CN104450606A - Eccrine sweat gland cell induction medium and application thereof - Google Patents
Eccrine sweat gland cell induction medium and application thereof Download PDFInfo
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Abstract
The invention discloses an eccrine sweat gland cell induction medium and an application thereof. The eccrine sweat gland cell induction medium comprises a DMEM/F12 cell medium, a keratinocyt medium, fetal calf serum, an epidermal growth factor, triiodothyronine, hydrocortisone, insulin-transferrin-sodium selenite, L-glutamine, penicillin, streptomycin, a hepatocyte growth factor and a bone morphogenetic protein 4. The eccrine sweat gland cell induction medium disclosed by the invention can be used for successfully inducing stem cells to be directionally differentiated to eccrine sweat gland-like cells so as to provide sufficient eccrine sweat gland cells for treating extensive burn patients, so that the living quality of the patients is greatly improved. The medium further provides a theoretical basis for researching differentiated development of the sweat gland, so that the medium has a potential clinical application prospect.
Description
Technical field
The present invention relates to a kind of sweat gland cells inducing culture and be the application in sweat gland like cell at differentiation of stem cells, belong to sweat gland cells culture technique field.
Background technology
China's burn annual morbidity is about 1.5% ~ 2%, namely about has ten million people to suffer to burn in various degree every year, nearly 10000 people serious burn, and wherein about 10% patient cannot save its life owing to lacking skin source.The large-area burns patient that is rescued makes it lose normal skin function throughout one's life because non-functional scar tissue replaces skin, has a strong impact on quality of life of patients.The treatment of large area skin serious burn is medical science urgently to be resolved hurrily at present and social concern.
Skin is the maximum organ of human body, has protection health, perspire, feels cold and hot and the function such as pressure.Wherein sweat gland is as important functional skin appendicle, in regulate body temperature, plays an important role in the process of secretion sweat and discharge human body parts meta-bolites.In human body, sweat gland is divided into two kinds, apocrine sweat gland and eccrine sweat gland.Apocrine sweat gland is comparatively large, has larger official jargon, has nothing to do with thermoregulation.Eccrine sweat gland is less, is distributed in Whole Body, participates in thermoregulation.
Eccrine sweat gland is divided into conduit part and secretory portion.Secreting section, in skin corium or subcutis, be curling tubule, and conduit part is opened on epidermal area, connects secretory portion and external environment.
Minor burn, the vessel cell of sweat gland can not be wound part for masterplate in its deep, is repaired completely by being differentiated to form its distinctive three-dimensional structure of stem cell.But full thickness skin extensive deep burn, the division growth that sweat gland then can not rely on stem cell breaks up carry out self and rebuild its complex construction with last eventually.
The sweat gland quantity existed in human body is certain, and growing due to it is a complicated process, once big area is impaired, cannot recover there is functional sweat gland fast and effectively.Though therefore the surface of a wound is through covering, and without perspiration functions, has a strong impact on quality of life of patients.
Skin tissue recovering and reconstruction are all core and the focus of biomedical sector research at home and in the world.At present, the developmental mechanism of sweat gland tissue is also indefinite, cannot realize clinical a large amount of transplanting.Research shows, stem cell directed differentiation can be and build the epidermal area tissue with covering protection ability; But for the functional skin appendicle in skin corium tissue, there is not been reported for current stem cell sweat gland cells directed differentiation.
Utilize collagenase digesting skin, treat that sweat gland individuality dissociates out to draw to isolate under inverted microscope and cultivate and can form sweat gland cells to culture dish.But at present some problems are existed to this research, as sweat gland separation and purification difficulty and subculture is limited in vitro, limit its application; Meanwhile, vitro culture sweat gland substratum used is not also determined at present, all undesirable for sweat gland cells vitro culture effect.
Now studies have found that, can sweat gland cells be divided into the stem cell of sweat gland cells Dual culture.Sweat gland cells is carried out heat-shocked process, the culture supernatant of sweat gland cells after collection heat-shocked, with mescenchymal stem cell Dual culture, find that mescenchymal stem cell can be divided into the cell with sweat gland phenotype.But this method also has problems, due to sweat gland cells separation and purification and Secondary Culture efficiency low, obtain mass propgation supernatant and have difficulties; And culture supernatant composition is unclear, its concrete mechanism of action is also failed to understand; Particularly can obtain that to have functional sweat gland cells also uncertain.
Therefore research and develop new inductive differentiation medium, set up new and effective stem cell sweat gland cells directed differentiation method very necessary.
Summary of the invention
The object of this invention is to provide a kind of inductive differentiation medium that stem cell directional can be induced to differentiate into sweat gland like cell; And disclose the method utilizing it stem cell to be induced to differentiate into sweat gland like cell, thus solve the demand to sweat gland in burn patients treatment process.
To achieve the above object of the invention, the technical solution used in the present invention is: a kind of sweat gland cells inducing culture, and described sweat gland cells inducing culture comprises volume ratio for (0.8 ~ 1): the DMEM/F12 cell culture medium of 1 and keratinocyte substratum; Also comprise following composition, the consumption of each composition is:
Foetal calf serum 3 ~ 6%
Urogastron 40 ~ 55 μ g/L
Trilute 0.5 ~ 1.5mmol/L
Hydrocortisone 0.1 ~ 0.3mg/L
Insulin-Transferrin-Sodium Selenite 0.5 ~ 2%
L-glutaminate 0.5 ~ 2mmol/L
Penicillin 0.02 ~ 0.08U/L
Streptomycin sulphate 0.02 ~ 0.09 μ g/L
PHGF 20 ~ 30 μ g/L
Bone morphogenetic protein 48 ~ 12 μ g/L.
Preferably, described sweat gland cells inducing culture comprises DMEM/F12 cell culture medium and the keratinocyte substratum that volume ratio is 0.9: 1; Also comprise following composition, the consumption of each composition is:
Foetal calf serum 5%
Urogastron 50 μ g/L
Trilute 1mmol/L
Hydrocortisone 0.2mg/L
Insulin-Transferrin-Sodium Selenite 1%
L-glutaminate 1mmol/L
Penicillin 0.05U/L
Streptomycin sulphate 0.05 μ g/L
PHGF 25 μ g/L
Bone morphogenetic protein 4 10 μ g/L.
In technique scheme, described keratinocyte substratum is KGM2 cell culture medium.
The invention also discloses above-mentioned sweat gland cells inducing culture at differentiation of stem cells is the application in sweat gland like cell.
In technique scheme, described stem cell is amniotic fluid stem cell or pluripotent stem cell.
Utilize the method that above-mentioned sweat gland cells inducing culture differentiation of stem cells is sweat gland like cell, comprise and get stem cell and be laid in culture dish, adherent culture; After described stem cell is adherent, add sweat gland cells inducing culture, start induced dry-cell; Then sweat gland cells inducing culture is changed every day; Stem cell growth after inducing is 70 ~ 80% to converging rate, carries out had digestive transfer culture, is seeded in new culture dish in the ratio of 1 biography 3, adds sweat gland Induction of committed differentiation substratum; Obtain sweat gland like cell after differentiation-inducing for some time, and the detection of sweat gland index is carried out to it.Quantity and the incubator of stem cell have pass, if too cellulous growth can cause the number of times of had digestive transfer culture to increase too soon, such as get 1 × 10
5stem cell kind is in 6 orifice plates, and density is proper.
In the present invention, amniotic fluid stem cell (Amniotic Fluid Stem Cell, AFS) has good proliferate efficiency and low immunogenicity, is a kind of novel stem cell.
Because technique scheme is used, the present invention compared with prior art has following advantages:
1. the invention discloses a kind of new sweat gland inducing culture, can effective differentiation of stem cells be sweat gland like cell; Use sweat gland inducing culture of the present invention well stem cell can be induced to differentiate in vitro the sweat gland like cell with sweat gland cells phenotype, and this sweat gland like cell can form the tubular structure being similar to sweat gland structure in glue.
2. the present invention's substratum based on DMEM/F12 cell culture medium and keratinocyte substratum configures new sweat gland inducing culture, culture device when being converted into sweat gland cells for induced dry-cell is simple, simple operating steps, fast, cost is low, be applicable to the differentiation-inducing of a large amount of stem cell, be easy to popularize.
Accompanying drawing explanation
Accompanying drawing 1 be human amniotic fluid stem cell in embodiment one, induction after sweat gland like cell, people's sweat gland cells aspect graph;
Accompanying drawing 2 is in embodiment one, mRNA level in-site detects the sweat gland indicatrix of inducing the sweat gland like cell obtained;
Accompanying drawing 3 is the differentiation ratio figure inducing the sweat gland like cell obtained in embodiment one;
Accompanying drawing 4 be human amniotic fluid stem cell in embodiment one, induction after sweat gland like cell, people's sweat gland cells transmission electron microscope picture;
Accompanying drawing 5 be people's sweat gland tissue in embodiment one, induction after sweat gland like cell, people's sweat gland cells sweat gland sample guide-tube structure figure;
Accompanying drawing 6 is that in embodiment one, human amniotic fluid stem cell sweat gland like cell of obtaining after induction forms the procedure chart of tube chamber in glue;
Accompanying drawing 7 be pluripotent stem cell in embodiment two, induction after sweat gland like cell, people's sweat gland cells aspect graph;
Accompanying drawing 8 is in embodiment two, mRNA level in-site detects the sweat gland indicatrix of inducing the sweat gland like cell obtained;
Accompanying drawing 9 is the differentiation ratio figure inducing the sweat gland like cell obtained in embodiment two;
Accompanying drawing 10 be pluripotent stem cell in embodiment two, induction after sweat gland like cell, people's sweat gland cells transmission electron microscope picture;
Accompanying drawing 11 be people's sweat gland tissue in embodiment two, induction after sweat gland like cell, people's sweat gland cells sweat gland sample guide-tube structure figure;
Accompanying drawing 12 is that in embodiment two, pluripotent stem cell sweat gland like cell of obtaining after induction forms the procedure chart of tube chamber in glue.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the invention will be further described:
Embodiment one: induction human amniotic fluid stem cell is divided into sweat gland like cell
Sweat gland cells inducing culture, comprise 450mL DMEM/F12 cell culture medium (Hyclone, SH30023.01B), 500mL keratinocyte substratum (KGM2) (Lonza, CC-3107), extra interpolation 50mL superfine foetal calf serum, 1mmol/L trilute, 0.2mg/L hydrocortisone, 1% Insulin-Transferrin-Sodium Selenite, 1mmol/L L-glutaminate, 0.05U/L penicillin and 0.05 μ g/L Streptomycin sulphate; Add 50 μ g/L Urogastrons (EGF) again, 25 μ g/L pHGFs (HGF) and 10 μ g/L bone morphogenetic protein 4s (BMP4).
Induction amniotic fluid stem cell is divided into sweat gland like cell, comprises the following steps:
(1) 5 × 10 are got
4the human amniotic fluid stem cell in individual/hole is laid in 6 orifice plates, uses amniotic fluid stem cell culture medium culturing;
Amniotic fluid stem cell substratum: add 15% foetal calf serum (FBS) in α-MEM substratum, 1 mM L-glutaminate and 0.05U/L penicillin, 0.05 μ g/L Streptomycin sulphate, 18%Chang B and 2%Chang C substratum;
(2) after 20h, after amniotic fluid stem cell is adherent, sweat gland cells inducing culture is added; Then change every day and cultivate sweat gland cells inducing culture;
(3) amniotic fluid stem cell after inducing grows to that to converge rate be 80%, suck substratum, clean with phosphate buffered saline buffer (PBS), use 1mL 0.25% pancreatin to cell dissociation 2 minutes, the substratum containing serum is used to stop the effect of pancreatin, the cell digested is sucked in centrifuge tube centrifugal, 1200 revs/min, centrifugal 5 minutes.Supernatant discarded, adds sweat gland cells inducing culture, is mixed by cell precipitation, passes the ratio inoculating cell of 3 in new culture dish in 1;
(4) repeating step (3), obtains sweat gland like cell after differentiation-inducing 28 days, and carries out the detection of sweat gland index to it.
Accompanying drawing 1 is the human amniotic fluid stem cell of vitro culture, the sweat gland like cell that human amniotic fluid stem cell is differentiation-inducing and people's sweat gland cells aspect graph under an optical microscope.Can find out human amniotic fluid stem cell, cell is less, in shuttle-type, arranges random; By the differentiation-inducing sweat gland like cell obtained of human amniotic fluid stem cell, cell becomes large, becomes epithelial cell sample, queueing discipline; People's sweat gland cells, cell is comparatively large, has obvious epithelial cell form, and grow in paving stone shape, cell arrangement is neat.The sweat gland like cell that human amniotic fluid stem cell obtains after sweat gland inducing culture is differentiation-inducing is morphologically close to people's sweat gland cells of vitro culture.
Accompanying drawing 2 is mRNA level in-site detects the sweat gland indicatrix of inducing the sweat gland like cell obtained.Realtime-PCR result shows, and human amniotic fluid stem cell, in the process of induction, expresses sweat gland cells index EDA, EDAR, K8 and CEA gradually, and moves closer to sweat gland cells in the process of differentiation.Human amniotic fluid stem cell (hAFS) is as negative control, and people's sweat gland cells (hSG) is as positive control.
Accompanying drawing 3 is the differentiation ratio figure inducing the sweat gland like cell obtained.Through the induction of 28 days, detect the expression of cell in sweat gland cells index after induction by low cytometric analysis.By analysis, the sweat gland like cell after human amniotic fluid stem cell induction expresses EDA about 36%, EDAR about 43%, K8 about 45%, CEA about 32%.Human amniotic fluid stem cell can well be induced to differentiate into sweat gland and support cell.
Accompanying drawing 4 detects human amniotic fluid stem cell for transmission electron microscope in organoid level, the sweat gland indicatrix of the sweat gland like cell that human amniotic fluid stem cell is differentiation-inducing and people's sweat gland cells.After human amniotic fluid stem cell induction, the sweat gland like cell that obtains is close to people's sweat gland cells in organoid level, and be in particular in the microvillus (black arrow) that the sweat gland like cell after induction has people's sweat gland cells and has, human amniotic fluid stem cell does not then have microvillus.
Accompanying drawing 5 is people's sweat gland tissue, the sweat gland sample guide-tube structure figure of the sweat gland like cell that human amniotic fluid stem cell is differentiation-inducing and people's sweat gland cells, and in figure, the cross section of visible sweat gland tubular structure, can observe sweat gland tube chamber; The sweat gland tubular structure that the sweat gland like cell obtained after human amniotic fluid stem cell induction is formed in glue, visible tubular structure cross section and tube chamber; The tubular structure that people's sweat gland cells is formed in glue, visible tubular structure cross section and tube chamber.
The accompanying drawing 6 sweat gland like cell that to be human amniotic fluid stem cell obtain after induction forms the process of tube chamber in glue.Sweat gland like cell is bred agglomerating in glue, middle cell apoptosis gradually, forms tube chamber.
Embodiment two: induced pluripotent stem cells is divided into sweat gland like cell
Sweat gland cells inducing culture, comprise 450mLDMEM/F12 cell culture medium, 450mL keratinocyte substratum (KGM2), extra interpolation 55mL superfine foetal calf serum, 1.5mmol/L trilute, 0.3mg/L hydrocortisone, 2% Insulin-Transferrin-Sodium Selenite, 1.5mmol/L L-glutaminate, 0.08U/L penicillin and 0.09 μ g/L Streptomycin sulphate; Add 55 μ g/L Urogastrons (EGF) again, 30 μ g/L pHGFs (HGF) and 8 μ g/L bone morphogenetic protein 4s (BMP4).
Induced pluripotent stem cells is divided into sweat gland like cell, comprises the following steps:
(1) 1 × 10 is got
5the pluripotent stem cell in individual/hole is laid in 6 orifice plates, uses pluripotent stem cell culture medium culturing;
Pluripotent stem cell substratum: containing the DMED F12 cell culture medium of 20%KnockOut serum, 1% non-essential amino acid, 0.1mmol/L β-2 mercapto ethanol, 4 μ g/L FGF-based, 2mmol/L L-glutaminate, 0.1U/L penicillin and 0.1ug/L Streptomycin sulphate.
(2) after 24h, after pluripotent stem cell is adherent, sweat gland cells inducing culture is added; Then change every day and cultivate sweat gland cells inducing culture;
(3) pluripotent stem cell after inducing grows to that to converge rate be 70%, suck substratum, clean with phosphate buffered saline buffer (PBS), use 0.8mL 0.25% pancreatin to cell dissociation 2 minutes, the substratum containing serum is used to stop the effect of pancreatin, the cell digested is sucked in centrifuge tube centrifugal, 1200 revs/min, centrifugal 5 minutes.Supernatant discarded, adds sweat gland cells inducing culture, is mixed by cell precipitation, passes the ratio inoculating cell of 4 in new culture dish in 1;
(4) repeating step (3), obtains sweat gland like cell after differentiation-inducing 21 days, and carries out the detection of sweat gland index to it, and utilizing sweat gland cells inducing culture successfully to be induced by pluripotent stem cell is sweat gland like cell.
Accompanying drawing 7 is the pluripotent stem cell of vitro culture, the sweat gland like cell that pluripotent stem cell is differentiation-inducing and people's sweat gland cells aspect graph under an optical microscope.Can find out pluripotent stem cell, cell is less, and in cloning growth, cell caryoplasm is higher; By the differentiation-inducing sweat gland like cell obtained of pluripotent stem cell, cell becomes large, and become epithelial cell sample, nuclear-cytoplasmic ratio diminishes; People's sweat gland cells, cell is comparatively large, has obvious epithelial cell form, and grow in paving stone shape, cell arrangement is neat.The sweat gland like cell that pluripotent stem cell obtains after sweat gland inducing culture is differentiation-inducing is morphologically close to people's sweat gland cells of vitro culture.
Accompanying drawing 8 is mRNA level in-site detects the sweat gland indicatrix of inducing the sweat gland like cell obtained.Realtime-PCR result shows, and pluripotent stem cell, in the process of induction, expresses sweat gland cells index EDA, EDAR, K8 and CEA gradually, and moves closer to sweat gland cells in the process of differentiation.Pluripotent stem cell (SG-iPS) is as negative control, and people's sweat gland cells (SG) is as positive control.
Accompanying drawing 9 is the differentiation ratio figure inducing the sweat gland like cell obtained.Through the induction of 21 days, detect the expression of cell in sweat gland cells index after induction by low cytometric analysis.By analysis, the sweat gland like cell after pluripotent stem cell induction expresses EDA about 63%, EDAR about 65%, K8 about 76%, CEA about 56%.Pluripotent stem cell can be supported cell by the sweat gland that is induced to differentiate into of high yield.
Accompanying drawing 10 detects pluripotent stem cell for transmission electron microscope in organoid level, the sweat gland indicatrix of the sweat gland like cell that pluripotent stem cell is differentiation-inducing and people's sweat gland cells.After pluripotent stem cell induction, the sweat gland like cell that obtains is close to people's sweat gland cells in organoid level, and be in particular in the microvillus (black arrow) that the sweat gland like cell after induction has people's sweat gland cells and has, pluripotent stem cell does not then have microvillus.
Accompanying drawing 11 is people's sweat gland tissue, the sweat gland sample guide-tube structure figure of the sweat gland like cell that pluripotent stem cell is differentiation-inducing and people's sweat gland cells, and in figure, the cross section of visible sweat gland tubular structure, can observe sweat gland tube chamber; The sweat gland tubular structure that the sweat gland like cell obtained after pluripotent stem cell induction is formed in glue, visible tubular structure cross section and tube chamber; The tubular structure that people's sweat gland cells is formed in glue, visible tubular structure cross section and tube chamber.
The accompanying drawing 12 sweat gland like cell that to be pluripotent stem cell obtain after induction forms the process of tube chamber in glue.Sweat gland like cell is bred agglomerating in glue, middle cell apoptosis gradually, forms tube chamber.
In sum, sweat gland cells inducing culture of the present invention success induced dry-cell directed differentiation can be used to be sweat gland like cell, and the treatment for burn patients provides the sweat gland cells of sufficient amount, greatly improves the quality of life of patient.Also be the theoretical foundation that the differentiation and development studying sweat gland provides, there is potential potential applicability in clinical practice.
Claims (5)
1. a sweat gland cells inducing culture, is characterized in that: described sweat gland cells inducing culture comprises volume ratio for (0.8 ~ 1): the DMEM/F12 cell culture medium of 1 and keratinocyte substratum; Also comprise following composition, the consumption of each composition is:
Foetal calf serum 3 ~ 6%
Urogastron 40 ~ 55 μ g/L
Trilute 0.5 ~ 1.5mmol/L
Hydrocortisone 0.1 ~ 0.3mg/L
Insulin-Transferrin-Sodium Selenite 0.5 ~ 2%
L-glutaminate 0.5 ~ 2mmol/L
Penicillin 0.02 ~ 0.08U/L
Streptomycin sulphate 0.02 ~ 0.09 μ g/L
PHGF 20 ~ 30 μ g/L
Bone morphogenetic protein 48 ~ 12 μ g/L.
2. sweat gland cells inducing culture according to claim 1, is characterized in that: described sweat gland cells inducing culture comprises DMEM/F12 cell culture medium and the keratinocyte substratum that volume ratio is 0.9: 1; Also comprise following composition, the consumption of each composition is:
Foetal calf serum 5%
Urogastron 50 μ g/L
Trilute 1mmol/L
Hydrocortisone 0.2mg/L
Insulin-Transferrin-Sodium Selenite 1%
L-glutaminate 1mmol/L
Penicillin 0.05U/L
Streptomycin sulphate 0.05 μ g/L
PHGF 25 μ g/L
Bone morphogenetic protein 4 10 μ g/L.
3. sweat gland cells inducing culture according to claims 1 or 2, is characterized in that: described keratinocyte substratum is KGM2 cell culture medium.
4. sweat gland cells inducing culture described in claims 1 or 2 is the application in sweat gland like cell at differentiation of stem cells.
5. application according to claim 4, is characterized in that: described stem cell is amniotic fluid stem cell or pluripotent stem cell.
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| CN104894053A (en) * | 2015-05-27 | 2015-09-09 | 苏州大学 | Method for forming sweat gland sample structure in gel |
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| CN107937333A (en) * | 2017-12-28 | 2018-04-20 | 广州润虹医药科技股份有限公司 | Induced fibroblast breaks up sweat gland cells culture medium and method |
| CN112725259A (en) * | 2021-01-07 | 2021-04-30 | 福州市皮肤病防治院 | Induction medium and induction method for differentiation of stem cells into sweat gland-like cells |
| CN113583940A (en) * | 2021-08-31 | 2021-11-02 | 成都以邦医药科技有限公司 | Liver oval cell immortalized culture medium and preparation method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104894053A (en) * | 2015-05-27 | 2015-09-09 | 苏州大学 | Method for forming sweat gland sample structure in gel |
| CN107267444A (en) * | 2017-08-24 | 2017-10-20 | 广州润虹医药科技股份有限公司 | The method that culture medium and application thereof is converted with mescenchymal stem cell to sweat gland like cell |
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| CN112725259A (en) * | 2021-01-07 | 2021-04-30 | 福州市皮肤病防治院 | Induction medium and induction method for differentiation of stem cells into sweat gland-like cells |
| CN112725259B (en) * | 2021-01-07 | 2023-08-29 | 福州市皮肤病防治院 | Induction medium and induction method for differentiation of stem cells into sweat gland-like cells |
| CN113583940A (en) * | 2021-08-31 | 2021-11-02 | 成都以邦医药科技有限公司 | Liver oval cell immortalized culture medium and preparation method and application thereof |
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| CN104450606B (en) | 2017-08-11 |
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