CN110129392A - A kind of preparation method of the umbilical cord mesenchymal stem cells factor and the purposes for being used to prepare stem cell Essence - Google Patents
A kind of preparation method of the umbilical cord mesenchymal stem cells factor and the purposes for being used to prepare stem cell Essence Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of umbilical cord mesenchymal stem cells factor and the purposes for being used to prepare stem cell Essence.Umbilical cord mesenchymal stem cells have very big advantage as the seed cell of stem cell anti-aging product, it is not related to ethics problem, are easily isolated culture, have huge amplification in vitro potential, have as necessary condition essential to elite seed cell.First, the umbilical cord mesenchymal stem cells factor that preparation method provided by the invention is prepared has effects that significant skin is anti-ageing, there is better cosmetic applications prospect in terms of reducing cicatrization, repairing, can be used for being prepared into the beauty products such as stem cell Essence；Secondly, umbilical cord mesenchymal stem cells amplification in vitro is high-efficient in the preparation method, the cell materials of sufficient amount can be provided to prepare the umbilical cord mesenchymal stem cells factor.
Description
Technical field
The present invention relates to stem cell beauty treatment fields, and in particular to a kind of preparation method of the umbilical cord mesenchymal stem cells factor and
It is used to prepare the purposes of stem cell Essence.
Background technique
Stem cell is a kind of multipotential cell with the of self-replication capacity, is known as " not old source ", is cured in regeneration
There is huge application prospect in field.Stem cell can be divided into embryonic stem cell and adult stem cell.The problems such as due to ethics, mesh
Preceding is clinically adult stem cell with wide application prospect, especially the mescenchymal stem cell in umbilical cord source.Stem cell
When growth, a large amount of cell factor can be secreted into the cell, wherein including a variety of growth factors, such as insulin-like growth factor, blood
Endothelial tube growth factor, transforming growth factor, skin growth factors, fibroblast growth factor and some relevant collagens
Substance, and these factors can all participate in the mechanism of different tissues and reaction.
Mescenchymal stem cell has very big advantage as the seed cell of stem cell anti-aging product, it is from a wealth of sources,
It is easily isolated culture, there is huge amplification in vitro potential, is had indispensable as " anti-aging skin care product " elite seed cell institute
Necessary condition.Wherein, it with the research of umbilical cord mesenchymal stem cells and is most widely used.
Patent CN106244652A discloses a kind of method of induction human mesenchymal stem cell production stem cell factor, the party
Freeze-dried powder prepared by method saves various biologically active in human mesenchymal stem cell culture supernatant to the full extent
Cytokine mixture, for human mesenchymal stem cell culture supernatant in terms of application lay a good foundation.
Patent CN106580851A discloses a kind of mescenchymal stem cell extract, its extracting method and application, the extraction
Include many potent anti-aging factors in object, can be applied in beauty product, plays to skin anti-aging reparation and beauty work
With.
Patent CN106318904A discloses purposes of the mescenchymal stem cell conditioned medium for cosmetic art, preferably
For the reparative regeneration of skin, whitening, crease-resistant and oxidation resistant purposes, which passes through following
Method preparation: (1) mescenchymal stem cell is obtained；(2) mesenchyma obtained in incubation step (1) is dry thin in complete medium
Born of the same parents；(3) mescenchymal stem cell obtained in incubation step (2) in basal medium；(4) centrifugation removal mescenchymal stem cell,
Supernatant liquid filtering is taken, filtrate is mescenchymal stem cell conditioned medium.
Patent CN108753713A discloses a kind of preparation method of umbilical cord mesenchymal stem cells liquid for cosmetology,
The following steps are included: step 1, obtains umbilical cord stem cells；Step 2, secondary culture；Step 3, the preparation of umbilical cord stem cells liquid.
Umbilical cord stem cells can secrete various kinds of cell growth factor in the technical solution, provide good micro-loop for skin epidermal cells
Border, such as fiber mother cell growth factor, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, blood
Pipe auxin etc. can activate the proliferation and migration of human keratinized cell in vitro, into promoting skin regeneration and union of wounded skin,
And skin epidermal cells can be promoted mature, increase skin elasticity, moisturizing smoothes away wrinkles and prevents color spot, has very
Good beauty functions.
Patent CN107488626A discloses a kind of umbilical cord mesenchymal stem cells extract and its application in terms of beauty,
The umbilical cord mesenchymal stem cells extract is extracted to obtain by following step: (1) isolated umbilical cord mesenchyma is dry thin from bleeding of the umbilicus
Born of the same parents；(2) umbilical cord mesenchymal stem cells, and separating funicle mesenchyme stem cell are obtained using serum free medium incubation step (1)
And culture solution；(3) umbilical cord mesenchymal stem cells are cracked, and culture solution is added and obtains protein suspension；(4) step (3) is obtained
Protein suspension concentration, obtain concentrate, as umbilical cord mesenchymal stem cells extract.Umbilical cord mesenchymal stem cells can have compared with
Strong immunoregulation effect, the histoorgan for promoting Radiation in jury function, repairing damage or lesion；Therefore between umbilical cord of the invention
Mesenchymal stem cells extract is in beauty product, and using rear excellent effect, effect is held time length.
As it can be seen that application of the mescenchymal stem cells such as umbilical cord mesenchymal stem cells in terms of beauty is more and more popular.
Summary of the invention
An object of the present disclosure is to provide a kind of preparation method of umbilical cord mesenchymal stem cells factor, and the second purpose is
Its purposes for being used to prepare stem cell Essence is provided.
The object of the invention is achieved through the following technical solutions:
A kind of preparation method of the umbilical cord mesenchymal stem cells factor, includes the following steps:
Step S1, it separating funicle mesenchyme stem cell and is cultivated from umbilical cord tissue；
Step S2, umbilical cord mesenchymal stem cells are collected, deionized water cracking are added, then it is broken to remove cell for low-temperature centrifugation
Piece, supernatant are freeze-dried to get the umbilical cord mesenchymal stem cells factor.
Further, step S1, further include external rapid amplifying step between step S2: taking the good umbilical cord of growth conditions
Mescenchymal stem cell is inoculated in sterile culture container, and culture medium is changed to the α-MEM containing fetal calf serum and steviolbioside
Culture medium is placed in 37 DEG C, 5%CO2, cultivate in saturated humidity incubator, digest, be centrifuged, wash, collect cell.
Preferably, fetal calf serum concentration expressed in percentage by volume is 10%.
Preferably, the concentration of steviolbioside is 100 μ g/mL.
Preferably, 37 DEG C, 5%CO are placed in2, cultivate 5d in saturated humidity incubator.
Preferably, every 5 × 1062ml deionized water is added in a cell.
A kind of external rapid amplifying method of umbilical cord mesenchymal stem cells takes the good umbilical cord mesenchyma of growth conditions dry thin
Born of the same parents are with 5 × 103/cm2Density be inoculated in the sterile petri dish of diameter 100mm, culture medium is changed to 15mL containing 10% tire ox
α-MEM the culture medium of serum and 100 μ g/mL steviolbiosides, is placed in 37 DEG C, 5%CO2, cultivate 5d in saturated humidity incubator,
Digestion, centrifugation, washing, collect cell.
Application of the steviolbioside in terms of umbilical cord mesenchymal stem cells amplification in vitro culture.
The aging of skin is a complicated biological process, relates generally to skin corium, such as matrix metalloproteinase level
Increase causes intercellular matrix collagen and elastomer degradation to increase, and TGF-1 signal beta conductive impairment causes collagen to close
At reducing etc., furthermore subcutaneous tissue blood vessel is reduced and structure change an important factor for being also skin aging, therefore passes through supplement
Cytokine activation skin corium cell viability has very big potentiality in skin anti-aging field.It is filled between being mainly from a variety of stem cells
The cell culture product that matter stem cell obtains has been shown to have skin anti-aging effect (Min Qian etc., mescenchymal stem cell and leaching
The senile-resistant efficacy research of bar cell secretion of cytokines, " Chinese medical beauty " in July, 2018 the 7th phase of volume 8).It is specific real
It applies in example, with human skin fibroblasts (HFF-1 cell) for cell model, by measuring umbilical cord mesenchymal stem cells factor pair
Whether HFF-1 cell there is Effect of promoting growth, which to evaluate it, promotes skin renewal anti-aging effects, as a result fills between 10 μ g/mL umbilical cords
The proliferation activity of HFF-1 cell can be improved (44.5 ± 5.9) % by matter stem cell factor, and 20 μ g/mL umbilical cord mesenchymas are dry thin
The proliferation activity of HFF-1 cell can be improved (79.7 ± 6.2) %, such as Fig. 3 by intracellular cytokine.It can be seen that umbilical cord mesenchyma is dry
Cell factor can remarkably promote the proliferation of human skin fibroblasts, and dose dependent is presented, above-mentioned preparation method preparation
The obtained umbilical cord mesenchymal stem cells factor can be used for preparing the beauty product of anti aging effect, wound healing, such as dry
Cell Essence.
The utility model has the advantages that
Umbilical cord mesenchymal stem cells have very big advantage as the seed cell of stem cell anti-aging product, it is not related to
Ethics problem is easily isolated culture, has huge amplification in vitro potential, has as necessity essential to elite seed cell
Condition.Firstly, the umbilical cord mesenchymal stem cells factor that preparation method provided by the invention is prepared is anti-with significant skin
Decline effect, with better cosmetic applications prospect in terms of inhibiting inflammation, reducing cicatrization, repair, can be used for
It is prepared into the beauty products such as stem cell Essence；Secondly, in the preparation method, umbilical cord mesenchymal stem cells amplification in vitro efficiency
Height can provide the cell materials of sufficient amount to prepare the umbilical cord mesenchymal stem cells factor.
Detailed description of the invention
Fig. 1 is that the inverted microscope of the 4th generation umbilical cord mesenchymal stem cells observes result；
Fig. 2 is the flow cytometer detection result of the 4th generation umbilical cord mesenchymal stem cells；
Fig. 3 is that the proliferation activity of HFF-1 cell can be increased percentage (%) by the umbilical cord mesenchymal stem cells factor.
Specific embodiment
Essentiality content of the invention is introduced below with reference to specific embodiment, but protection of the present invention cannot be limited with this
Range.
Embodiment 1: the preparation of the umbilical cord mesenchymal stem cells factor
One, experimental material and instrument
Umbilical cord tissue is derived from healthy Cesarean esction pregnant woman, 4 DEG C of preservations, for 24 hours in carry out the separation of umbilical cord mesenchymal stem cells with
Culture.Puerpera's signed informed consent form states that taken umbilical cord can only be used to scientific research.
The sterile petri dish of diameter 100mm is U.S. Corning product.
Cell incubator is Thermo Fisher Scientific product.
Flow cytometer is U.S. BD Biosciences product.
Fetal calf serum is U.S. Gibco Products, and α-MEM culture medium is Hyclone Products, and pancreatin is purchased from green cloud
It.
Steviolbioside is purchased from Xiamen Hui Jia Biotechnology Co., Ltd, and purity is not less than 95%.
HFF-1 cell is Shanghai Hu Zhen Biotechnology Co., Ltd product.
Streaming identification use fluorescent marker streaming antibody CD14-PE, CD31-FITC, CD34-PE, CD44-FIT,
CD45-FITC, CD90-FITC, CD105-PE, HLA-DR-PE are Beckman product.
Deionized water is made by oneself using MERCK MILLIPORE.
Low temperature refrigerator is Thermo Fisher Scientific product.
Two, experimental method and result
1, the separation, culture and identification of umbilical cord mesenchymal stem cells
According to this field conventional practices, umbilical cord is cut into 3-5cm segment in aseptic operating platform, it is abundant with physiological saline
Washing removes umbilical vein and endarterial remaining blood, rejects vein, artery.Umbilical cord jelly of Wharton is shredded into 0.5-1.0mm3
Tissue block, it is evenly laid out in the sterile petri dish of diameter 100mm, be placed in 37 DEG C, 5%CO2, in saturated humidity incubator,
After about 1~2h when tissue block slightly drying can be securely affixed on culture dish wall, it is slowly added to the α-MEM containing 10% fetal calf serum and trains
Base is supported, so that culture solution is impregnated tissue block, continues to cultivate, be changed liquid 1 time every 3~4d, it is long to 80%-90% fusion to cell clone
When, with 0.05% pancreatin had digestive transfer culture into new sterile petri dish.Streaming identification method is as follows:
Take the good 4th generation umbilical cord mesenchymal stem cells of growth conditions, washed, filtering, fixed, fluorescence antibody label,
It washs again, flow cytometer loading, detects surface marker CD14, CD31, CD34, CD44, CD45, CD90, CD105 and HLA-
The expression rate of DR.
Fig. 1 is the inverted microscope observation of the 4th generation umbilical cord mesenchymal stem cells as a result, can be observed in fibre-like, raw
Long status is good；Fig. 2 is the flow cytometer detection of the 4th generation umbilical cord mesenchymal stem cells as a result, the equal strongly expressed of CD44, CD90, CD105,
Expression rate is not less than 98%；The equal weak expression of CD14, CD31, CD34, CD45 and HLA-DR, expression rate is less than 2%.Inverted microscope
The characteristics of observation result and flow cytometer detection result meet human umbilical cord mesenchymal stem cells.
2, the rapid amplifying of umbilical cord mesenchymal stem cells
Take the good 4th generation umbilical cord mesenchymal stem cells of growth conditions with 5 × 103/cm2Density be inoculated in diameter 100mm
Sterile petri dish in, culture medium is changed to α-MEM of the 15mL containing 10% fetal calf serum and 100 μ g/mL steviolbiosides and is trained
Base is supported, 37 DEG C, 5%CO are placed in2, 5d is cultivated in saturated humidity incubator, cell is collected in digestion, centrifugation, washing.
Cell count is carried out with the three parts of sterile petri dish expanded in parallel, count results are indicated with mean value ± standard deviation.
After steviolbioside intervention culture 5d, the umbilical cord mesenchymal stem cells quantity in sterile petri dish is (8.9 ± 0.7) × 106。
The control group for not adding steviolbioside is provided in research process simultaneously, i.e., each sterile petri dish is contained with 15mL
α-MEM the culture medium of 10% fetal calf serum is placed in 37 DEG C, 5%CO2, cultivate in saturated humidity incubator, inoculum density and other
Experiment parameter is identical, and after cultivating 5d, the umbilical cord mesenchymal stem cells quantity in sterile petri dish is (1.8 ± 0.4) × 106。
The phenotype of the umbilical cord mesenchymal stem cells of steviolbioside intervention culture 5d is determined in research process with streaming method,
As a result almost the same with Fig. 2, the still equal strongly expressed of CD44, CD90, CD105, expression rate is not less than 98%；CD14,CD31,
The still equal weak expression of CD34, CD45 and HLA-DR, expression rate illustrate that steviolbioside is intervened rapid amplifying and will not be changed less than 2%
Become the phenotype of umbilical cord mesenchymal stem cells.
3, the cracking, centrifugation and freeze-drying of umbilical cord mesenchymal stem cells
After the umbilical cord mesenchymal stem cells for collecting amplification, deionized water is added and cracks, every 5 × 106A cell is added 2ml and goes
Ion water-splitting, then 4 DEG C of low temperature 1500rpm centrifugation 5min remove cell fragment, and supernatant freeze-drying is filled to get between umbilical cord
Matter stem cell factor, -20 DEG C of preservations.5 times that every ware yield is about the every ware yield of control group are cultivated in steviolbioside intervention, and thin
Born of the same parents' quantity multiple coincide substantially.
Embodiment 2: the anti-aging effects of the umbilical cord mesenchymal stem cells factor
The aging of skin is a complicated biological process, relates generally to skin corium, such as matrix metalloproteinase level
Increase causes intercellular matrix collagen and elastomer degradation to increase, and TGF-1 signal beta conductive impairment causes collagen to close
At reducing etc., furthermore subcutaneous tissue blood vessel is reduced and structure change an important factor for being also skin aging, therefore passes through supplement
Cytokine activation skin corium cell viability has very big potentiality in skin anti-aging field.It is filled between being mainly from a variety of stem cells
The cell culture product that matter stem cell obtains has been shown to have skin anti-aging effect (Min Qian etc., mescenchymal stem cell and leaching
The senile-resistant efficacy research of bar cell secretion of cytokines, " Chinese medical beauty " in July, 2018 the 7th phase of volume 8).
(senile-resistant efficacy of Min Qian etc., mescenchymal stem cell and lymphocytic emiocytosis cell factor is ground bibliography method
Study carefully, " Chinese medical beauty " in July, 2018 the 7th phase of volume 8), with human skin fibroblasts (HFF-1 cell) for cell membrane
Type, by measuring whether umbilical cord mesenchymal stem cells factor pair HFF-1 cell there is Effect of promoting growth to evaluate its promotion skin
Update anti-aging effects.The specific method is as follows:
1, cell culture
HFF-1 cell culture is in the DMEM/F12 culture solution of 10% fetal calf serum, in 37 DEG C of temperature, concentration 5%CO2's
Carry out routine culture in saturated humidity incubator, cell it is long to 85% when with pancreatin digestion, secondary culture.
2, CCK-8 measures cell-proliferation activity
The HFF-1 cell of logarithmic growth phase, every hole 1 × 104A cell inoculation is in 96 well culture plates.After 12 hours, more
Be changed to containing 0 μ g/mL of final concentration, 10 μ g/mL, the 20 μ g/mL umbilical cord mesenchymal stem cells factors (preparation of 1 method of embodiment) it is complete
Full culture medium continues after cultivating 48h, and every hole is added the CCK-8 solution 20 μ L of 5mg/ml, 37 DEG C of temperature be protected from light continue to be incubated for 4 it is small
When, every hole culture solution is then removed, 150 μ l of DMSO is added, concussion mixes, and sufficiently dissolves.Microplate reader detects at 570nm wavelength
Absorbance OD value, using 0 μ g/mL as control wells, according to following formula calculate:
Proliferation activity increases percentage (%)=(OD experimental port-OD control wells)/OD control wells × 100%.
As a result the 10 μ g/mL umbilical cord mesenchymal stem cells factors proliferation activity of HFF-1 cell can be improved (44.5 ±
5.9) proliferation activity of HFF-1 cell can be improved (79.7 ± 6.2) % by %, the 20 μ g/mL umbilical cord mesenchymal stem cells factors,
Such as Fig. 3.It can be seen that the umbilical cord mesenchymal stem cells factor can remarkably promote the proliferation of human skin fibroblasts, and present
Dose dependent.It also measured were in research process and does not add steviolbioside control group umbilical cord mesenchymal stem cells factor pair HFF-
1 Effect of promoting growth, 10 μ g/mL, 20 μ g/mL the proliferation activity of HFF-1 cell can be respectively increased (47.1 ±
6.5) %, (75.5 ± 6.9) %.
A kind of embodiment 3: stem cell Essence
It is made of the raw material of following percentage composition: the umbilical cord mesenchymal stem cells factor, 0.8%；Pyrrolidone sodium carboxylate,
0.5%；Glycerol, 3.5%；1,3-PD, 3.5%；Glycine betaine, 1%；Glutathione, 0.05%；Ethylenediamine tetra-acetic acid two
Sodium, 0.05%；Dodecanamide propyl hydroxy sulfo lycine, 0.5%；Allantoin, 0.1%；Ascorbic acid, 0.1%；Hyaluronic acid
Sodium, 0.1%；Xanthan gum, 0.5%；Essence, 0.05%；Surplus is deionized water.
Umbilical cord mesenchymal stem cells have very big advantage as the seed cell of stem cell anti-aging product, it is not related to
Ethics problem is easily isolated culture, has huge amplification in vitro potential, has as necessity essential to elite seed cell
Condition.Firstly, the umbilical cord mesenchymal stem cells factor that preparation method provided by the invention is prepared is anti-with significant skin
Decline effect, with better cosmetic applications prospect in terms of inhibiting inflammation, reducing cicatrization, repair, can be used for
The beauty products such as stem cell Essence are prepared into for anti aging effect, wound healing；Secondly, in the preparation method, navel
Band mescenchymal stem cell amplification in vitro is high-efficient, can provide the cell original of sufficient amount to prepare the umbilical cord mesenchymal stem cells factor
Material.
Above-described embodiment is used to introduce essentiality content of the invention, but cannot limit the scope of the present invention with this.
Claims (10)
1. a kind of preparation method of the umbilical cord mesenchymal stem cells factor, which comprises the steps of:
Step S1, it separating funicle mesenchyme stem cell and is cultivated from umbilical cord tissue；
Step S2, umbilical cord mesenchymal stem cells are collected, deionized water cracking is added, then low-temperature centrifugation removes cell fragment, on
Clear liquid is freeze-dried to get the umbilical cord mesenchymal stem cells factor.
2. preparation method according to claim 1, which is characterized in that further include step S1, external quick between step S2
Amplification step: it takes the good umbilical cord mesenchymal stem cells of growth conditions to be inoculated in sterile culture container, culture medium is changed to
α-MEM culture medium containing fetal calf serum and steviolbioside, is placed in 37 DEG C, 5%CO2, cultivate in saturated humidity incubator, disappear
Change, centrifugation, washing, collects cell.
3. preparation method according to claim 2, it is characterised in that: fetal calf serum concentration expressed in percentage by volume is 10%.
4. preparation method according to claim 2, it is characterised in that: the concentration of steviolbioside is 100 μ g/mL.
5. preparation method according to claim 2, it is characterised in that: be placed in 37 DEG C, 5%CO2, in saturated humidity incubator
Cultivate 5d.
6. preparation method according to claim 1, it is characterised in that: every 5 × 1062ml deionized water is added in a cell.
7. a kind of external rapid amplifying method of umbilical cord mesenchymal stem cells, it is characterised in that: take the good umbilical cord of growth conditions
Mescenchymal stem cell is with 5 × 103/cm2Density be inoculated in the sterile petri dish of diameter 100mm, culture medium is changed to 15mL
α-MEM culture medium containing 10% fetal calf serum and 100 μ g/mL steviolbiosides, is placed in 37 DEG C, 5%CO2, saturated humidity culture
5d is cultivated in case, cell is collected in digestion, centrifugation, washing.
8. application of the steviolbioside in terms of umbilical cord mesenchymal stem cells amplification in vitro culture.
9. the umbilical cord mesenchymal stem cells factor that any preparation method of claim 1~6 is prepared is in preparing beauty product
Application, the beauty product be used for anti aging effect, wound healing.
10. application according to claim 9, it is characterised in that: the beauty product is stem cell Essence.
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| CN110812308A (en) * | 2019-09-09 | 2020-02-21 | 浙江工业大学 | Traditional Chinese medicine composition and application thereof in preparation of skin care products |
| CN111067857A (en) * | 2020-01-17 | 2020-04-28 | 广州博科健康科技集团有限公司 | Scar-removing ointment containing umbilical cord extract and preparation method thereof |
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| CN110812308A (en) * | 2019-09-09 | 2020-02-21 | 浙江工业大学 | Traditional Chinese medicine composition and application thereof in preparation of skin care products |
| CN110755453A (en) * | 2019-12-05 | 2020-02-07 | 伯仕利生物科技发展（盐城）有限公司 | Preparation method of freeze-dried powder rich in hEGF and used for wound skin repair |
| CN111067857A (en) * | 2020-01-17 | 2020-04-28 | 广州博科健康科技集团有限公司 | Scar-removing ointment containing umbilical cord extract and preparation method thereof |
| CN112220734A (en) * | 2020-11-16 | 2021-01-15 | 郑州源创吉因实业有限公司 | Skin-care spray containing stem cell active factors and preparation method thereof |
| CN112545975A (en) * | 2020-11-16 | 2021-03-26 | 郑州大学 | Stem cell essence containing hydrophilic nano zinc oxide and preparation method thereof |
| CN113384517A (en) * | 2021-07-16 | 2021-09-14 | 天津汉青生物科技有限公司 | Preparation method of stem cell cytokine and application of stem cell cytokine in preparation of medicines or cosmetics |
| CN113384517B (en) * | 2021-07-16 | 2021-12-31 | 上海汉氏方舟生物科技有限公司 | Preparation method of stem cell cytokine and application of stem cell cytokine in preparation of medicines or cosmetics |
| CN113827618A (en) * | 2021-10-20 | 2021-12-24 | 上海交通大学医学院附属仁济医院 | Application of stem cell conditioned medium in preparation of medicine for treating inflammatory skin |
| CN113827618B (en) * | 2021-10-20 | 2023-09-26 | 上海交通大学医学院附属仁济医院 | Use of stem cell conditioned medium in preparation of medicament for treating inflammatory skin |
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