CN101864395B - In-vitro inducing differentiation of umbilical cord mesenchymal stem cells into tissue engineering skin seed cells - Google Patents
In-vitro inducing differentiation of umbilical cord mesenchymal stem cells into tissue engineering skin seed cells Download PDFInfo
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Abstract
The invention relates to in-vitro inducing differentiation of umbilical cord mesenchymal stem cells into skin tissue engineering seed cells. The invention discloses a new research direction for human umbilical cord mesenchymal stem cells, i.e. in-vitro inducting differentiation into skin tissue engineering seed cells. The invention also discloses an experimental method thereof, which comprises the following steps: collecting the caesarean umbilical cords in aseptic environment; culturing original-generation umbilical cord mesenchymal stem cells (UCMSCs) by an enzyme digestion or tissue-piece adherent method; after purifying and subculturing to the third generation, inducing by using different in-vitro condition induction liquids to differentiate into epidermis stem cells and fibroblasts; and inducing and differentiating epidermis stem cells and fibroblasts into sudoriferous gland cells by using coculture mode with the normal sudoriferous cells. After the induced cells are digested, liquid nitrogen is stored for later use. The invention provides seed cells for skin tissue engineering, and has the advantages of strong multiplication capacity, easy inducing differentiation, low immunogenicity, abundant sources and easy obtainment.
Description
Technical field
The present invention relates to the new purposes of umbilical cord mesenchymal stem cells, relate in particular to research as skin tissue engineering seed cells.The invention still further relates to the external evoked experimental technique that is divided into skin seed cells of umbilical cord mesenchymal stem cells.
Background technology
Aspect seed cell, be traditionally with from the inoblast of body or allosome and keratinocyte as the seed cell organization engineering skin that becomes to assign to make up, though be applied to clinical, but there is cell proliferation capacity lowly to reach limitation such as immunological rejection, there is result of study to show that the representative products Apligraf of corium-epiderm substitute also only can be used as the interim coverture of the surface of a wound, can not become permanent Graftskin abroad.Therefore, seek key point and the emphasis that the ideal skin tissue engineering seed cells is research.Mesenchymal stem cells MSCs (mesenchymal stem cells MSCs) because of its powerful multiplication capacity, multidirectional differentiation potential, becomes the research focus of present tissue engineering seed cell.And Cord blood and tissue are another main sources of mescenchymal stem cell, but mescenchymal stem cell content is abundanter in the umbilical cord tissue.Compare with bone marrow MSCs, umbilical cord MSCs has wide material sources, obtain conveniently, antigenicity is low, can evade more superiority such as Bioethics problem better, and umbilical cord MSCs is promoting also to be better than into somatocyte aspect the wound healing near embryonic cell.Have and studies confirm that marrow MSC can be induced to differentiate into epidermic cell, inoblast and sweat gland cells under given conditions.The separation and Culture technology of umbilical cord tissue MSCs is day by day ripe at present, so umbilical cord tissue MSCs is expected to become the new source of tissue engineering skin seed cells, its theory significance and using value are more important.
The relevant umbilical cord mesenchymal stem cells separation and Culture of Gong Buing and the patent of cell therapy aspect only limited to cell cultures and single cell therapy in the past, and the curative effect instability.The present invention at the external evoked skin seed cells that is divided into, and is used for making up organization engineering skin with umbilical cord mesenchymal stem cells, but directly transplanting covers the skin injury surface of a wound.
Summary of the invention
One of task of the present invention is to be divided into epidermal stem cells, inoblast, sweat gland cells at external evoked umbilical cord mesenchymal stem cells, promptly as skin tissue engineering seed cells.
Another task of the present invention provides the external evoked experimental technique that is divided into skin tissue engineering seed cells of umbilical cord mesenchymal stem cells.
The external evoked experimental technique that is divided into skin tissue engineering seed cells of umbilical cord mesenchymal stem cells of the present invention, the caesarean umbilical cord of aseptic collection, cultivate former generation umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells by enzymic digestion or tissue block adherent method, UCMSCs), after purifying is passaged to the third generation, by different conditions in vitro induced liquids, it is induced to differentiate into epidermal stem cells and inoblast, and, it is induced to differentiate into sweat gland cells by being total to best cultivation with normal sweat gland cells.Liquid nitrogen is preserved standby behind the cell dissociation after inducing.
The external evoked experimental technique that is divided into skin tissue engineering seed cells of umbilical cord mesenchymal stem cells of the present invention, wherein said dermal fibroblast is induced system, promptly contain high sugared DMEM substratum, 5% foetal calf serum, the 15ng/mL transforminggrowthfactor-, the 20ng/mL Prostatropin, 1% pancreas islet plain sheet selenium transferrin, 0.1 μ mol/L dexamethasone is induced to differentiate into inoblast under the corium inductive condition substratum effect of 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates.On the basis of perfect medium, add the 20ng/mL epithelical cell growth factor, the 20ng/mL Prostatropin, 10 μ g/mL Regular Insulin, 8 μ g/mL vitamin A acids, 16 μ g/ml calcium chloride are induced, and make it be divided into epidermal stem cells.By cultivating altogether 5 days, make it be induced to differentiate into sweat gland cells with the sweat gland cells of in-vitro separation cultivation.
The external evoked experiment effect that is divided into skin tissue engineering seed cells of umbilical cord mesenchymal stem cells is proved below by experimental studies results for a better understanding of the present invention.
After external application conditions induces basic directional induction umbilical cord MSC to be divided into inoblast to induce 3 days, observation group's cell fission is active, molecular marker for increased proliferation, cellular form begins to occur changing. and kytoplasm inwardly shrinks, and is irregular shape or trilateral. and blank group cell quantity also increases. but cellular form changes little (Fig. 1).The immunofluorescence dyeing result shows that blank group cell expressing collagen protein I, III type and S100A4 are weak positive expression, is that strong positive is expressed (Fig. 2) and induce the group cell.Real-time PCR result shows, the mRNA level of inducing group cell I, III collagen type apparently higher than control group (P＜0.05, Fig. 3)
External application conditions induce basic directional induction umbilical cord MSC be divided into epidermal stem cells induce the group cell under inverted phase contrast microscope, observe, cell appearance changes oblate or irregular shape into by spindle shape, the growth of whirlpool shape seldom appears, slabbing or irregular growth, proliferative ability obviously weakens (Fig. 4).Immunofluorescence dyeing shows, induces the most of CK19 of cell after the differentiation, and β 1-integrin stained positive (Fig. 5) .Real-timePCR result shows, induce group cell β 1-integrin mRNA level apparently higher than control group (P＜0.05, Fig. 6)
After directional induction umbilical cord MSC is divided into sweat gland cells co-cultivation 5d, cytogamy, effective transfer material, fused cell keeps both features.Part umbilical cord MSCs presents sweat gland epithelial cell morphological specificity (Fig. 7).Anti-with anti-BrdU and anti-CEA monoclonal antibody respectively as one, detect cultured cells altogether with the two methods of dying of immunocytochemistry, the sweat gland cells individual layer has BrdU+ cell (karyon, black-and-blue) mix, part BrdU+ cell is expressed CEA (endochylema, redness) simultaneously, form and sweat gland cells on every side do not have obvious difference (Fig. 8), show the co-culturing, inducing success.
Description of drawings
Fig. 1 umbilical cord mesenchymal stem cells is induced the variation of 1 week of differentiation back morphocytology to inoblast
Fig. 2 umbilical cord mesenchymal stem cells is induced 1 week of differentiation back immunofluorescence dyeing to inoblast
Fig. 3 umbilical cord mesenchymal stem cells is induced the variation of 1 week of differentiation back related gene expression to inoblast
Fig. 4 umbilical cord mesenchymal stem cells is induced the variation of 1 week of differentiation back morphocytology to epidermal stem cells
Fig. 5 umbilical cord mesenchymal stem cells is induced 1 week of differentiation back immunofluorescence dyeing to epidermal stem cells
Fig. 6 umbilical cord mesenchymal stem cells is induced the variation of 1 week of differentiation back β 1-integrin genetic expression to epidermal stem cells
Fig. 7 umbilical cord mesenchymal stem cells is morphological observation after sweat gland cells breaks up 5 days
Fig. 8 umbilical cord mesenchymal stem cells is immunofluorescence dyeing after sweat gland cells breaks up 5 days
Embodiment
Under the informed consent that obtains the c-section pregnant woman, aseptic collection umbilical cord, adopt enzymic digestion or tissue block adherent method to cultivate former generation umbilical cord MSCs, after purifying is passaged to the third generation, (contain high sugared DMEM in the dermal fibroblast system of inducing, 5%FBS, 15ng/mL transforminggrowthfactor-(TGF-β 1, R﹠amp; D), 20ng/mLbFGF, 1%ITS, 0.1 μ mol/L dexamethasone, the corium inductive condition substratum of 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates) induces differentiation under the effect, inverted phase contrast microscope observation of cell morphological change adopts the Tagman probe method to inducing and cellular control unit carries out Real-time PCR, induces noble cells I, III collagen mRNA level to change with detection.Under specific inductive condition, induce umbilical cord MSCs to break up to epidermal stem cells.The 3rd generation umbilical cord MSCs is collected in digestion, divide two groups promptly to induce group and control group, control group adds perfect medium, and (LG-DMEM contains 10% calf serum, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates), induce group on the basis of perfect medium, to add 20ng/mLEGF, 20ng/mLbFGF, 10 μ g/mL Insul in, 8 μ g/mL Retinoic Acid, 16 μ g/ml CaCl
2Induce, change liquid every other day, cultivate 7d, respectively two groups of cells are carried out morphological observation, the expression that application cell immunochemistry and Real-timePCR detect epidermal stem cells directed differentiation sign changes.Be total to best cultivation by sweat gland cells and induce differentiation with the in-vitro separation cultivation.The former foster sweat gland epithelial cell of being commissioned to train is put into 47 ℃ of environment 40min after reaching the 70%-80% fusion, causes the external heat-shocked state of sweat gland epithelial cell, puts 37 ℃ of cooling 1-2h then, adds (1-2) * 10 approximately
5The umbilical cord MSCs of Brdu mark cultivates altogether, observation of cell morphological change under the inverted phase contrast microscope behind the 5d.Anti-with anti-CEA and anti-BrdU as one, adopt the two cells that dye method detection co-culturing, inducing of immunofluorescence cell chemistry.
Claims (1)
1. external evoked experimental technique that is divided into skin tissue engineering seed cells of umbilical cord mesenchymal stem cells, it is characterized in that: the caesarean umbilical cord of aseptic collection, cultivate former generation umbilical cord mesenchymal stem cells by enzymic digestion or tissue block adherent method, after purifying is passaged to the third generation, third generation umbilical cord mesenchymal stem cells is induced system at dermal fibroblast, promptly contain high sugared DMEM substratum, 5% foetal calf serum, 15 ng/mL transforming growth factor-beta l, 20 ng/mL Prostatropins, l% pancreas islet plain sheet selenium transferrin, 0.1 μ mol/L dexamethasone is induced to differentiate into inoblast under the corium inductive condition substratum effect of 100 U/mL penicillin and 100 μ g/mL Streptomycin sulphates; On the basis of perfect medium, add 20 ng/mL epithelical cell growth factors, 20 ng/mL Prostatropins, 10 μ g/mL Regular Insulin, 8 μ g/mL vitamin A acids, 16 μ g/ml calcium chloride are induced, make it be divided into epidermal stem cells, wherein said perfect medium is that LG-DMEM contains 10% calf serum, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates; By in the sweat gland cells of handling through 47 ℃ of heat-shockeds, add (1-2) * 10 approximately
5The human umbilical cord mesenchymal stem cells of BrdU mark places 37.0 ° of C to cultivate 5 days, makes it be induced to differentiate into sweat gland cells.
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| CN102250837B (en) * | 2011-06-28 | 2013-03-06 | 江苏省北科生物科技有限公司 | Digital automatic production method for umbilical cord mesenchymal stem cells |
| CN102284082B (en) * | 2011-07-01 | 2014-03-26 | 董萍 | Facial fibrous protein composite filled and positioned in subcutaneous soft tissues, and preparation method thereof |
| CN103041453A (en) * | 2013-01-18 | 2013-04-17 | 新乡医学院 | Double-layer skin covering material compositely built by collagen/fibrin glue-VEGF (vascular endothelial growth factor) and mesenchymal stem cell as well as preparation method and application of double-layer skin covering material |
| CN103194425B (en) * | 2013-04-02 | 2014-12-31 | 中国人民解放军第二军医大学 | Method for promoting epidermal cell proliferation |
| CN104667353B (en) * | 2015-03-06 | 2017-03-15 | 广州赛莱拉干细胞科技股份有限公司 | A kind of organization engineering skin and its application |
| CN105744457B (en) * | 2016-04-18 | 2019-04-09 | 格云特自动化科技（深圳）有限公司 | Audio parts frequency response test device and method |
| CN106399229A (en) * | 2016-10-25 | 2017-02-15 | 浙江译美生物科技有限公司 | Inductive agent and method for preparing epidermal stem cells by adoption of inductive agent |
| CN107937333B (en) * | 2017-12-28 | 2020-09-15 | 广州润虹医药科技股份有限公司 | Culture medium and method for inducing fibroblast to differentiate sweat gland cells |
| CN111040984A (en) * | 2018-10-15 | 2020-04-21 | 美奇创新生物科技(北京)有限公司 | Method for forming skin fibroblasts by inducing differentiation of umbilical cord mesenchymal stem cells |
| CN109971703B (en) * | 2019-04-04 | 2021-02-26 | 上海科医联创生物科技有限公司 | Culture method and culture medium for autologous tissue engineering epidermis |
| CN111662864A (en) * | 2020-06-19 | 2020-09-15 | 天晴干细胞股份有限公司 | Method for differentiating umbilical cord mesenchymal stem cells into dermal stem cells through in-vitro induction |
| CN112011505B (en) * | 2020-09-09 | 2021-07-30 | 陕西中港万海生命科学研究院有限公司 | Umbilical cord mesenchymal stem cell separation method |
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| US20080118477A1 (en) * | 2006-11-09 | 2008-05-22 | Rush University Medical Center | Umbilical cord mesenchymal stem cells support cord blood hematopoiesis |
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