CN106581760A - Special treatment method for enhancing cell activity - Google Patents
Special treatment method for enhancing cell activity Download PDFInfo
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- CN106581760A CN106581760A CN201610983606.0A CN201610983606A CN106581760A CN 106581760 A CN106581760 A CN 106581760A CN 201610983606 A CN201610983606 A CN 201610983606A CN 106581760 A CN106581760 A CN 106581760A
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Abstract
The invention provides a special treatment method for enhancing cell activity. The method includes the steps of: performing first contact to a mesenchymal stem cell with a collagen protein stimulating factor; performing second contact to a target cell with the mesenchymal stem cell, after the first contact, and soft tissue filler being selected from at least one of collagen protein, hyaluronic acid, calcium hydroxyapatite, polylactic acid, fat, and platelet-rich plasma. The method can improve cell activity, for example, the activity of a fibroblast. Compared with the prior art, the method is low in immunogenicity, has quicker effects, is natural, has good curative effects and more durable effects, and is low in risk. The special treatment method can effectively used for removing wrinkles, resisting senility and scar, and repairing wounds.
Description
Technical field
The present invention relates to biomedicine field, specifically, the present invention relates to the method for improving cytoactive.
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 method that can be used for and prevent skin from relaxing the raising cytoactive generated with wrinkle is naturally gone out, the method has immunity
Originality is low, instant effect, naturality, good effect, the advantage that effect is lasting, risk is low.
In a first 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：Mescenchymal stem cell and collagen protein stimulating factor are carried out into the first contact；And by target cell with
Mescenchymal stem cell and soft tissue filler carry out the second contact after first contact, and the soft tissue filler is included selected from glue
At least one of former albumen, hyaluronic acid, calcium hydroxyapatite, polylactic acid, fat and platelet rich plasma.Using according to this
The method of the raising cytoactive of bright embodiment, for smoothing away wrinkles, beauty, repair in trauma, compared to prior art, overcome
The unstable defect of autologous fibroblasts transplanting slowly effects, effect, overcomes the adipose cell at a specified future date of autologous fat transplantation
Survival rate 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 factor such as fat stem cell transplant operation risk is high, range of application is limited.Method according to embodiments of the present invention, takes effect more
Hurry up, immunogenicity is low, instant effect, with naturality, effect persistently, safely and effectively.Method according to embodiments of the present invention, it is special
The addition of the factor can stimulate mescenchymal stem cell secretion collagen, fibronectin etc..Mesenchyme after pre-stimulation is dry thin
Born of the same parents, are more beneficial for the secretion of collagen protein when being expelled to skin corium with subcutaneous layer of fat, more effectively activate autogenous cell and pierce
The generation of sharp blood vessel so that more nutrient substance are transported to the skin at wrinkle position.Additionally, according to embodiments of the present invention
The method for improving cytoactive, soft tissue filler is used with mesenchyma stem cell combined, and soft tissue filler can fill by between
Matter stem cell is maintained in certain space and volume range, not only can play a part of protection and lubricant cell, additionally it is possible to
The activity of efficiency factor in the range of this is improved, the increase of target cell activity is more efficiently promoting.
Embodiments in accordance with the present invention, the method for above-mentioned raising cytoactive can further include following supplementary technology
At least one feature：
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 further secrete collagen protein, hyaluronic acid and elastic fiber.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 method for the embodiment of the present invention is used to improve
Cytoactive 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 collagen protein stimulating factor is included selected from least one of TGF-β 1, bFGF.
Inventor has found that mescenchymal stem cell is contacted in advance with least one of collagen protein stimulating factor TGF-β 1, bFGF, can be stimulated
Mescenchymal stem cell secretion collagen, fibronectin etc., including it is above-mentioned contact with collagen protein stimulating factor in advance between fill
The pharmaceutical composition of matter stem cell is used to improve cytoactive, and drug effect is more preferable.
Embodiments in accordance with the present invention, the concentration of the TGF-β 1 is 2-20ng/m, and optionally, the concentration of the bFGF is
5-30ng/ml.Inventor has found that TGF-β 1 and bFGF stimulate mescenchymal stem cell secretion collagen, fiber to connect under above-mentioned concentration
The effect for connecing albumen is more significantly.
Embodiments in accordance with the present invention, the mescenchymal stem cell carries out 1～5 day with the collagen protein stimulating factor
First contact.Inventor has found that TGF-β 1 and bFGF stimulate mescenchymal stem cell secretion glue under above-mentioned time of contact
The effect of former, fibronectin is more significantly.
Embodiments in accordance with the present invention, mescenchymal stem cell is being suspended in culture medium, be suspended in life after first contact
What the form of reason saline or adhere-wall culture in culture medium was provided.Embodiments in accordance with the present invention, the mescenchymal stem cell with
The one of which of above-mentioned three kinds of forms is provided, and the good cell state of stem cell is filled between can keeping.
Embodiments in accordance with the present invention, mescenchymal stem cell is being suspended in culture medium or be suspended in life after first contact
Form in reason saline is provided, and the density of the mescenchymal stem cell is 0.1*106-10*106Individual/ml.Inventor is by experiment
It was found that, suspension density of the mescenchymal stem cell in culture medium or normal saline is 0.1*106-10*106Individual/ml, can be further
Ensure the kilter of mescenchymal stem cell and the volume ratio of suitable size is provided, and then the method for the embodiment of the present invention is used
In the better of raising cytoactive.
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's discovery, suspension of the mescenchymal stem cell in culture medium or normal saline after first contact
Density 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 embodiment of the present invention for improve cytoactive method it is better.
Embodiments in accordance with the present invention, shape of the mescenchymal stem cell with adhere-wall culture in culture medium after first contact
Formula is provided, and the adherent density of mescenchymal stem cell is 1*10 after first contact4～3*104Individual/cm2.Inventor is by experiment
It was found that, in culture medium, adherent density is 1*10 to mescenchymal stem cell adhere-wall culture4～3*104Individual/cm2, can be further ensured that
The kilter of mescenchymal stem cell, and then cause the method for 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, methods described is improving cell
Activity on effect it is more significantly.
Embodiments in accordance with the present invention, the number ratio of mescenchymal stem cell is after the target cell, first contact
1：0.1～1:10.Inventor has found that the number ratio of mescenchymal stem cell is 1 after target cell, the first contact:0.1～1：10 models
In enclosing, the activity to improving target cell is more quick, effective.
Description of the drawings
Fig. 1 is the aspect graph of according to embodiments of the present invention 2 umbilical cord mesenchymal stem cells；
Fig. 2 is according to embodiments of the present invention 2 fluidic cell qualification figure；
Fig. 3 is the aspect graph before and after according to embodiments of the present invention 3 umbilical cord mesenchymal stem cells induction；And
Fig. 4 is the sxemiquantitative that collage synthesis amount increases after according to embodiments of the present invention 3 umbilical cord mesenchymal stem cells are induced
RT-PCR result figures.
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.
The method for improving cytoactive
In a first 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：Mescenchymal stem cell and collagen protein stimulating factor are carried out into the first contact；And by target cell with
Mescenchymal stem cell and soft tissue filler carry out the second contact after first contact, and the soft tissue filler is included selected from glue
At least one of former albumen, hyaluronic acid, calcium hydroxyapatite, polylactic acid, fat and platelet rich plasma.Using according to this
The method of the raising cytoactive of bright embodiment, for smoothing away wrinkles, beauty, compared to prior art, overcome it is autologous into
The unstable defect of fibrocyte transplanting slowly effect, effect, overcomes the adipose cell survival rate at a specified future date of autologous fat transplantation not
It is stable, and be easily affected by gravity and sagging, fat absorption is uneven, the not good defect of therapeutic effect, overcomes fat dry thin
The unfavorable factor such as born of the same parents' transplant operation risk is high, range of application is limited.Method according to embodiments of the present invention, the addition of specific factor
The low dosage addition of mescenchymal stem cell secretion collagen, fibronectin etc., such as bFGF can be stimulated can just promote cell
I type and III Collagen Type VI are secreted, TGF-β 1 can increase the generation of collagen protein in cell.By exclusive use TGF-β 1, individually
Using bFGF or both be used in combination, pre-stimulation is carried out to mescenchymal stem cell, the mesenchyme after pre-stimulation done
Cell, is more beneficial for the secretion of collagen protein, more effectively activation autogenous cell simultaneously when being expelled to skin corium with subcutaneous layer of fat
Vasostimulant generation so that more nutrient substance are transported to the skin at wrinkle position.The soft tissue filler, with saturating
As a example by bright matter acid, it has water absorption, high negative charge property, biocompatibility and the biological activity in wound healing etc., by
Gradually it is applied to the skin wound treatment such as Wound dressing and organizational project field.The inflammation of hyaluronic acid participation skin wound healing,
Propagation and reconstruction phase.Wherein in proliferation period, short chain hyaluronic acid (containing 6～20 monosaccharide) can promote migration of fibroblast cells to wound
Injured tissue, fibroblast produces collagen and glycosaminoglycans (GAG), builds new extracellular matrix (ECM)；Additionally, hyalomitome
Acid may additionally facilitate propagation and the migration of angiogenesis and keratinocyte, further promote the healing of skin trauma.According to this
The method of inventive embodiments, take effect faster, immunogenicity it is low；Mescenchymal stem cell and including selected from collagen protein, hyaluronic acid,
At least one of calcium hydroxyapatite, polylactic acid, fat and platelet rich plasma are composite filled, and plastotype is plentiful, instant effect；It is repaiied
Multiple cicatrix and wrinkle, do not destroy skin script structure, and the somatomedin activation secreted with mescenchymal stem cell is autologous into fiber finer
The function of born of the same parents, and further accelerate, reaching filling improves skin activity state, with naturality；Additionally, being expelled to skin
Fault location is included selected from collagen protein, hyaluronic acid, calcium hydroxyapatite, polylactic acid, fat and platelet rich plasma at least
One of the effect with smoothing wrinkle, repair the smooth of skin, gloss and elasticity, effect is lasting；Finally, the method has evaded operation
Risk of failure, is a kind of very safe method.In addition, inventor has found, the method for improving cytoactive disclosed by the invention
With the advantage for being substantially better than the exclusive use of both mescenchymal stem cell, soft tissue filler --- soft tissue filler can be by
Mescenchymal stem cell is maintained in certain space and volume range, not only can play a part of protection and lubricant cell, also
The activity of efficiency factor in the range of this can be improved, the increase of target cell activity is more efficiently promoting.With hyaluronic acid
As a example by, it is used in combination in addition to having above-mentioned advantage with mescenchymal stem cell, and hyaluronic acid may also operate as being filled between keeping
Matter stem cell moisture, protects 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, 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.
Embodiments in accordance with the present invention, the collagen protein stimulating factor is included selected from least one of TGF-β 1, bFGF.
Inventor has found that mescenchymal stem cell is contacted in advance with least one of collagen protein stimulating factor TGF-β 1, bFGF, can be stimulated
Mescenchymal stem cell secretion collagen, fibronectin etc., including it is above-mentioned contact with collagen protein stimulating factor in advance between fill
The pharmaceutical composition of matter stem cell is used to improve cytoactive, and drug effect is more preferable.
Embodiments in accordance with the present invention, the concentration of the TGF-β 1 is 2-20ng/m, and optionally, the concentration of the bFGF is
5-30ng/ml.Inventor has found that TGF-β 1 and bFGF stimulate mescenchymal stem cell secretion collagen, fiber to connect under above-mentioned concentration
The effect for connecing albumen is more significantly.
Embodiments in accordance with the present invention, the mescenchymal stem cell carries out 1～5 day with the collagen protein stimulating factor
First contact.Inventor has found that TGF-β 1 and bFGF stimulate mescenchymal stem cell secretion glue under above-mentioned time of contact
The effect of former, fibronectin is more significantly.
Specific example of the invention, mescenchymal stem cell is being suspended in culture medium, be suspended in after first contact
What the form of normal saline or adhere-wall culture in culture medium was provided.Inventor is by experiment it was unexpectedly observed that mesenchyme is dry thin
Born of the same parents being suspended in culture medium, be suspended in normal saline or adhere-wall culture in the culture medium in the form of provide, it is ensured that mesenchyme is done
The good growth conditions of cell.
According to a particular embodiment of the invention, after first contact mescenchymal stem cell being suspended in culture medium or suspension
Form in normal saline is provided, and the density of the mescenchymal stem cell is 0.1*106-10*106Individual/ml.Be conducive to keeping
The activity of mescenchymal stem cell, is conducive to secretion somatomedin and the collagen protein for keeping mescenchymal stem cell higher, and then
So that the effect that the method for the embodiment of the present invention 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, it is described first contact after mescenchymal stem cell with adhere-wall culture in culture
Form in base is provided, and density of the mescenchymal stem cell in the culture medium is 1*104～3*104Individual/cm2.Inventor
It was found that, cell growth has a Density dependence effect, overstocked or excessively dilute can all affect cell state, density to be less than 1*104Individual/cm2
When, cell state is poor；Higher than 3*104Individual/cm2When, cell does not have enough Growth In Spaces, and consuming excessively for culture medium is cellular
State is poor.According to a particular embodiment of the 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 the secretion life for keeping mescenchymal stem cell higher
The long factor 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 number ratio of mescenchymal stem cell is after the target cell, first contact
1:0.1～1：10.Inventor has found that the number ratio of mescenchymal stem cell is higher than 1 after target cell, the first contact:0.1, fill
The quantity of matter stem cell is relatively fewer, and the factor amount of its secretion is low, is not enough to promote the increase of substantial amounts of target cell activity；Mesh
Mark cell, the number ratio of mescenchymal stem cell are less than 1:10, the quantity of target cell is very few, cause mescenchymal stem cell secretion because
The saturation of son, causes to a certain extent the waste of certain cell and factor.Target cell, the mescenchymal stem cell
Number is than being 1:0.1～1：In 10, the activity to improving target cell is more quick, effective.
Pharmaceutical composition
Another aspect of the present invention, the present invention proposes a kind of pharmaceutical composition, and the pharmaceutical composition is used to improve cell
Activity.Embodiments in accordance with the present invention, the pharmaceutical composition includes：Mescenchymal stem cell；And soft tissue filler, it is described soft
Tissue filling agent is included selected from collagen protein, hyaluronic acid, calcium hydroxyapatite, polylactic acid, fat and platelet rich plasma
At least one, wherein, the mescenchymal stem cell is contacted in advance with collagen protein stimulating factor.Using according to embodiments of the present invention
Pharmaceutical composition improve cytoactive, smooth away wrinkles, beauty, compared to prior art, overcome autologous fibroblasts shifting
The unstable defect of slowly effect, effect is planted, the adipose cell survival rate at a specified future date for overcoming autologous fat transplantation is unstable, and easily
It is affected by gravity and sagging, fat absorption is uneven, the not good defect of therapeutic effect, overcomes fat stem cell transplant operation wind
The unfavorable factor such as danger is high, range of application is limited.Pharmaceutical composition according to embodiments of the present invention, the addition of specific factor can stimulate
Mescenchymal stem cell secretion collagen, fibronectin etc., the low dosage addition of such as bFGF can just promote cell to secrete I type
With III Collagen Type VI, TGF-β 1 can increase the generation of collagen in cell.By be used alone TGF-β 1, be used alone bFGF or
It is both be used in combination, pre-stimulation is carried out to mescenchymal stem cell, the mescenchymal stem cell after pre-stimulation is expelled to true
The secretion of collagen protein is more beneficial for when cortex is with subcutaneous layer of fat, autogenous cell and vasostimulant is more effectively activated
It is raw so that more nutrient substance are transported to the skin at wrinkle position.The pharmaceutical composition immunogenicity of the embodiment of the present invention
Low, effect is more preferable；Mescenchymal stem cell and including selected from collagen protein, hyaluronic acid, calcium hydroxyapatite, polylactic acid, fat
Composite filled with least one of platelet rich plasma, plastotype is plentiful, instant effect；It repairs cicatrix and wrinkle, does not destroy skin
Script structure, the somatomedin secreted with mescenchymal stem cell activates the function of autologous fibroblasts, and further increases number
Amount, reaching filling improves skin activity state, with naturality；Additionally, be expelled at skin defect include selected from collagen protein,
The effect of at least one of hyaluronic acid, calcium hydroxyapatite, polylactic acid, fat and platelet rich plasma with smoothing wrinkle, repairs
The smooth of skin, gloss and elasticity, effect is lasting；Finally, the pharmaceutical composition can adopt injection fillers, evade the mistake of operation
Risk is lost, is a kind of very safe drugs.In addition, inventor has found, pharmaceutical composition disclosed by the invention has substantially excellent
In the advantage that the two is used alone --- mescenchymal stem cell can be maintained certain space and volume by soft tissue filler
In the range of, protection and lubricant cell not only can be played a part of, additionally it is possible to improve the activity of efficiency factor in the range of this,
It is more efficiently promoting the increase of target cell activity.By taking hyaluronic acid as an example, its with mescenchymal stem cell be used in combination except
Have outside above-mentioned advantage, hyaluronic acid may also operate as keeping mescenchymal stem cell moisture, protect mescenchymal stem cell not by disease
Infringement of opportunistic pathogen etc. is acted on.
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.The selection of the collagen protein stimulating factor is not particularly limited, according to the present invention
Specific embodiment, the collagen protein stimulating factor at least one of may include selected from TGF-β 1, bFGF.Inventor's discovery,
Mescenchymal stem cell is contacted in advance with least one of collagen protein stimulating factor TGF-β 1, bFGF, and mesenchyme can be stimulated dry thin
Intracrine collagen, fibronectin etc., including the above-mentioned mescenchymal stem cell for contacting with collagen protein stimulating factor in advance
Pharmaceutical composition is used to improve cytoactive, and drug effect is more preferable.
Still another embodiment of the invention, the concentration of the TGF-β 1 is 2-20ng/m, optionally, the bFGF
Concentration be 5-30ng/ml.Inventor has found that TGF-β 1 and bFGF stimulate mescenchymal stem cell secretion glue under above-mentioned concentration
The effect of former, fibronectin is more significantly.
According to a particular embodiment of the invention, the mescenchymal stem cell is carried out in advance with the collagen protein stimulating factor
The contact of 1～5 day.Inventor has found that TGF-β 1 and bFGF stimulate mescenchymal stem cell secretion glue under above-mentioned time of contact
The effect of former, fibronectin is more significantly.
Specific example of the invention, mescenchymal stem cell is being suspended in culture medium, be suspended in after first contact
What the form of normal saline or adhere-wall culture in culture medium was provided.Inventor is by experiment it was unexpectedly observed that mesenchyme is dry thin
Born of the same parents being suspended in culture medium, be suspended in normal saline or adhere-wall culture in the culture medium in the form of provide, it is ensured that mesenchyme is done
The good growth conditions of cell.
According to a particular embodiment of the invention, after first contact mescenchymal stem cell being suspended in culture medium or suspension
Form in normal saline is provided, and the density of the mescenchymal stem cell is 0.1*106-10*106Individual/ml.Be conducive to keeping
The activity of mescenchymal stem cell, is conducive to secretion somatomedin and the collagen protein for keeping mescenchymal stem cell higher, and then
So that the effect that the pharmaceutical composition of the embodiment of the present invention 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, it is described first contact after mescenchymal stem cell with adhere-wall culture in culture
Form in base is provided, and density of the mescenchymal stem cell in the culture medium is 1*104～3*104Individual/cm2.Inventor
It was found that, cell growth has a Density dependence effect, overstocked or excessively dilute can all affect cell state, density to be less than 1*104Individual/cm2
When, cell state is poor；Higher than 3*104Individual/cm2When, cell does not have enough Growth In Spaces, and consuming excessively for culture medium is cellular
State is poor.According to a particular embodiment of the 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 the secretion life for keeping mescenchymal stem cell higher
The long factor and collagen protein, and then cause the pharmaceutical composition of the embodiment of the present invention to be used to improve the effect of cytoactive 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 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
Include the order of severity and the drug type as active component of the age of people to be processed, sex, body weight and cicatrix etc..Root
According to some embodiments of the present invention, dosage can be divided into 1 dose, 2 doses of suitable form or multi-agent, with the whole time period with 1 time,
2 times or multiple dosing, as long as reaching beauty effective dose.
Term " beauty effective dose " refers to that pharmaceutical composition be enough to significantly improve the amount of some symptoms, also as given symptom
The amount of cosmetic result is provided.For example, in beauty, the medicine for reducing, prevent, delay, suppress or blocking any symptom should be
Beauty is effective.The medicine of beauty effective dose need not eliminate symptom, but will provide beauty for symptom so that individual symptom send out
It is delayed, prevent or prevents, or symptom is alleviated, or the time limit of symptom is changed.Term " beauty " is obtained for finger
Obtain desired pharmacology and/or physiologic effect.The effect can be preventative for preventing symptom wholly or in part.
" beauty " used herein covers the symptom of mammal, particularly people." beauty " used herein is covered and gives medicine
Body will including but not limited to contain medicine described herein to cure, alleviate, improve, mitigate or suppress any medication of individual symptom
Compositions give individuality in need.
Embodiments in accordance with the present invention, the pharmaceutical composition of the present invention can be in combination with conventional cosmetic method and/or therapy
Use, or can be used separately with conventional cosmetic method and/or therapy.When the pharmaceutical composition of the present invention is being adopted and other medicines
When being administered in the conjoint therapy of thing, they can sequentially or simultaneously give individuality.Or, the pharmaceutical composition of the present invention can be wrapped
Combination containing pharmaceutically acceptable carrier or pharmaceutically acceptable excipient and other beauty medicines known in the art.
The purposes of pharmaceutical composition
In another aspect of the 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.Mescenchymal stem cell secretion bag in the pharmaceutical composition of the embodiment of the present invention
BFGF, VEGF, IGF etc. are included in interior cytokine, nutrition is played, is stimulated fibroblast survival and propagation in skin corium, entered
One step secretion collagen protein, hyaluronic acid and elastic fiber, fill corium Rotating fields, and smooth out wrinkles fill up depression；Inherently may be used
Collagen protein is produced to secrete, further increases the thickness and elasticity of skin, unfolded female, filled and led up wrinkle and lack
Damage, recover the elasticity and gloss of skin.Mescenchymal stem cell in the pharmaceutical composition of the embodiment of the present invention is in advance and collagen
The mescenchymal stem cell of albumen stimulating factor contact, is more beneficial for the secretion of collagen protein, better efficacy.Further it is combined simultaneously
Including collagen, hyaluronic acid, calcium hydroxyapatite (crystallite porcelain), polylactic acid, fat and platelet rich plasma at least it
One feeds back to patient skin fault location can carry out effective wrinkle elimination, cicatrix reparation, opposing aging etc..The embodiment of the present invention
Medicine is used to resist aging, smooth away wrinkles, with low excellent of instant effect, lasting medicine, low immunogenicity, naturality and risk
Gesture.
It should be noted that herein described " beauty " " wrinkle removing " " cicatrix reparation " " repair in trauma " etc. is to be with non-treatment
Purpose.
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：Cord Blood-Derived, derived from peripheral blood, umbilical cord source, Placenta Hominiss source, amnion-derived, derived from bone marrow, periosteum
It is source, adipose-derived, tooth source etc..Inventor has found that the mescenchymal stem cell in umbilical cord source comes compared to other
The mescenchymal stem cell in source, itself belongs to Biohazard Waste, and cell immunogenicity is lower, with certain versatility, is obtained
Cell quality it is high, quantity is more, purity is also higher.And then in subsequent experimental, inventor selects to include that umbilical cord fills between originating
Matter stem cell and collagen protein or hyaluronic acid or calcium hydroxyapatite (crystallite porcelain) or polylactic acid or fat or rich platelet blood
The pharmaceutical composition of slurry carries out subsequent detection.Hereinafter, by taking the pharmaceutical composition of mescenchymal stem cell and hyaluronic acid as an example, carry out
Labor and description.
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.
Streaming identification is carried out to obtained umbilical cord mesenchymal stem cells, as a result shown in Fig. 2, be can be seen that from Fig. 2 results
Umbilical cord mesenchymal stem cells express CD90 (positive cell rate：100%), CD105 (positive cell rate：100%), hemopoietic is not expressed
And endothelial cell marker --- CD34 and CD31.
Embodiment 3
In the present embodiment, inventor further to the umbilical cord mesenchymal stem cells that obtained using TGF-β 1, bFGF or
Person is that both be used in combination carries out pre-stimulation.By the mescenchymal stem cell after pre-stimulation, mice skin corium and skin are expelled to
Lower fat deposit.Inventor has found that this is more beneficial for the secretion of collagen protein, and more effective activation autogenous cell is simultaneously vasostimulant
Occur so that more nutrient substance are transported to the skin at wrinkle position.Specifically used dosage in the present embodiment is 2-
The bFGF of the TGF-β 1,5-30ng/ml of 20ng/ml, or both are while addition use is carried out to umbilical cord mesenchymal stem cells
Pretreatment, the time of pretreatment is 1-5 days.
Inventor has found that the shape of 3D is broken up in umbilical cord mesenchymal stem cells induction in the culture medium of addition TGF-β 1 and bFGF
State is illustrated in fig. 3 shown below, and as can be seen from the results, compared with the mescenchymal stem cell of normal morphology, induces the cell of differentiation whole
Body is bigger than normal, and, in fusiformis or triangle, after cell confluency, arrangement is tight, in typical swirling, radial traveling for cell space.
Further, inventor is using NTx before and after Semiquatitative RT-PCR assay detection umbilical cord mesenchymal stem cells induction
The expression of mRNA level in-site, as a result as shown in figure 4, result shows：Expressions of the chains of the α 1 (COL1A1) of NTx before and after induction
Have no substantially change；Expressions of the chains of the α 2 (COL1A2) of NTx after induction is 3 times of expression before induction.This shows
After induction differentiation, collage synthesis increased.
Embodiment 4
In the present embodiment, inventor is dry to the selected umbilical cord mesenchyma through collagen protein stimulating factor pre-stimulation
Screened on cell and the consumption of hyaluronic acid.
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 (100ul) (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 100ul.
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 100ul fresh, and adds 10ul 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 5
In the present embodiment, inventor using through collagen protein stimulating factor pre-stimulation umbilical cord mesenchymal stem cells and
Hyaluronic acid is co-cultured with fibroblast, 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.Above-mentioned the results show is dry thin through the umbilical cord mesenchyma of collagen protein stimulating factor pre-stimulation
Born of the same parents and soft tissue filler can improve the activity of target cell, such as fibroblast.
Embodiment 6
In the present embodiment, inventor further demonstrates the raising cytoactive of the embodiment of the present invention by zoopery
Method effect.
Umbilical cord mesenchymal stem cells after pre-stimulation is processed are with 1 × 106The density (being suspended in normal saline) of/mL and note
Penetrate with hyaluronic acid (hyaluronic acid) (auspicious blue No. 2) according to 1：2 ratio is mixed before the experiments were performed, shape after mix homogeneously
Into cell-hyaluronic acid complex.Carry out zoopery afterwards, (scale of construction is about for packet implantation male BLAB/c nude mices for concrete operations
20g, purchased from dimension tonneau China, production licence number：SCXK (capital) 2012-0001).Experimental mouse itself is matched group and experiment
Group, respectively by injection hyaluronic acid (left side), umbilical cord mesenchymal stem cells-hyaluronic acid complex (right side), by each sample
0.3mL is injected in the left side and right side of nude mice back waist buttockss skin, and per group of each 1 point observes again injection site after 6-10 is all
Form.
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 the μ l hyaluronic acid of waist skin of buttock intradermal injection 300, right side waist buttockss on the left of nude mice
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 0.3cm, and radius is about
The circle (being close to circle, substantially oval) of 0.4cm 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. it is a kind of improve cytoactive method, it is characterised in that include：
Mescenchymal stem cell and collagen protein stimulating factor are carried out into the first contact；And
Mescenchymal stem cell and soft tissue filler carry out the second contact, the soft tissue after target cell is contacted with first
Filler is included selected from collagen protein, hyaluronic acid, calcium hydroxyapatite, polylactic acid, fat and platelet rich plasma at least
One of.
2. method according to claim 1, it is characterised in that the mescenchymal stem cell is umbilical cord mesenchymal stem cells,
Optionally, the mescenchymal stem cell is P1～P6 for mescenchymal stem cell.
3. method according to claim 1, it is characterised in that the collagen protein stimulating factor include selected from TGF-β 1,
At least one of bFGF,
Optionally, the concentration of the TGF-β 1 is 2-20ng/ml,
Optionally, the concentration of the bFGF is 5-30ng/ml.
4. method according to claim 1, it is characterised in that the mescenchymal stem cell and the collagen protein stimulate because
Son carries out first contact of 1～5 day.
5. method according to claim 1, it is characterised in that mescenchymal stem cell is being suspended in training after first contact
Foster base, it is suspended in what form in culture medium of normal saline or adhere-wall culture was provided.
6. method according to claim 5, it is characterised in that mescenchymal stem cell is being suspended in training after first contact
Foster base or the form being suspended in normal saline are provided, and the density of the mescenchymal stem cell is 0.1*106-10*106Individual/ml.
7. method according to claim 6, it is characterised in that the culture medium or the normal saline and the soft tissue
The volume ratio of filler is 1:0.5～1:5.
8. method according to claim 5, it is characterised in that mescenchymal stem cell is with adhere-wall culture after first contact
Form in culture medium is provided, and the adherent density of the mescenchymal stem cell is 1*104～3*104Individual/cm2。
9. method according to claim 8, it is characterised in that the volume ratio of the culture medium and the soft tissue filler
For 1:0.5～1:5.
10. method according to claim 1, it is characterised in that mescenchymal stem cell after the target cell, the first contact
Number ratio be 1:0.1～1：10.
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| CN201610983606.0A CN106581760A (en) | 2016-11-08 | 2016-11-08 | Special treatment method for enhancing cell activity |
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Application publication date: 20170426 |