CN104450606B - Sweat gland cells inducing culture and its application - Google Patents
Sweat gland cells inducing culture and its application Download PDFInfo
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Abstract
The invention discloses a kind of sweat gland cells inducing culture and its application, the sweat gland cells inducing culture includes DMEM/F12 cell culture mediums, keratinocyte culture medium, hyclone, EGF, trilute, hydrocortisone, insulin transferrins sodium selenite, L glutamine, penicillin, streptomysin, HGF and bone morphogenetic protein 4.It can successfully induce stem cell directional to be divided into sweat gland like cell using the sweat gland cells inducing culture of the present invention, provide sufficient amount of sweat gland cells for the treatment of burn patients, substantially improve the quality of life of patient.Also to study the theoretical foundation that the differentiation and development of sweat gland is provided, with potential potential applicability in clinical practice.
Description
Technical field
The present invention relates to a kind of sweat gland cells inducing culture and its in differentiation of stem cells is sweat gland like cell
Using belonging to sweat gland cells culture technique field.
Background technology
China's burn annual morbidity is about 1.5%~2%, i.e., there are about million people every year and burnt in various degree, nearly 10000 people
Serious burn, wherein about 10% patient can not save its life due to lacking skin source.Large-area burns are rescued patient due to nonfunctional
Scar tissue substitution skin it is lost normal skin function throughout one's life, have a strong impact on quality of life of patients.Large area skin is tight
The treatment of reheating wound is medical science and social concern urgently to be resolved hurrily at present.
Skin is the maximum organ of human body, with protection body, is perspired, and feels the functions such as cold and hot and pressure.Wherein sweat gland
As important functional skin appendicle, in regulation body temperature, during secretion sweat and discharge human body parts metabolite
Play an important role.Sweat gland is divided into two kinds in human body, apocrine sweat gland and eccrine sweat gland.Apocrine sweat gland is larger, there is larger
Official jargon, it is unrelated with body heat regulation.Eccrine sweat gland is smaller, is distributed in Whole Body, participates in body heat regulation.
Eccrine sweat gland is divided into conduit part and secretory portion.Secreting section in skin corium or hypodermis, be curling tubule,
And conduit part is opened on epidermis, connection secretory portion and external environment.
Mild burn, the vessel cell of sweat gland can not be wound part for masterplate with its deep, pass through the differentiation shape of stem cell
Repaired completely into its distinctive three-dimensional structure.But, full thickness skin extensive deep burn, sweat gland can not then rely on stem cell
Division growth and terminal differentiation carry out self-renewing and rebuild its labyrinth.
The sweat gland quantity existed in human body is certain, because its development is a complicated process, once large area by
Damage, will be unable to fast and effectively recover the sweat gland of functional property.Therefore though the surface of a wound, without perspiration functions, has a strong impact on disease through covering
Living quality of patients.
Skin histology reparation and reconstruction are all the core and focus of biomedical sector research at home and in the world.
At present, the developmental mechanism of sweat gland tissue is also indefinite, it is impossible to realize clinical a large amount of transplanting.Research shows that stem cell can orient
It is divided into and builds the epidermis layer tissue with covering protection ability；But it is attached for the functional skin in corium layer tissue
Device, there is not been reported for current stem cell sweat gland cells directed differentiation.
Using collagenase digesting skin, treat that the individual separate out of sweat gland is drawn under inverted microscope and isolate to culture dish
Middle culture can form sweat gland cells.But there are problems that at present to this research, such as sweat gland isolates and purifies difficult and in vitro
Subculture is limited, limits its application；Meanwhile, the culture medium used in vitro culture sweat gland is there is presently no determination, for sweat gland
Cell injuring model effect is undesirable.
It is existing studies have found that, the stem cell co-cultured with sweat gland cells can be divided into sweat gland cells.Sweat gland cells are carried out
Heat shock processing, collects the culture supernatant of sweat gland cells after heat shock, is co-cultured with mescenchymal stem cell, it is found that mesenchyma is dry thin
Born of the same parents can be divided into the cell with sweat gland phenotype.But this method is there is also problem, isolated and purified due to sweat gland cells and
Secondary Culture efficiency is low, obtains mass propgation supernatant and has difficulties；And culture supernatant composition is unclear, its specific mechanism of action
Also fail to understand；It can particularly obtain and not known also with functional sweat gland cells.
Therefore new inductive differentiation medium is researched and developed, sets up new and effective stem cell sweat gland cells directed differentiation method ten
Divide necessity.
The content of the invention
It is an object of the invention to provide a kind of induction differentiation that stem cell directional can be induced to differentiate into sweat gland like cell
Culture medium；And the method that stem cell is induced to differentiate into sweat gland like cell using it is disclosed, so as to solve large-area burns disease
To the demand of sweat gland during people's treatment.
To achieve the above object of the invention, the technical solution adopted by the present invention is：A kind of sweat gland cells inducing culture, it is described
Sweat gland cells inducing culture is including volume ratio（0.8~1）: 1 DMEM/F12 cell culture mediums and keratinocyte culture
Base；Also include following component, the consumption of each composition is：
Hyclone 3~6%
The μ g/L of EGF 40~55
0.5~1.5mmol/L of trilute
0.1~0.3mg/L of hydrocortisone
Insulin-Transferrin-sodium selenite 0.5~2%
0.5~2mmol/L of Glu
0.02~0.08U/L of penicillin
The μ g/L of streptomysin 0.02~0.09
The μ g/L of HGF 20~30
The μ g/L of bone morphogenetic protein 4 8~12.
It is preferred that, the sweat gland cells inducing culture include volume ratio for 0.9: 1 DMEM/F12 cell culture mediums and
Keratinocyte culture medium；Also include following component, the consumption of each composition is：
Hyclone 5%
The μ g/L of EGF 50
Trilute 1mmol/L
Hydrocortisone 0.2mg/L
Insulin-Transferrin-sodium selenite 1%
Glu 1mmol/L
Penicillin 0.05U/L
The μ g/L of streptomysin 0.05
The μ g/L of HGF 25
The μ g/L of bone morphogenetic protein 4 10.
In above-mentioned technical proposal, the keratinocyte culture medium is KGM2 cell culture mediums.
The invention also discloses above-mentioned sweat gland cells inducing culture in differentiation of stem cells is sweat gland like cell
Using.
In above-mentioned technical proposal, the stem cell is amniotic fluid stem cell or pluripotent stem cell.
It is the method for sweat gland like cell using above-mentioned sweat gland cells inducing culture differentiation of stem cells, including takes dry thin
Born of the same parents are laid in culture dish, adhere-wall culture；After the stem cell is adherent, sweat gland cells inducing culture is added, starts induction dry
Cell；Then sweat gland cells inducing culture is changed daily；Stem cell growth after inducing is 70 ~ 80% to rate is converged, and is carried out
Had digestive transfer culture, is seeded in new culture dish in 1 ratio for passing 3, adds sweat gland Induction of committed differentiation culture medium；Induction differentiation one
Sweat gland like cell is obtained after the section time, and the detection of sweat gland index is carried out to it.The quantity of stem cell has with incubator closes, such as
The growth of the too many cell of fruit can cause very much the number of times of had digestive transfer culture to increase soon, such as take 1 × 105Stem cell kind in 6 orifice plates,
Density is proper.
In the present invention, amniotic fluid stem cell（Amniotic Fluid Stem Cell, AFS）With good propagation efficiency
And low immunogenicity, it is a kind of new stem cell.
Because above-mentioned technical proposal is used, the present invention has following advantages compared with prior art:
1. can differentiation of stem cells be effectively that sweat gland sample is thin the invention discloses a kind of new sweat gland inducing culture
Born of the same parents；Stem cell can be induced to differentiate into sweat gland cells phenotype in vitro well with the sweat gland inducing culture of the present invention
Sweat gland like cell, and the sweat gland like cell can form the tubular structure similar to sweat gland structure in glue.
2. present invention culture medium based on DMEM/F12 cell culture mediums and keratinocyte culture medium configures new sweat
Gland inducing culture, it is simple operating steps, quick for inducing culture device when stem cell transformation is sweat gland cells simple,
Cost is low, it is adaptable to the induction differentiation of a large amount of stem cells, it is easy to popularize.
Brief description of the drawings
Fig. 1 is the sweat gland like cell after human amniotic fluid stem cell, induction in embodiment one, the aspect graph of people's sweat gland cells；
Fig. 2 induces the sweat gland indicatrix of obtained sweat gland like cell for detection in mRNA level in-site in embodiment one；
Fig. 3 is to induce the differentiation ratio figure of obtained sweat gland like cell in embodiment one；
Fig. 4 is the sweat gland like cell after human amniotic fluid stem cell, induction in embodiment one, the transmission electron microscope of people's sweat gland cells
Figure；
Fig. 5 is the sweat gland like cell after people's sweat gland tissue, induction in embodiment one, the sweat gland sample conduit knot of people's sweat gland cells
Composition；
Fig. 6 forms tube chamber for the sweat gland like cell that human amniotic fluid stem cell in embodiment one is obtained after induction in glue
Procedure chart；
Fig. 7 is the sweat gland like cell after pluripotent stem cell, induction in embodiment two, the aspect graph of people's sweat gland cells；
Fig. 8 induces the sweat gland indicatrix of obtained sweat gland like cell for detection in mRNA level in-site in embodiment two；
Fig. 9 is to induce the differentiation ratio figure of obtained sweat gland like cell in embodiment two；
Figure 10 is the sweat gland like cell after pluripotent stem cell, induction in embodiment two, the transmission electron microscope of people's sweat gland cells
Figure；
Figure 11 is the sweat gland like cell after people's sweat gland tissue, induction in embodiment two, the sweat gland sample conduit of people's sweat gland cells
Structure chart；
Figure 12 forms tube chamber for the sweat gland like cell that pluripotent stem cell in embodiment two is obtained after induction in glue
Procedure chart.
Embodiment
With reference to embodiment, the invention will be further described with accompanying drawing:
Embodiment one：Induction human amniotic fluid stem cell is divided into sweat gland like cell
Sweat gland cells inducing culture, including 450mL DMEM/F12 cell culture mediums（Hyclone, SH30023.01B）,
500mL keratinocyte culture mediums（KGM2）（Lonza, CC-3107）, the extra superfine hyclones of addition 50mL, 1mmol/L tri-
Iodine thyronine, 0.2mg/L hydrocortisones, 1% Insulin-Transferrin-sodium selenite, 1mmol/L L- glutamy
Amine, 0.05U/L penicillin and 0.05 μ g/L streptomysins；Add 50 μ g/L EGFs（EGF）, the life of 25 μ g/L liver cells
The long factor（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）Take 5 × 104The human amniotic fluid stem cell in individual/hole is laid in 6 orifice plates, uses amniotic fluid stem cell medium culture；
Amniotic fluid stem cell culture medium：15% hyclone is added in α-MEM culture mediums（FBS）, 1 mM Glus and
0.05U/L penicillin, 0.05 μ g/L streptomysins, 18%Chang B and 2%Chang C culture mediums；
（2）After 20h, after amniotic fluid stem cell is adherent, sweat gland cells inducing culture is added；Then culture sweat is changed daily
Gland cell inducing culture；
（3）Amniotic fluid stem cell after inducing, which is grown to, converges rate for 80%, sucks culture medium, uses phosphate buffer
（PBS）Cleaning, using the pancreatin of 1mL 0.25% to cell dissociation 2 minutes, the effect of pancreatin is terminated using the culture medium containing serum,
The cell digested is sucked in centrifuge tube and centrifuged, 1200 revs/min, is centrifuged 5 minutes.Supernatant discarding, adds sweat gland cells
Inducing culture, cell precipitation is mixed, in the ratio inoculating cell of 1 biography 3 into new culture dish；
（4）Repeat step（3）, induction differentiation obtains sweat gland like cell after 28 days, and the detection of sweat gland index is carried out to it.
Accompanying drawing 1 is the human amniotic fluid stem cell of in vitro culture, the sweat gland like cell of human amniotic fluid stem cell induction differentiation and people
The aspect graph of sweat gland cells under an optical microscope.It can be seen that human amniotic fluid stem cell, cell is smaller, in shuttle-type, arrangement is random
Then；The sweat gland like cell that differentiation is obtained is induced by human amniotic fluid stem cell, cell becomes big, into epithelial cell sample, queueing discipline；People's sweat
Gland cell, cell is larger, with obvious epithelial cell form, is grown in paving stone shape, cell arrangement is neat.People's amniotic fluid is dry thin
People sweat gland of the sweat gland like cell that born of the same parents obtain after sweat gland inducing culture induces differentiation morphologically close in vitro culture is thin
Born of the same parents.
Accompanying drawing 2 is the sweat gland indicatrix of the sweat gland like cell that detection induction is obtained in mRNA level in-site.Realtime-PCR is tied
Fruit show, human amniotic fluid stem cell during induction, gradually express sweat gland cells index EDA, EDAR, K8 and CEA, and
Sweat gland cells are moved closer to during differentiation.Human amniotic fluid stem cell（hAFS）It is used as negative control, people's sweat gland cells（hSG）Make
For positive control.
The differentiation ratio figure for the sweat gland like cell that accompanying drawing 3 obtains for induction.By the induction of 28 days, flow cytometer skill is used
Expression of the cell in sweat gland cells index after art detection induction.By analysis, the sweat gland sample after human amniotic fluid stem cell induction
Cell expression EDA about 36%, EDAR about 43%, K8 about 45%, CEA about 32%.Human amniotic fluid stem cell can be very good by
It is induced to differentiate into sweat gland and supports cell.
Accompanying drawing 4 is that transmission electron microscope detects human amniotic fluid stem cell in organelle level, human amniotic fluid stem cell induction differentiation
The sweat gland indicatrix of sweat gland like cell and people's sweat gland cells.The sweat gland like cell obtained after human amniotic fluid stem cell induction is in cell
Close to people's sweat gland cells in device level, the microvillus that there are the sweat gland like cell after induction people's sweat gland cells to have is in particular in
（Black arrow）, and human amniotic fluid stem cell is then without microvillus.
Accompanying drawing 5 is people's sweat gland tissue, the sweat gland like cell of human amniotic fluid stem cell induction differentiation and the sweat of people's sweat gland cells
The cross section of visible sweat gland tubular structure in adenoid guide-tube structure figure, figure, it may be observed that sweat gland tube chamber；Human amniotic fluid stem cell is induced
The sweat gland tubular structure that the sweat gland like cell obtained afterwards is formed in glue, it is seen that tubular structure cross section and tube chamber；People's sweat gland
The tubular structure that cell is formed in glue, it is seen that tubular structure cross section and tube chamber.
Accompanying drawing 6 is the process that the sweat gland like cell that human amniotic fluid stem cell is obtained after induction forms tube chamber in glue.Sweat gland
Like cell breeds agglomerating, middle cell gradually apoptosis in glue, forms tube chamber.
Embodiment two：Induced pluripotent stem cells are divided into sweat gland like cell
Sweat gland cells inducing culture, including 450mLDMEM/F12 cell culture mediums, 450mL keratinocyte culture mediums
（KGM2）, the extra superfine hyclones of addition 55mL, 1.5mmol/L trilutes, 0.3mg/L hydrocortisones,
2% Insulin-Transferrin-sodium selenite, 1.5mmol/L Glus, 0.08U/L penicillin and 0.09 μ g/L strepto-s
Element；Add 55 μ g/L EGFs（EGF）, 30 μ g/L HGFs（HGF）And 8 μ g/L Bones morphologies occur
Albumen 4（BMP4）.
Induced pluripotent stem cells are divided into sweat gland like cell, comprise the following steps：
（1）Take 1 × 105The pluripotent stem cell in individual/hole is laid in 6 orifice plates, uses pluripotent stem cell medium culture；
Pluripotent stem cell culture medium：Serum containing 20%KnockOut, 1% nonessential amino acid, 0.1mmol/L β -2- sulfydryls
Ethanol, 4 μ g/L FGF-based, 2mmol/L Glus, the DMED of 0.1U/L penicillin and 0.1ug/L streptomysins
F12 cell culture mediums.
（2）After 24h, after pluripotent stem cell is adherent, sweat gland cells inducing culture is added；Then culture is changed daily
Sweat gland cells inducing culture；
（3）Pluripotent stem cell after inducing, which is grown to, converges rate for 70%, sucks culture medium, uses phosphate buffer
（PBS）Cleaning, using the pancreatin of 0.8mL 0.25% to cell dissociation 2 minutes, uses the culture medium containing serum to terminate the work of pancreatin
With, the cell digested is sucked in centrifuge tube and centrifuged, 1200 revs/min, centrifugation 5 minutes.Supernatant discarding, adds sweat gland thin
Born of the same parents' inducing culture, cell precipitation is mixed, in the ratio inoculating cell of 1 biography 4 into new culture dish；
（4）Repeat step（3）, induction differentiation obtains sweat gland like cell after 21 days, and carries out the detection of sweat gland index to it,
Can be successfully sweat gland like cell by pluripotent stem cell induction using sweat gland cells inducing culture.
Accompanying drawing 7 is the pluripotent stem cell of in vitro culture, the sweat gland like cell of pluripotent stem cell induction differentiation and people
The aspect graph of sweat gland cells under an optical microscope.It can be seen that pluripotent stem cell, cell is smaller, in cloning growth, carefully
Karyon matter is higher；The sweat gland like cell that differentiation is obtained is induced by pluripotent stem cell, cell becomes big, into epithelial cell sample, core
Matter ratio diminishes；People's sweat gland cells, cell is larger, with obvious epithelial cell form, is grown in paving stone shape, cell arrangement is whole
Together.The sweat gland like cell that pluripotent stem cell is obtained after sweat gland inducing culture induces differentiation is morphologically close to external training
Foster people's sweat gland cells.
Accompanying drawing 8 is the sweat gland indicatrix of the sweat gland like cell that detection induction is obtained in mRNA level in-site.Realtime-PCR is tied
Fruit show, pluripotent stem cell during induction, gradually express sweat gland cells index EDA, EDAR, K8 and CEA, and
Sweat gland cells are moved closer to during differentiation.Pluripotent stem cell（SG-iPS）It is used as negative control, people's sweat gland cells（SG）
It is used as positive control.
The differentiation ratio figure for the sweat gland like cell that accompanying drawing 9 obtains for induction.By the induction of 21 days, flow cytometer skill is used
Expression of the cell in sweat gland cells index after art detection induction.By analysis, the sweat gland sample after pluripotent stem cell induction
Cell expression EDA about 63%, EDAR about 65%, K8 about 76%, CEA about 56%.Pluripotent stem cell can be by high yield
Be induced to differentiate into sweat gland support cell.
Accompanying drawing 10 is that transmission electron microscope detects pluripotent stem cell in organelle level, pluripotent stem cell induction differentiation
The sweat gland indicatrix of sweat gland like cell and people's sweat gland cells.The sweat gland like cell obtained after pluripotent stem cell induction is in cell
Close to people's sweat gland cells in device level, the microvillus that there are the sweat gland like cell after induction people's sweat gland cells to have is in particular in
（Black arrow）, and pluripotent stem cell is then without microvillus.
Accompanying drawing 11 is people's sweat gland tissue, the sweat gland like cell of pluripotent stem cell induction differentiation and the sweat of people's sweat gland cells
The cross section of visible sweat gland tubular structure in adenoid guide-tube structure figure, figure, it may be observed that sweat gland tube chamber；Pluripotent stem cell is induced
The sweat gland tubular structure that the sweat gland like cell obtained afterwards is formed in glue, it is seen that tubular structure cross section and tube chamber；People's sweat gland
The tubular structure that cell is formed in glue, it is seen that tubular structure cross section and tube chamber.
Accompanying drawing 12 is the process that the sweat gland like cell that pluripotent stem cell is obtained after induction forms tube chamber in glue.Sweat
Adenoid cell breeds agglomerating, middle cell gradually apoptosis in glue, forms tube chamber.
In summary, the sweat gland cells inducing culture of the present invention can be used successfully to induce stem cell directional to be divided into sweat
Adenoid cell, provides sufficient amount of sweat gland cells for the treatment of burn patients, substantially improves the quality of life of patient.
Also to study the theoretical foundation that the differentiation and development of sweat gland is provided, with potential potential applicability in clinical practice.
Claims (5)
1. a kind of sweat gland cells inducing culture, it is characterised in that：The sweat gland cells inducing culture is including volume ratio
（0.8~1）: 1 DMEM/F12 cell culture mediums and keratinocyte culture medium；Also include following component, the consumption of each composition
For：
Hyclone 3~6%
The μ g/L of EGF 40~55
0.5~1.5mmol/L of trilute
0.1~0.3mg/L of hydrocortisone
Insulin-Transferrin-sodium selenite 0.5~2%
0.5~2mmol/L of Glu
0.02~0.08U/L of penicillin
The μ g/L of streptomysin 0.02~0.09
The μ g/L of HGF 20~30
The μ g/L of bone morphogenetic protein 4 8~12.
2. sweat gland cells inducing culture according to claim 1, it is characterised in that：The sweat gland cells inducing culture bag
Include DMEM/F12 cell culture mediums and keratinocyte culture medium that volume ratio is 0.9: 1；Also include following component, each composition
Consumption is：
Hyclone 5%
The μ g/L of EGF 50
Trilute 1mmol/L
Hydrocortisone 0.2mg/L
Insulin-Transferrin-sodium selenite 1%
Glu 1mmol/L
Penicillin 0.05U/L
The μ g/L of streptomysin 0.05
The μ g/L of HGF 25
The μ g/L of bone morphogenetic protein 4 10.
3. the sweat gland cells inducing culture according to claims 1 or 2, it is characterised in that：The keratinocyte culture
Base is KGM2 cell culture mediums.
4. the answering in differentiation of stem cells is sweat gland like cell of sweat gland cells inducing culture described in claims 1 or 2
With.
5. application according to claim 4, it is characterised in that：The stem cell is that amniotic fluid stem cell or pluripotency are dry thin
Born of the same parents.
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| CN104894053B (en) * | 2015-05-27 | 2019-02-26 | 苏州大学 | A method of forming sweat gland spline structure in glue |
| CN107267444B (en) * | 2017-08-24 | 2020-06-12 | 广州润虹医药科技股份有限公司 | Culture medium and application thereof, and method for transforming mesenchymal stem cells into sweat gland-like cells |
| 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 |
| 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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| WO2005087287A1 (en) * | 2004-03-05 | 2005-09-22 | University Of Florida Research Foundation Inc. | Novel tissue engineered scaffolds derived from copper capillary alginate gels |
| CN101906401A (en) * | 2009-06-08 | 2010-12-08 | 中国人民解放军总医院第一附属医院 | Method for culturing and inducing adult bone mesenchymal stem cells to be converted into sweat gland cells under a noncontact condition |
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