CN105267243A - Stem cell extract product for eliminating skin striae gravidarum - Google Patents
Stem cell extract product for eliminating skin striae gravidarum Download PDFInfo
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- CN105267243A CN105267243A CN201410365950.4A CN201410365950A CN105267243A CN 105267243 A CN105267243 A CN 105267243A CN 201410365950 A CN201410365950 A CN 201410365950A CN 105267243 A CN105267243 A CN 105267243A
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Abstract
The invention provides a stem cell extract product for eliminating skin striae gravidarum, and belongs to the field of stem cell technology. The stem cell extract product derives from stem cells of normal placenta or umbilical cord, and solves the technology problems existing in acquirement of stem cells from embryo and umbilical cord as well as separation of the needed stem cells and the like. The stem cell extract product contains a lot of bioactive factors which are good for skin regeneration and repair, products using the stem cell extract product or using the stem cell extract product as a main component can effectively mitigate or even eliminate skin striae gravidarum and do not irritate skin and do not have adverse reactions.
Description
Technical field
The present invention relates to stem cells technology field, relate to preparation method and the purposes of stem cell extract particularly.
Background technology
Striae gravidarum (stretchmarks) is some width difference, pink different in size or mauve wavy decorative patterns that woman gestationperiod appears at the area skin such as stomach wall, thigh interior outside, buttocks, low back, chest; Divide the puerperium, the color of these decorative patterns can fade away, and leaves white or argenteous glossiness cicatrix strain line.The formation of striae gravidarum is mainly due to the impact of pregnancy hormones, and abdominal tympanites makes the elastic fibers of skin and collagen fiber damage or rupture in addition, and thinning of skin attenuates.Striae gravidarum is particularly evident primipara, once occur many can not Lock-out, and normal with cutis laxa, affect figure and the physical and mental health in women puerperal.
At present, striae gravidarum lacks effective minimizing technology.Market having the method for laser, quintessence oil and endo-medicine of employing, how because effect is not significantly or with reasons such as apparent side effects, is not majority's acceptance.
The allelotaxis that Placenta Hominis is fetal development period is fetus and grow up and provide the place of multiple nutrients material is also the initial hemopoietic organ of fetus.Placenta Hominis has the ability of active synthesis of biologically active material, supports the growth of fetus, comprises multiple hormone (protein hormones and steroid hormone) and enzyme.Research in recent years shows, and placenta tissue contains multiple stem cell; In addition, also stem cell is contained in the colloid of umbilical cord.
Summary of the invention
The object of this invention is to provide a kind of preparation eliminating striae gravidarum safely and effectively and preparation method thereof.
Preparation of the present invention contains the material of Placenta Hominis or umbilical cord stem cells secretion, is called stem cell extract.Inventor find, this extract itself, or with the compositions that other material is formed, can significantly alleviate or eliminate striae gravidarum.This extract is produced, to no skin irritation by normal (people) cell.
The invention provides a kind of method preparing stem cell extract.The stem cell of the present invention's preparation used stem cell extract, is separated the Placenta Hominis from normal person or umbilical cord tissue, through cultured and amplified in vitro.This stem cell has common feature: can adherent growth, and cell surface expression CD140a, CD105, CD73 and CD29, do not express CD45.
The stem cell of the present invention's preparation used stem cell extract is by obtaining in conjunction with cell attachment characteristic and surface molecular character separation; Or directly obtain according to the sorting of cell surface molecule feature.
Method for separating stem cell in conjunction with cell attachment characteristic and surface molecular feature is, the first step is separated Placenta Hominis or umbilical cord cells according to cell attachment characteristic.The adherent separation of placenta cells, method is cut into small pieces by placenta tissue, free cell is washed away with cold phosphate buffer (PBS), then 0.25% trypsin/HBSS is used to carry out sequential digestion twice in 37 DEG C of water-baths, each 10 minutes, collect the Digestive system of twice, and neutralize with the DMEM of equivalent containing 10% human serum, wash and be seeded in Tissue Culture Dish, change liquid after 24 hours and remove suspension cell.Within every 3 days later, change liquid once, 7-10 days visible stem cell colonies, continue to be cultured to trypsinization collecting cell when cell 70% is saturated in culture dish.
Umbilical cord cells adherent method is separated: repeatedly rinsed by normal person's umbilical cord PBS, divest blood vessel and adventitia, collect colloid (Wharton ' sjelly), shear into about 1mm fritter, be planted on Tissue Culture Dish, with the DMEM containing 10% human serum at 37 DEG C and 5%CO
2cultivate in incubator, within 3-5 days, carefully change liquid afterwards, within 5-7 days, cell grows from organizing periphery, with collecting cell after trypsinization.
DMEM in above-mentioned culture fluid can substitute with IMDM or α-MEM.
Second step is according to cell surface molecule feature, is separated Placenta Hominis and umbilical cord stem cells: the Placenta Hominis obtained from said method or umbilical cord cells, separation of C D140a positive cell.Method is first by the Placenta Hominis of cultivation or umbilical cord cells trypsinization, forms single cell suspension, with the immunomagnetic beads (Miltenyibiotee) or the selected by flow cytometry apoptosis CD140a positive cell that carry anti-CD140a antibody.
Placenta stem-cell also directly can be separated according to placenta stem-cell surface molecular feature, method is the single cell suspension above-mentioned placenta tissue digestion obtained, the cell of the CD45 positive is first removed with the immunomagnetic beads of anti-CD45, then with carrying the immuno magnetic cell separation CD140a positive cell of anti-CD140a antibody; Or with the cell mass of the direct sorting CD45 feminine gender/CD140a positive of flow cytometer.The cell sub-elected carries out cultivating and going down to posterity with above-mentioned serum-free medium.
The serum-free culture amplification of Placenta Hominis or umbilical cord stem cells: the Placenta Hominis sub-elected or umbilical cord stem cells serum-free medium carry out cultivating and going down to posterity, serum-free medium can select commercially available culture medium as MesenGro (StemRD), UltraCULTURE (Lonza) etc., or the serum-free medium containing blood serum substituting composition and cytokine (as bFGF) of autogamy.
The Placenta Hominis of the present invention's preparation used stem cell extract and stem cell also available Maitland culture carry out cultivation amplification.The Placenta Hominis or umbilical cord stem cells that obtain is separated, with 10 by said method
4the concentration of/ml is inoculated into containing gelatin bag by the culture medium of microcarrier (Cultispher), under 37 DEG C and 5%CO2 condition, carry out amplification cultivation with stir culture bottle.
According to the embodiment of the present invention, surface expression CD140a, CD105, CD73 and CD29 of Placenta Hominis or umbilical cord stem cells, do not express CD45.
According to the embodiment of the present invention, Placenta Hominis or umbilical cord stem cells, after suitably inducing, can be divided into skin keratin epithelial cell, adipose cell etc.
The invention provides a kind of preparation method of stem cell extract, method is that employment skin keratinocytes conditioned medium inducing human fetal dish or umbilical cord stem cells produce the material being conducive to skin regeneration technique.In Human plactnta or umbilical cord stem cells culture medium, add a certain amount of application on human skin horn cell conditioned medium, inducing culture 1-10 days, then with the culture fluid containing 0-20% serum or phosphate buffer at hypoxia (O
2< 5%) cultivate 0.1-120 hour under condition, collect culture fluid, 4 DEG C of centrifugal segregation cell debriss, cryopreservation is for subsequent use.
According to the embodiment of the present invention, stem cell extract contains tens of kinds of secreted proteins, comprises the somatomedin that IGF-1, bFGF, EGF, PDGF, EPO etc. promote skin regeneration technique.Inventor finds, thisly comprises Placenta Hominis (or umbilical cord) the stem cell secretion thing (stem cell extract) of multiple bioactie agent itself or effectively can alleviate with the compositions that other material is formed or eliminate striae gravidarum.Because these bioactive molecules produced by normal cell, its concentration and mutual ratio more meet needed for the physiology of skin, contribute to skin regeneration technique.
According to the embodiment of the present invention, the stem cell extract of the present invention's elimination used striae gravidarum can also have following additional technical feature:
According to embodiments of the invention, in stem cell extract, add collagen protein, collagen hydrolysate, hyaluronic acid and albumin, or one wherein, be beneficial to the stable of active component in stem cell extract, or play synergism.
Accompanying drawing explanation
Fig. 1 shows the present invention's Placenta Hominis used and umbilical cord stem cells adhere-wall culture microscope figure below;
Fig. 2 shows the expression of embodiment of the present invention placenta stem-cell surface character used molecule;
Fig. 3 shows embodiment of the present invention placenta stem-cell used and be differentiated to form adipose cell after induction;
Fig. 4 shows embodiment of the present invention placenta stem-cell used and be differentiated to form skin epithelial cell after induction;
Fig. 5 shows the secreted protein that stem cell extract of the present invention contains;
Fig. 6 shows the embodiment of the present invention alleviates striae gravidarum design sketch with stem cell extract.
Detailed description of the invention
Embodiment 1
The separation of placenta stem-cell and amplification cultivation, specific as follows:
(1) normal Human plactnta is selected, placenta tissue is cut into small pieces, free cell is fully washed away with cold phosphate buffer (PBS), then use 0.25% trypsin/HBSS 37 DEG C of water-bath vibrations digestion twice, each 10 minutes, collect the Digestive system of twice, and neutralize containing 10% human serum DMEM by equivalent.Centrifugal rear use 10% human serum DMEM suspension cell, and be planted on Tissue Culture Dish, change liquid after 24 hours and remove suspension cell, within later every 3 days, change liquid once, 7-10 days visible stem cell colonies, continue to be cultured to culture dish cell 70% saturated time trypsinization collecting cell.
(2) with immunomagnetic beads method sorting CD140a positive cell, basic step following (method that specifically can refer to Miltenyibiotec provides is carried out).First the cell of the cell 40um micropore collected above sieve (BDBioscience) filtration cell is centrifugal, cell is resuspended in the PBS containing 1% human serum, add Biotin-anti-humanCD140a antibody (Biolegend), hatch 30 minutes on ice after, fully wash with PBS and (add the PBS of 20 times of volumes, centrifugal 10 minutes of 300g, removes supernatant).With PBS re-suspended cell (every≤10 of 80 μ l containing 1% serum
7individual cell), add 20 μ lMACSAnti-BiotinMicroBeads (Miltenyibiotec), 15 minutes are hatched at 6 ~ 12 DEG C after mixing, after PBS fully washes, with the PBS re-suspended cell of 500 μ l containing 1% serum, cross cell detached dowel (Miltenyibiotec); As cell > 2x10
8select the cell separation post of other model).After rinsing with the PBS containing 1% human serum, collect the CD140a cell of absorption.
(3) placenta stem-cell is cultivated with serum-free medium.CD140a positive cell (placenta stem-cell) MesenGro serum-free medium is suspended, is inoculated on CorningCellBIND Tissue Culture Dish, at 37 DEG C and 5%CO
2cultivate in incubator, every 2-3 days changes liquid once, when cell density is saturated to 70-80%, goes down to posterity with trypsinization.With MesenGro serum-free medium cultured cells form to containing serum free culture system liquid cultured cells similar (Fig. 1).
(4) flow cytometry analysis showed, the placenta stem-cell expression characteristic surface marker obtained, as CD105, CD73, CD29; Do not express CD45 (Fig. 2).
(5) fat Analytical Chemical Experiment is become: the placenta stem-cell getting cultivation 3 generation is inoculated in six well culture plates, and density is 1x10
5/ hole, when Growth of Cells to 80% is saturated, is changed to Adipogenic induction culture fluid (containing DMEM, 100 μ g/mlIBMX, the 50 μ g/ml insulins, 10 of 10% hyclone
-6m dexamethasone), within every 3 days, change liquid 1 time, in 2-3 pericyte, visible obviously fat drips (Fig. 3).
(6) become epithelial differentiation experiment: be seeded on the cultivation chamber slides of two Room by the skin keratin epithelial cell that people's foreskin is originated, be cultured to K-SFM (Gibco, LifeTechnologies) 80% saturated, then irradiate with 10Gy Co 60.After 24 hours, every room inoculation 10
4the human placenta stem cell of labeled with green fluorescent protein gene, to skin epithelial cell layer, continues cultivation 7 days with the K-SFM containing 1% hyclone, within every 3 days, changes liquid 1 time.Finally, by cell with 1% glutaraldehyde fix, with the antibody incubation of anti-full keratoprotein, then resist with two of Cy3 labelling and hatch, confocal microscopy.See that part placenta stem-cell is except expressing green fluorescent protein, also express keratoprotein (red fluorescence) (Fig. 4), illustrate that placenta stem-cell is to Eponychium cell differentiation.
Embodiment 2
The separation of umbilical cord stem cells and amplification cultivation, specific as follows:
(1) umbilical cord cells adherent method is separated: repeatedly rinsed by normal person's umbilical cord PBS, divest blood vessel and adventitia, collect colloid (Wharton ' sjelly), shear into about 1mm fritter, suspendible is in the DMEM containing 10% human serum, be inoculated on Tissue Culture Dish, the amount of culture fluid does not make piece of tissue suspend as degree, at 37 DEG C and 5%CO
2cultivate in incubator, within 3-5 days, carefully change liquid afterwards, within 5-7 days, cell grows from organizing periphery, with collecting cell after trypsinization.
(2) with immunomagnetic beads method sorting CD140a positive cell, the separation of step with embodiment 1 placenta stem-cell and the step (2) of amplification cultivation.
(3) umbilical cord stem cells is cultivated with serum-free medium, the separation of step with embodiment 1 placenta stem-cell and the step (3) of amplification cultivation.
Embodiment 3
The preparations and applicatio of stem cell extract
(1) preparation of Eponychium cell conditioned medium: the horn cell of being originated by people's foreskin is cultured to 70% saturated full K-SFM culture medium (LifeTechnologies), the K-SFM culture medium be changed to without additive continues cultivation 24 hours, collect culture fluid, at 4 DEG C, under 2095G condition centrifugal 30 minutes, abandon bottom sludge collection supernatant ,-80 DEG C store for future use.
(2) by placenta stem-cell with containing 10% Eponychium cell conditioned medium and 90%MesenGro serum-free medium at 37 DEG C and 5%CO
2in incubator cultivate 3 days saturated to 80-90%, trypsinization goes down to posterity.
(3) cell that goes down to posterity above is continued containing 10% Eponychium cell conditioned medium and 90%MesenGro serum-free medium at 37 DEG C and 5%CO
2in incubator cultivate 2 days saturated to 70%, remove former culture medium, add PBS or DMEM being equivalent to former culture medium half amount, 37 DEG C, continue cultivation 24 hours under 5%CO2 and 1%O2 condition, collect culture medium, at 4 DEG C, under 2095G condition centrifugal 30 minutes, abandon bottom sludge collection supernatant (stem cell extract) ,-80 DEG C stored for future use.
(4) analysis of secreted protein contained by extract: the composition and the content that measure the secreted protein in stem cell extract with the protein chip (antibody-basedproteinarray) based on antibody in conjunction with ELISA method.Based on antibody protein chip (
humanCytokineAntibodyArrayGSeries) purchased from RayBiotech, Inc., USA, ELISA kit is purchased from R & D, and the method that concrete assay method provides by manufacturer is carried out.The merocrine secretion type albumen of accompanying drawing 5 contained by placenta stem-cell extract.
(5) in stem cell extract, add 1% (w/v) collagen hydrolysate, 1% (w/v) hyaluronic acid and 1% (w/v) albumin, 4 DEG C store for future use.
(6) stem cell extract (1) or the compositions (3) that is mixed with other composition are coated in striae gravidarum place skin, once a day, use one month continuously; Can be extended working time increase result of use.If desired before painting, first use exfoliation cream to remove aging horn cell, be convenient to the absorption of active component.Generally after use 2-3 week, striae gravidarum has and obviously alleviates (Fig. 6).
Claims (12)
1. for eliminating a stem cell extract for skin striae gravidarum, it is characterized in that, extract originates from placenta stem-cell or umbilical cord stem cells; And this extract can be combined to form multiple mixture with other compositions.
2. stem cell according to claim 1, is characterized in that, source of human stem cell is in Placenta Hominis or umbilical cord tissue.
3. stem cell according to claim 1, is characterized in that, stem cell grows with adherent manner, or grows under condition of suspension culture after attaching microcarrier.
4. stem cell according to claim 1, is characterized in that, cell surface expression CD140a, CD105, CD73 and CD29, do not express CD45.
5. stem cell according to claim 1, is characterized in that, Placenta Hominis or umbilical cord tissue derive from human or animal.
6. stem cell according to claim 1, is characterized in that, Placenta Hominis or umbilical cord stem cells can be passed through genetic modification (as artificial method process LAN telomerase etc.), make its immortalization, or character stable for extended periods of time.
7. stem cell extract according to claim 1, it is characterized in that, Placenta Hominis or umbilical cord stem cells are at the conditioned medium being subject to producing under special physics or electrochemical conditions stimulation, and incentive condition includes but not limited to following citing: chemical inducer is as cytokine or the material deriving from the release of another cell; Low nutrition (as low serum or low somatomedin culture medium); Low pH; Hypoxia.
8. stem cell extract according to claim 1, is characterized in that, it stores and occupation mode is powder after liquid or cold drying.
9. compositions according to claim 1, is characterized in that, except stem cell extract, can containing being conducive to the stable material of stem cell extract active component, and as glycerol, albumin etc.
10. compositions according to claim 1, it is characterized in that, except stem cell extract, other nutrient being conducive to skin health and reparation and biological active matter can be added, as collagen protein and hydrolysate, hyaluronic acid, fibroin, soybean extract, yeast extract, synthetic peptide, epidermal growth factor, fibroblast growth factor, insulin like growth factor etc.
11. compositionss according to claim 1, is characterized in that, except stem cell extract, containing the material promoting bioactive substance and nutrient Transdermal absorption.
Content described in 12. any one of claim 1-11 is used for alleviating and eliminating of skin striae gravidarum.
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| CN105963156A (en) * | 2016-06-23 | 2016-09-28 | 娇时日化（杭州）股份有限公司 | Pregnancy line massage lotion and method for preparing same |
| CN107823110A (en) * | 2017-12-06 | 2018-03-23 | 广州赛莱拉干细胞科技股份有限公司 | Mild repair essence |
| CN110652491A (en) * | 2019-09-30 | 2020-01-07 | 广州赛琅生物技术有限公司 | Skin care product with effect of removing striae gravidarum and preparation method and application thereof |
| CN110934814A (en) * | 2019-12-06 | 2020-03-31 | 杭州恩格生物医疗科技有限公司 | Skin care essence containing stem cell active factors and application thereof |
| CN111214433A (en) * | 2020-03-24 | 2020-06-02 | 山东兴瑞生物科技有限公司 | A composition for treating striae gravidarum and scar, and its preparation method |
| CN111358749A (en) * | 2020-05-09 | 2020-07-03 | 山东兴瑞生物科技有限公司 | Composition for promoting skin wound healing and preparation method thereof |
| CN112190531A (en) * | 2019-06-19 | 2021-01-08 | 湖北医药学院 | Anti-cell-aging preparation, preparation method and application thereof, cell and construction method thereof |
| CN112294847A (en) * | 2020-10-28 | 2021-02-02 | 陕西中鸿科瑞再生医学研究院有限公司 | Striae gravidarum removing product and preparation method and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105963156A (en) * | 2016-06-23 | 2016-09-28 | 娇时日化（杭州）股份有限公司 | Pregnancy line massage lotion and method for preparing same |
| CN105963156B (en) * | 2016-06-23 | 2019-03-05 | 虞琼斐 | Pregnant line massage lotion and preparation method thereof |
| CN107823110A (en) * | 2017-12-06 | 2018-03-23 | 广州赛莱拉干细胞科技股份有限公司 | Mild repair essence |
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| CN110652491A (en) * | 2019-09-30 | 2020-01-07 | 广州赛琅生物技术有限公司 | Skin care product with effect of removing striae gravidarum and preparation method and application thereof |
| CN110652491B (en) * | 2019-09-30 | 2021-09-21 | 广州赛琅生物技术有限公司 | Skin care product with effect of removing striae gravidarum and preparation method and application thereof |
| CN110934814A (en) * | 2019-12-06 | 2020-03-31 | 杭州恩格生物医疗科技有限公司 | Skin care essence containing stem cell active factors and application thereof |
| CN111214433A (en) * | 2020-03-24 | 2020-06-02 | 山东兴瑞生物科技有限公司 | A composition for treating striae gravidarum and scar, and its preparation method |
| CN111358749A (en) * | 2020-05-09 | 2020-07-03 | 山东兴瑞生物科技有限公司 | Composition for promoting skin wound healing and preparation method thereof |
| CN111358749B (en) * | 2020-05-09 | 2023-01-03 | 山东兴瑞生物科技有限公司 | Composition for promoting skin wound healing and preparation method thereof |
| CN112294847A (en) * | 2020-10-28 | 2021-02-02 | 陕西中鸿科瑞再生医学研究院有限公司 | Striae gravidarum removing product and preparation method and application thereof |
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