individual/ml.Inventor is found through experiments, mesenchyme
Suspension density of the stem cell in culture medium or normal saline is 0.1*106-10*106Individual/ml, can be further ensured that mesenchyme is done
The kilter of cell simultaneously provides the volume ratio of suitable size, and then causes the pharmaceutical composition of the embodiment of the present invention to be used to improve
Cytoactive it is better.
The volume ratio of embodiments in accordance with the present invention, the culture medium or the normal saline and the soft tissue filler
For 1:0.5～1:5.Inventor has found that the suspension density in culture medium or normal saline of mescenchymal stem cell is 0.1*
106-10*106Individual/ml, culture medium or normal saline are 1 with the volume ratio of soft tissue filler:0.5～1:5 so that the present invention
The pharmaceutical composition of embodiment is used to improve the better of cytoactive.
Embodiments in accordance with the present invention, the mescenchymal stem cell is provided in the form of adhere-wall culture is in the culture medium, institute
The adherent density for stating mescenchymal stem cell is 1*104～3*104Individual/cm2.Inventor is found through experiments, mescenchymal stem cell patch
Wall is incubated in culture medium, and adherent density is 1*104～3*104Individual/cm2, the good shape of mescenchymal stem cell can be further ensured that
State, and then cause the pharmaceutical composition of the embodiment of the present invention to be used to improve the better of cytoactive.
Embodiments in accordance with the present invention, the culture medium is 1 with the volume ratio of the soft tissue filler:0.5～1:5.
Inventor has found that the volume ratio of culture medium and soft tissue filler is 1:0.5～1:In the range of 5, described pharmaceutical composition is being carried
Effect is more significantly in the activity of high cell.
Embodiments in accordance with the present invention, the dosage form of described pharmaceutical composition is injection.Pharmaceutical composition is with injection
Dosage form is provided, and pharmaceutical composition can more accurately arrive at site to be treated, the therapeutic effect of pharmaceutical composition can more added with
Effect, persistently.
In a second aspect of the present invention, the present invention proposes purposes of the pharmaceutical composition noted earlier in medicine is prepared,
The medicine is used to resist aging, smooth away wrinkles and repair in trauma.The medicine of the embodiment of the present invention is used to resist aging, eliminates wrinkle
Stricture of vagina, with the low advantage of instant effect, lasting medicine, low immunogenicity, naturality and risk.
Description of the drawings
Fig. 1 is the aspect graph of umbilical cord mesenchymal stem cells according to embodiments of the present invention；And
Fig. 2 is according to embodiments of the present invention to identify selected umbilical cord mesenchymal stem cells by FCM analysis
The result figure of surface marker.
Specific embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this
It is bright, and be not considered as limiting the invention.
Pharmaceutical composition
A first aspect of the present invention, the present invention proposes a kind of pharmaceutical composition, and the pharmaceutical composition is used to improve cell
Activity, is the new pharmaceutical composition for beauty.Embodiments in accordance with the present invention, the pharmaceutical composition includes：Mesenchyme is done
Cell；And soft tissue filler, wherein, the soft tissue filler is included selected from collagen protein, hyaluronic acid, calcium hydroxyl phosphorus
At least one of lime stone (crystallite porcelain), polylactic acid, fat and platelet rich plasma.Using medicine group according to embodiments of the present invention
Compound improve cell viability, smooth away wrinkles, beauty, compared to prior art, overcome autologous fibroblasts transplanting slowly effect,
The unstable defect of effect, the adipose cell survival rate at a specified future date for overcoming autologous fat transplantation is unstable, and easily receives gravity shadow
Loud and sagging, fat absorption is uneven, the not good defect of therapeutic effect, overcomes fat stem cell transplant operation risk height, answers
With the unfavorable factor such as scope is limited.Pharmaceutical composition according to embodiments of the present invention adopts mescenchymal stem cell, and immunogenicity is low.
Pharmaceutical composition according to embodiments of the present invention, the soft tissue filler, by taking hyaluronic acid as an example, it has water absorption, height
Negative charge property, biocompatibility and the biological activity in wound healing etc., are gradually applied to Wound dressing and organizational project
Etc. skin wound treatment field.Hyaluronic acid participates in inflammation, propagation and the reconstruction phase of skin wound healing.Wherein in proliferation period,
Short chain hyaluronic acid (containing 6～20 monosaccharide) can promote migration of fibroblast cells to wound tissue, and fibroblast produces collagen
With glycosaminoglycans (GAG), new extracellular matrix (ECM) is built；Additionally, hyaluronic acid may additionally facilitate angiogenesis and cutin shape
Propagation and migration into cell, further promotes the healing of skin trauma.Mescenchymal stem cell and including collagen, hyalomitome
At least one of acid, calcium hydroxyapatite (crystallite porcelain), polylactic acid, fat and platelet rich plasma are composite filled, plastotype is plentiful,
Instant effect；It repairs cicatrix and wrinkle, does not destroy skin script structure, and the somatomedin secreted with mescenchymal stem cell is activated certainly
The fibroblastic function of body, and further accelerate, reaching filling improves skin vitality state, with naturality；Additionally,
It is expelled to collagen protein at skin defect, hyaluronic acid, calcium hydroxyapatite (crystallite porcelain), polylactic acid, fat and Fu Xue little
Plate blood plasma has the effect of smoothing wrinkle, repairs the smooth of skin, gloss and elasticity, and effect is lasting；Finally, the pharmaceutical composition can be adopted
With injection fillers, the risk of failure of operation is evaded, has been a kind of very safe drugs.In addition, inventor has found, the present invention is public
The pharmaceutical composition opened has the advantage for being substantially better than the two exclusive use --- and soft tissue filler can be dry thin by mesenchyme
Born of the same parents are maintained in certain space and volume range, not only can play a part of protection and lubricant cell, additionally it is possible to which improving should
In the range of efficiency factor activity, be more efficiently promoting target cell activity increase.By taking hyaluronic acid as an example, its with
Mesenchymal stem cells are used in combination in addition to having above-mentioned advantage, and hyaluronic acid may also operate as keeping mescenchymal stem cell water
Point, protect mescenchymal stem cell not receive the effect such as infringement of pathogen.
Another specific embodiment of the invention, the mescenchymal stem cell is umbilical cord mesenchymal stem cells.Between umbilical cord
Mesenchymal stem cells wide material sources, it can be adopted from Biohazard Waste --- separating mesenchymal stem cell in umbilical cord, it is not necessary to from trouble
Draw materials with person, remove the risk and pain of corrective surgery from, and umbilical cord mesenchymal stem cells immunogenicity is lower, medication is more pacified
Entirely, umbilical cord mesenchymal stem cells can then secrete collagen protein, and can further secrete including SCF, bFGF, VEGF, IGF etc.
The interior factor, stimulates fibroblast in skin corium to survive with propagation and further secretes collagen protein, hyaluronic acid and elasticity
Fiber, moreover umbilical cord mesenchymal stem cells can also further promote the generation of vascularization, further function as nutrition corium
And the effect of subcutaneous relevant cell, drug effect is stronger.
Specific example of the invention, the mescenchymal stem cell is P1～P6 for mescenchymal stem cell.Inventor is led to
Cross experiment to find, the cell state of P6 mescenchymal stem cells more than generation is not as the cell of early generation, the cytokine amount of secretion
Also reduced, P1～P6 is good for the cell state of mescenchymal stem cell, cytoactive and cytokine secretion ability are stronger, for carrying
The active effect of high cell is more significantly.
Specific example of the invention, the mescenchymal stem cell being suspended in culture medium, be suspended in normal saline or
Form of the adhere-wall culture in culture medium is provided.Inventor is by testing it was unexpectedly observed that mescenchymal stem cell is to be suspended in
Culture medium, it is suspended in form in culture medium of normal saline or adhere-wall culture and provides, it is ensured that mescenchymal stem cell is good
Growth conditions.
According to a particular embodiment of the invention, the mescenchymal stem cell is being suspended in culture medium or be suspended in normal saline
In form provide, the density of the mescenchymal stem cell is 0.1*106-10*106Individual/ml.Be conducive to keeping mesenchyme dry thin
The activity of born of the same parents, is conducive to secretion somatomedin and the collagen protein for keeping mescenchymal stem cell higher, and then causes the present invention
The effect that the pharmaceutical composition of embodiment is used to improve cytoactive is more significantly.
The volume ratio of embodiments in accordance with the present invention, the culture medium or the normal saline and the soft tissue filler
For 1:0.5～1:5.Inventor has found that the suspension density in culture medium or normal saline of mescenchymal stem cell is 0.1*
106-10*106Individual/ml, culture medium or normal saline are 1 with the volume ratio of soft tissue filler:0.5～1:5, both can ensure that
The good cell state of mescenchymal stem cell, can ensure that the strong positive physiological effect of soft tissue filler again, such as external mould
Intend or be applied to decree stricture of vagina, corners of the mouth dimpled grain, volume glabellar frown lineses to fill up, it might even be possible to the repairing nose bridge of the nose, rich lip, repair down
The recessed scar amendment of bar, comedo and chickenpox, so as to produce smooth, full, resilient skin.Culture medium or normal saline with soft group
The volume ratio for knitting filler is 1:0.5～1:5 so that the pharmaceutical composition of the embodiment of the present invention is used to improve the effect of cytoactive
Fruit is more preferably.
Some specific embodiments of the invention, the mescenchymal stem cell is in the form of adhere-wall culture is in the culture medium
There is provided, density of the mescenchymal stem cell in the culture medium is 1*104～3*104Individual/cm2.Inventor's discovery, cell
Growth has a Density dependence effect, overstocked or excessively dilute can all affect cell state, density to be less than 1*104Individual/cm2When, cell state
Difference；Higher than 3*104Individual/cm2When, cell do not have enough Growth In Spaces, and culture medium is consumed excessively, and cell state is poor.According to this
The specific embodiment of invention, in culture medium, its adherent density is 1*10 to mescenchymal stem cell adhere-wall culture4～3*104Individual/
cm2, be conducive to keeping the activity of mescenchymal stem cell, be conducive to keeping the higher secretion somatomedin of mescenchymal stem cell and
Collagen protein, so cause the embodiment of the present invention pharmaceutical composition be used for improve cytoactive effect it is more significantly.
Some specific embodiments of the invention, the culture medium is 1 with the volume ratio of the soft tissue filler:
0.5～1:5.Inventor has found that the volume ratio of culture medium and soft tissue filler is 1:0.5～1:In the range of 5, the medicine group
Compound effect in the activity for improving cell is more significantly.
Embodiments in accordance with the present invention, the dosage form of described pharmaceutical composition is injection.The pharmaceutical composition of the present invention can
To be administered by any common approach, as long as it can reach expected tissue, the main approach administration by injection.Give
The various modes of medicine can be it is contemplated that including subcutaneous, cortex, locally, but the invention is not restricted to these administrations illustrated
Mode.Preferably, compositionss of the invention are provided with the dosage form of injection, and the pharmaceutical composition of the present invention can be more accurately
Site to be treated is arrived at, the therapeutic effect of pharmaceutical composition can be more efficient, lasting.Additionally, the pharmaceutical composition of the present invention can
To be administered using the particular instrument that active component is sent to target cell.
The administration frequency and dosage of the pharmaceutical composition of the present invention can be determined by multiple correlative factors, the factor bag
Including will be by the type of beauty, route of administration, by the age of medicine, sex, body weight and the order of severity and the medicine as active component
Species type.Some embodiments of the invention, dosage can be divided into 1 dose, 2 doses of suitable form or multi-agent, with the whole time
With 1 time, 2 times or multiple dosing in section, as long as reaching beauty effective dose.
Term " beauty effective dose " refers to that compound be enough to significantly improve the amount of some related symptoms, also as given disease
Disease and dosage regimen provide the amount of cosmetic result.For example, in beauty, reduce, prevent, delay, suppress or block any symptom
Medicine or compound should be therapeutically effective.The medicine or compound of beauty effective dose need not cure symptom, but will be
Symptom provides treatment so that the outbreak of individual symptom is delayed, prevents or prevents, or symptom is alleviated, or symptom
Time limit be changed, or such as symptom becomes not serious, or fast track rehabilitation.
Term " beauty " is used to refer to the desired pharmacology of acquisition and/or physiologic effect.The effect is just wholly or in part
Can be preventative for prevention symptom." beauty " used herein covers the symptom of mammal, particularly people.Make herein
" beauty " to be covered and give individuality by medicine or compound to cure, alleviate, improve, mitigate or suppress appointing for the symptom of individuality
What medication, will including but not limited to contain pharmaceutical composition described herein and give individuality in need.
Embodiments in accordance with the present invention, the pharmaceutical composition of the present invention can be in combination with conventional treatments and/or therapy
Use, or can be used separately with conventional treatments and/or therapy.When the mescenchymal stem cell or pharmaceutical composition of the present invention
When being administered in using the conjoint therapy with other medicines, they can sequentially or simultaneously give individuality.Or, it is of the invention
Pharmaceutical composition can comprising pharmaceutically acceptable carrier or pharmaceutically acceptable excipient and it is known in the art other
The combination of curative or preventive drug.
The purposes of pharmaceutical composition
In a second aspect of the present invention, the present invention proposes purposes of the pharmaceutical composition noted earlier in medicine is prepared,
The medicine be used for resist aging, smooth away wrinkles, repair in trauma.Mesenchyme in the pharmaceutical composition of the embodiment of the present invention is dry thin
Born of the same parents not only can secrete the cytokine including including bFGF, VEGF, IGF etc., play nutrition, stimulate in skin corium into fiber finer
Born of the same parents are survived and propagation, further secrete collagen protein, hyaluronic acid and elastic fiber, fill corium Rotating fields, and smooth out wrinkles are filled out
Mend depression；In addition, mescenchymal stem cell can secrete generation collagen protein, further increase the thickness and elasticity of skin, make recessed
Sunken part is unfolded, and fills and leads up wrinkle and defect, recovers the elasticity and gloss of skin.The further composite transparent of mescenchymal stem cell
Matter acid, collagen protein etc. feed back to patient skin fault location and can carry out effective wrinkle elimination, cicatrix reparation, opposing aging etc..
The medicine of the embodiment of the present invention is used to resist aging, smooth away wrinkles, with instant effect, lasting medicine, immunogenicity be low, naturality
The advantage low with risk.
The method for improving cell viability
In a third aspect of the present invention, the present invention proposes a kind of method of raising cytoactive.Reality of the invention
Example is applied, the method includes：Target cell is contacted with mescenchymal stem cell and soft tissue filler, wherein, it is described soft
Tissue filling agent is included selected from collagen protein, hyaluronic acid, calcium hydroxyapatite (crystallite porcelain), polylactic acid, fat and Fu Xue little
At least one of plate blood plasma.Using the method for raising cytoactive according to embodiments of the present invention, for smoothing away wrinkles, beautiful
Hold, compared to prior art, overcome the unstable defect of autologous fibroblasts transplanting slowly effect, effect, overcome autologous
The adipose cell survival rate at a specified future date of fat transplantation is unstable, and is easily affected by gravity and sagging, and fat absorption is uneven, treatment
The defect of effect on driving birds is not good, overcomes the unfavorable factors such as fat stem cell transplant operation risk is high, range of application is limited.According to this
The pharmaceutical composition of bright embodiment adopts mescenchymal stem cell, and immunogenicity is low；Mescenchymal stem cell and including collagen, thoroughly
Composite filled, the plastotype of at least one of bright matter acid, calcium hydroxyapatite (crystallite porcelain), polylactic acid, fat and platelet rich plasma
Plentiful, instant effect；It repairs cicatrix and wrinkle, does not destroy skin script structure, the somatomedin secreted with mescenchymal stem cell
The function of activation autologous fibroblasts, and further accelerate, reaching filling improves skin activity state, with nature
Property；Additionally, the collagen protein being expelled at skin defect, hyaluronic acid, calcium hydroxyapatite (crystallite porcelain), polylactic acid, fat
With the effect that platelet rich plasma has smoothing wrinkle, the smooth of skin, gloss and elasticity are repaired, effect is lasting；Finally, party's regulation
The risk of failure of operation is kept away, has been a kind of very safe method.In addition, inventor has found, raising cell disclosed by the invention
Activity method have be substantially better than the two exclusive use advantage --- soft tissue filler can tie up mescenchymal stem cell
Hold in certain space and volume range, protection and lubricant cell not only can be played a part of, additionally it is possible to improve the scope
The activity of interior efficiency factor, is more efficiently promoting the increase of target cell activity.By taking hyaluronic acid as an example, itself and mesenchyme
Stem cell is used in combination in addition to having above-mentioned advantage, and hyaluronic acid may also operate as keeping mescenchymal stem cell moisture, protect
Shield mescenchymal stem cell does not receive the effect such as infringement of pathogen.
Another specific embodiment of the invention, the mescenchymal stem cell is umbilical cord mesenchymal stem cells.Between umbilical cord
Mesenchymal stem cells wide material sources, it can be adopted from Biohazard Waste --- separating mesenchymal stem cell in umbilical cord, it is not necessary to from trouble
Draw materials with person, remove the risk and pain of corrective surgery from, and umbilical cord mesenchymal stem cells immunogenicity is lower, it is safer,
Umbilical cord mesenchymal stem cells can further secrete the factor including including SCF, bFGF, VEGF, IGF etc., stimulate in skin corium into
Fibrocyte is survived with propagation and further secretes collagen protein, hyaluronic acid and elastic fiber, moreover umbilical cord mesenchyma
Stem cell can also further promote the generation of vascularization, further function as the effect of nutrition corium and subcutaneous relevant cell, medicine
Effect is stronger.
Specific example of the invention, the mescenchymal stem cell is P1～P6 for mescenchymal stem cell.Inventor is led to
Cross experiment to find, the cell state of P6 mescenchymal stem cells more than generation is not as the cell of early generation, the cytokine amount of secretion
Also reduced, P1～P6 is good for the cell state of mescenchymal stem cell, cytoactive and cytokine secretion ability are stronger, for carrying
The active effect of high cell is more significantly.
Specific example of the invention, the mescenchymal stem cell being suspended in culture medium, be suspended in normal saline or
Form of the adhere-wall culture in culture medium is provided.Inventor is by testing it was unexpectedly observed that mescenchymal stem cell is to be suspended in
Culture medium, it is suspended in form in culture medium of normal saline or adhere-wall culture and provides, it is ensured that mescenchymal stem cell is good
Growth conditions.
According to a particular embodiment of the invention, the mescenchymal stem cell is being suspended in culture medium or be suspended in normal saline
In form provide, the density of the mescenchymal stem cell is 0.1*106-10*106Individual/ml.Be conducive to keeping mesenchyme dry thin
The activity of born of the same parents, is conducive to secretion somatomedin and the collagen protein for keeping mescenchymal stem cell higher, and then causes the present invention
The effect that the method for embodiment is used to improve cytoactive is more significantly.
The volume ratio of embodiments in accordance with the present invention, the culture medium or the normal saline and the soft tissue filler
For 1:0.5～1:5.Inventor has found that the suspension density in culture medium or normal saline of mescenchymal stem cell is 0.1*
106-10*106Individual/ml, culture medium or normal saline are 1 with the volume ratio of soft tissue filler:0.5～1:5, both can ensure that
The good cell state of mescenchymal stem cell, can ensure that the strong positive physiological effect of soft tissue filler again, such as external mould
Intend or be applied to decree stricture of vagina, corners of the mouth dimpled grain, volume glabellar frown lineses to fill up, it might even be possible to the repairing nose bridge of the nose, rich lip, repair down
The recessed scar amendment of bar, comedo and chickenpox, so as to produce smooth, full, resilient skin.Culture medium or normal saline with soft group
The volume ratio for knitting filler is 1:0.5～1:5 so that the embodiment of the present invention for improve cytoactive method effect more
It is good.
Some specific embodiments of the invention, the mescenchymal stem cell is in the form of adhere-wall culture is in the culture medium
There is provided, density of the mescenchymal stem cell in the culture medium is 1*104～3*104Individual/cm2.Inventor's discovery, cell
Growth has a Density dependence effect, overstocked or excessively dilute can all affect cell state, density to be less than 1*104Individual/cm2When, cell state
Difference；Higher than 3*104Individual/cm2When, cell do not have enough Growth In Spaces, and culture medium is consumed excessively, and cell state is poor.According to this
The specific embodiment of invention, in culture medium, its adherent density is 1*10 to mescenchymal stem cell adhere-wall culture4～3*104Individual/
cm2, be conducive to keeping the activity of mescenchymal stem cell, be conducive to keeping the higher secretion somatomedin of mescenchymal stem cell and
Collagen protein, so cause the embodiment of the present invention method be used for improve cytoactive effect it is more significantly.
Some specific embodiments of the invention, the culture medium is 1 with the volume ratio of the soft tissue filler:
0.5～1:5.Inventor has found that the volume ratio of culture medium and soft tissue filler is 1:0.5～1:In the range of 5, methods described exists
Improve effect in the activity of cell more significantly.
Embodiments in accordance with the present invention, the target cell, the number ratio of the mescenchymal stem cell are 1:0.1～1：
10.Inventor has found that target cell, the number ratio of mescenchymal stem cell are higher than 1:0.1, the quantity of mescenchymal stem cell is relatively
Few, the factor amount of its secretion is low, is not enough to promote the increase of substantial amounts of target cell activity；Target cell, mescenchymal stem cell
Number ratio be less than 1:10, the quantity of target cell is very few, the saturation of mescenchymal stem cell excreted factor is caused, to a certain degree
On cause the waste of certain cell and factor.Target cell, the number ratio of the mescenchymal stem cell are 1:0.1～1：10
Interior, the activity to improving target cell is more quick, effective.
It should be noted that herein described " beauty " " wrinkle removing " " cicatrix reparation " " repair in trauma etc. " etc. is with non-treatment
For the purpose of.
It should be noted that described herein, " target cell enters with mescenchymal stem cell and soft tissue filler
Row contact ", both can be the cytokine of contact between cell individual and filler, or mescenchymal stem cell secretion
Contact between target cell and soft tissue filler.
It should be noted that " cytoactive " described herein refers to the physiological statuss and function of cell, including it is thin
The maintenance of born of the same parents' basic status, ability of cell proliferation (sometimes referred to as vigor), the ability of excreted factor and regular growth function.
It should be noted that " cell state is good " described herein refers to that cell maintains form during normal culture good
(by taking mescenchymal stem cell as an example, cell attachment, cell is in fusiformis, lamellar growth), cell is bright, clear-cut margin, cell refractive power
Property preferably, contamination-free in whole cell cultivation process (fill including mycoplasma, antibacterial, funguses, black glue, protozoon etc.) occurs.
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this
It is bright, and be not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according to text in the art
Offer described technology or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument,
For can pass through city available from conventional products.
Embodiment 1
In the present embodiment, inventor has screened the mescenchymal stem cell and collagen protein or hyalomitome including separate sources
The pharmaceutical composition of acid or calcium hydroxyapatite (crystallite porcelain) or polylactic acid or fat or platelet rich plasma is lived to improving cell
Property impact, in the present embodiment, wait to carry highly active cell for fibroblast, the mescenchymal stem cell bag of separate sources
Include：It is that umbilical cord is originated, Placenta Hominiss source, derived from bone marrow, periosteum source, adipose-derived, tooth source etc..Inventor
It was found that, the mescenchymal stem cell in umbilical cord source itself belongs to Biohazard Waste, carefully compared to the mescenchymal stem cell that other are originated
Born of the same parents' immunogenicity is lower, and with certain versatility, the cell quality for being obtained is high, quantity is more, purity is also higher.And then rear
In continuous experiment, inventor selects to include the mescenchymal stem cell and collagen protein or hyaluronic acid or calcium hydroxy-apatite that umbilical cord is originated
The pharmaceutical composition of stone (crystallite porcelain) or polylactic acid or fat or platelet rich plasma carries out subsequent detection.Hereinafter, with mesenchyme
As a example by the pharmaceutical composition of stem cell and hyaluronic acid, labor and description are carried out.
Embodiment 2
In the present embodiment, inventor to the form and phenotype of selected umbilical cord mesenchymal stem cells carried out observation and
Identification.As a result it is as described below：
The morphologic appearance of umbilical cord mesenchymal stem cells is cell attachment, in fusiformis, lamellar growth.Concrete form such as Fig. 1 institutes
Show.Under the such as growth conditions of Fig. 1, the multiplication capacity of umbilical cord mesenchymal stem cells is strong, and secretion includes for umbilical cord mesenchymal stem cells
BFGF, VEGF, IGF etc. are strong in the multiplication capacity of interior cytokine and collagen protein, are suitably selected for as medicine of the present invention
The umbilical cord mesenchymal stem cells of compositionss.
Inventor identifies that selected umbilical cord mesenchymal stem cells express CD90 (positive cells by FCM analysis
Rate：99%), CD105 (positive cell rate：99%), CD73 (positive cell rate：99%), not expression of HLA-DR, CD14, CD19,
CD34 and CD45, as a result as shown in Figure 2.
Embodiment 3
In the present embodiment, on consumption of the inventor to the mescenchymal stem cell in selected umbilical cord source with hyaluronic acid
Screened.
Inventor has carried out the Cell viability experiment of mescenchymal stem cell, and experimentation is as described below：
1st, umbilical cord mesenchymal stem cells are digested：The umbilical cord mesenchymal stem cells that one bottle grows to 80%～90% are used into pancreas
Enzymic digestion, in culture medium with after, is centrifuged 5min under conditions of 1200rpm；
2nd, re-suspended cell：Supernatant is abandoned after centrifugation, is carried out using umbilical cord mesenchymal stem cells culture medium resuspended；
3rd, inoculating cell：Cell is inoculated with according to same volume (100 μ l) (dry thin per 2000, hole mesenchyme
Born of the same parents), each hole is repeated 5 times, and experiment 3 days, i.e. each hole are carried out altogether 15 repetition samples.Afterwards by postvaccinal cell in 37
DEG C, 5%CO2Incubated overnight in incubator.
4th, hyaluronic acid/collagen protein is added：Old culture medium is abandoned after cell pellet overnight culture is adherent, and is added into fresh
Culture medium.Wherein, matched group only adds culture medium, and experimental group adds hyaluronic acid, wherein culture medium according to following volume ratio：
The volume ratio of hyaluronic acid is：1：0.01,1：0.1,1：0.5,1：1,1：5,1：10, cumulative volume is 100 μ l.
5th, detect：Culture medium and hyaluronic acid/collagen protein are lost with pipette tips after 24 hours, 48 hours and 72 hours, it
Afterwards PBS is washed once, the culture medium for adding 100 μ l fresh, and adds 10 μ l CCK8, soft to mix, it is to avoid the generation of bubble, it
37 DEG C afterwards, 5%CO2In incubator stand 3-4 hours, after OD560 at detection absorption photometric value.All Time point has been detected
Bi Hou, carries out the analysis of statistical data.
Experimental result shows that the volume ratio of culture medium and hyaluronic acid is 1：0.5-1:Fill between can keeping in the range of 5
The primary activity of matter stem cell, mescenchymal stem cell it is active good.It is 1 in volume ratio：0.01 and 1：When 0.1, hyaluronic acid mistake
It is dilute, affect the performance of the effect of hyaluronic acid；Volume ratio is 1：10, then mescenchymal stem cell cell state be deteriorated, mesenchyme
Stem cell mortality rate substantially rises.Therefore preferred culture medium is 1 with the volume ratio of hyaluronic acid：0.5-1:5.
Embodiment 4
In the present embodiment, inventor is using the mescenchymal stem cell and hyaluronic acid in selected umbilical cord source and into fibre
Dimension co-culture of cells, and fibroblastic activity is detected.
Experimentation is as described below：
1st, fibroblast is digested：The fibroblast that one bottle grows to 80%～90% is digested using pancreatin, is cultivated
In base with after, 5min is centrifuged under conditions of 1200rpm.
2nd, it is fibroblastic simultaneously in digestion, digest umbilical cord mesenchymal stem cells.Concrete operations are as described below：By one bottle
The umbilical cord mesenchymal stem cells for growing to 80%～90% are digested using pancreatin, in culture medium with after, under conditions of 1200rpm
Centrifugation 5min.
3rd, re-suspended cell：Supernatant is abandoned after centrifugation, using culture medium by cell according to 1*105The density of individual/ml carries out resuspended.
4th, inoculating cell：By 1*104Individual fibroblast is inoculated in 24 orifice plates, and each hole is repeated 3 times that (one has 3
Detection time point, i.e. each hole have 9 repetition samples), the Transwell cells that aperture is 1 μm are placed afterwards, and (suspension type cell is trained
Foster ware) and by 0.7*104Individual mescenchymal stem cell is inoculated in cell, culture medium cumulative volume be on close cell along be it is optimal, it
Afterwards by postvaccinal cell in 37 DEG C, 5%CO2Incubated overnight in incubator.
5th, hyaluronic acid is added：Abandon old culture medium after cell pellet overnight culture is adherent, PBS wash add afterwards for 1 time it is fresh
Serum-free medium.Wherein, matched group only adds culture medium, and experimental group is according to (the suspension type cell culture of Transwell cells
Ware) culture volume and hyaluronic acid volume ratio be 1：1 addition hyaluronic acid.
6th, detect：Only retain fibroblast after 12 hours, 24 hours and 48 hours and washed once using PBS, add 1mL
Fresh serum-free medium, and 100 μ l CCK8 are added, it is soft to mix, it is to avoid the generation of bubble, 37 DEG C afterwards, 5%CO2Training
In foster case stand 3-4 hours, after OD560 at detection absorption photometric value.After the detection of All Time point is finished, statistical number is carried out
According to analysis.
Experimental result shows, cell culture 12 and has what mescenchymal stem cell and soft tissue filler were located after 24 hours
It is basis set that the OD values of experimental group, cell state and detection will be substantially better than exclusive use serum-free culture；After 48 hours, due to cell
In the state of being chronically at serum-free culture, integrality is not so good as 12 hours and 24 hours, but also observes that experimental group
Result be better than matched group.It is thin that above-mentioned the results show, mescenchymal stem cell and soft tissue filler can improve target
The activity of born of the same parents, such as fibroblast.
Embodiment 5
In the present embodiment, inventor further demonstrates the pharmaceutical composition of the embodiment of the present invention by zoopery
Medication effect.
By umbilical cord mesenchymal stem cells with 1 × 106The density (being suspended in normal saline) of/mL is with injection hyaluronic acid (thoroughly
The acid of bright matter) (auspicious blue No. 2) according to 1：2 ratio is mixed before the experiments were performed, and cell-hyaluronic acid is formed after mix homogeneously
Complex.Carry out zoopery afterwards, concrete operations are the packet implantation male BLAB/c nude mices (scale of construction about 20g, purchased from dimension tonneau
China, production licence number：SCXK (capital) 2012-0001).Experimental mouse itself is matched group and experimental group, respectively by injection
Hyaluronic acid (left side), umbilical cord mesenchymal stem cells-hyaluronic acid complex (right side) are injected in the nude mice back of the body by each sample 0.3mL
The left side and right side of portion's waist buttockss skin, per group of each 1 point, after 6-10 is all injection site form is observed again.
Concrete outcome is as follows：
(1) observation before not injecting：Preparation carries out zooperal male BLAB/c nude mices before not injecting, its back
Skin is regular, the form without abnormal projection.
(2) observation after injecting is completed：By nude mice left side waist skin of buttock intradermal injection 300ul hyaluronic acid, right side waist buttockss
The μ l umbilical cord mesenchymal stem cells of the injection of skin 300-hyaluronic acid complex at the parallel position in portion.From in form, Mouse Lumbar buttocks
Respectively there is a sizable projection left and right, and bump height is about 0.5cm, and radius is about the circle of 0.4cm or so and (is close to circle
Shape, substantially oval) certain hardness is felt, it is able to maintain that certain form.
(3) observation in 6-10 weeks after completing to inject：After experiment development 6-10 is all, zoomorphism is observed again.Left side is individually
The cutaneous protuberance state that injection hyaluronic acid group is formed is gradually lowered (size can be ignored substantially), but between right side injection umbilical cord
There is certain protuberance form the side of mesenchymal stem cells-hyaluronic acid complex, and bump height is about 0.25cm, and radius is about
The circle (being close to circle, substantially oval) of 0.35cm or so, protuberance amplitude is apparently higher than individually injection hyaluronic acid group.This table
Bright umbilical cord mesenchymal stem cells-hyaluronic acid complex is more permanent to the maintenance after moulding.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.And, the specific features of description, structure, material or feature can be with office
Combine in an appropriate manner in one or more embodiments or example.Additionally, in the case of not conflicting, the skill of this area
Art personnel can be tied the feature of the different embodiments or example described in this specification and different embodiments or example
Close and combine.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (10)
1. a kind of pharmaceutical composition, described pharmaceutical composition is used to improve cytoactive, it is characterised in that include：
Mescenchymal stem cell；And
Soft tissue filler,
Wherein, the soft tissue filler include selected from collagen protein, hyaluronic acid, calcium hydroxyapatite, polylactic acid, fat and
At least one of platelet rich plasma.
2. pharmaceutical composition according to claim 1, it is characterised in that the mescenchymal stem cell is dry for umbilical cord mesenchyma
Cell.
3. pharmaceutical composition according to claim 1, it is characterised in that the mescenchymal stem cell is to fill between P1～P6 generations
Matter stem cell.
4. pharmaceutical composition according to claim 1, it is characterised in that the mescenchymal stem cell is being suspended in culture
Base, it is suspended in what form in culture medium of normal saline or adhere-wall culture was provided.
5. pharmaceutical composition according to claim 4, it is characterised in that the mescenchymal stem cell is being suspended in culture medium
Or the offer of the form in normal saline is provided, the density of the mescenchymal stem cell is 0.1*106-10*106Individual/ml.
6. pharmaceutical composition according to claim 5, it is characterised in that the culture medium or the normal saline with it is described
The volume ratio of soft tissue filler is 1:0.5～1:5.
7. pharmaceutical composition according to claim 1, it is characterised in that the mescenchymal stem cell is with adhere-wall culture in training
Form in foster base is provided, and the adherent density of the mescenchymal stem cell is 1*104～3*104Individual/cm2。
8. pharmaceutical composition according to claim 7, it is characterised in that the culture medium and the soft tissue filler
Volume ratio is 1:0.5～1:5.
9. pharmaceutical composition according to claim 1, it is characterised in that the dosage form of described pharmaceutical composition is injection.
10. purposes of any one of claim 1～9 described pharmaceutical composition in medicine is prepared, the medicine declines for opposing
Always, smooth away wrinkles.
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