CN107828716A - A kind of method and its culture medium group that differentiation sweat gland cells are induced by epidermal stem cells - Google Patents
A kind of method and its culture medium group that differentiation sweat gland cells are induced by epidermal stem cells Download PDFInfo
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Abstract
The invention discloses a kind of method that differentiation sweat gland cells are induced by epidermal stem cells, comprise the following steps：1) epidermal stem cells are separated from isolated skin tissue, Secondary Culture is carried out in epidermal stem cells culture medium；2) epidermal stem cells that step 1) obtains are subjected to Secondary Culture, addition sweat gland cells inducing culture carries out induction differentiation；With 3) passed on and bred using sweat gland cells culture medium.Invention also provides corresponding culture medium group.This method cell source efficiently avoid ethics dispute, and the preparation method helps that the sweat gland cells that cell directional breaks up, proliferative conditions are more uniform are made.
Description
Technical field
The present invention relates to a kind of biological tissue's culture technique field, more particularly to a kind of induced by epidermal stem cells to break up sweat
The method and its culture medium group of gland cell.
Background technology
Skin is the maximum organ of human body, has protection body, perspires, and feels the functions such as cold and hot and pressure.Sweat gland secretion
Sweat, distribute body heat.The burn patient of large area, after wound repair, sweat gland missing, cause body heat regulation function impacted.People
The work skin repair surface of a wound, only with epidermis, corium Rotating fields, no sweat gland and other cutaneous appendageses.Tissue engineering skin is difficult
It is to have problem to be solved at present to build sweat gland.Simulate sweat gland mechanism come induce stem cell to sweat gland cells orient
Differentiation, it may be possible to rebuild the unique channel of sweat gland.Differentiation sweat gland cells are induced with epidermal stem cells, and reach sweat gland cells expansion
Increase the purpose of culture, there is preferable application prospect, but the cell differentiation of epidermal stem cells, the mechanism of metabolism are still unintelligible, it is fixed
Need to overcome more uncertain factor to differentiation.
The content of the invention
For overcome the deficiencies in the prior art, an object of the present invention is to provide a kind of stabilization, directed differentiation
The method that differentiation sweat gland cells are induced by epidermal stem cells.
The second object of the present invention is to provide the above-mentioned culture medium group for inducing differentiation sweat gland cells by epidermal stem cells.
An object of the present invention adopts the following technical scheme that realization：
A kind of method that differentiation sweat gland cells are induced by epidermal stem cells, comprises the following steps：
1) epidermal stem cells are separated from isolated skin tissue, Secondary Culture is carried out in epidermal stem cells culture medium；
The epidermal stem cells culture medium is to contain hydrocortisone, insulin, adenine, penicillin, human epidermal growth
The factor, transferrins, glutamic acid, the DMEM culture mediums of Y-27632 and carboxymethyl chitosan；
2) epidermal stem cells for obtaining step 1) carry out Secondary Culture, when its degrees of fusion reaches 80%, discard culture
Base, cleaned with DMEM/F12, add sweat gland cells inducing culture and carry out induction differentiation；
The sweat gland cells Fiber differentiation be containing hEGF, cholera toxin, trilute and
The DMEM culture mediums of acecoline；
3) after cultivating some days, treat occur polygon, the as cell of paving stone sample, sweat gland cells in culture medium, will train
Foster base changes sweat gland cells culture medium into, after cultivating some days, is passed on and is bred；
The sweat gland cells culture medium is the DMEM culture mediums containing EGF, ox pituitary extract, penicillin and streptomysin.
Further, in step 1), the isolated skin is organized as the foreskin abandoned after healthy infants foreskin stereotomy.
Further, in step 1), from isolated skin tissue separate epidermal stem cells concrete operations be：3-6 year is taken to be good for
The foreskin abandoned after Kang child's foreskin stereotomy, under aseptic condition, chloramphenicol cleaning skin histology, add dispase in 4 DEG C of effects
18-24h, separate epidermal tissue；Sterile PBS epidermal tissue, 0.25% pancreas enzyme -EDTA digestion 4h, adds epidermal stem cells
Culture medium terminates, 1000r/min, centrifuges 5min, collects epidermal cell, sterile PBS three times, adds epidermal stem cells culture
Base is resuspended, and is cultivated as the coated culture plates of Matrigel, obtains epidermal stem cells.
Further, in step 2), when induction is broken up, sweat gland cells inducing culture is changed daily.
Further, the epidermal stem cells culture medium is the DMEM cultures containing the following components in terms of ultimate density
Base：0.1-2ng/mL hydrocortisones, 0.01-1ng/mL insulin, 1-5 × 10-4mol/L adenines, 50-200IU/mL are blue or green
Mycin, 5-50ng/mL hEGFs, 2-50 μ g/mL transferrins, 1-10 μ g/mL glutamic acid, 1-20 μM of Y-27632
With 0.01-1mg/mL carboxymethyl chitosans.
Further, the sweat gland cells inducing culture is the DMEM cultures containing the following components in terms of ultimate density
Base：25-100ng/mL hEGFs, 0.1-2 × 10-10mol/L cholera toxins, 0.5-5 × 10-7mol/L triiodo first
Shape gland original ammonia acid, 2-8 × 10-5mol/L acecolines.
Further, the sweat gland cells culture medium is the DMEM culture mediums containing the following components in terms of ultimate density：
10-100ng/mL EGF, 10-50mg/mL ox pituitary extracts, 50-200U/mL penicillin and 50-200 μ g/mL streptomysins.
The second object of the present invention adopts the following technical scheme that realization：
A kind of culture medium group that differentiation sweat gland cells are induced by epidermal stem cells, including following culture medium：
Epidermal stem cells culture medium：Containing hydrocortisone, insulin, adenine, penicillin, hEGF,
Transferrins, glutamic acid, the DMEM culture mediums of Y-27632 and carboxymethyl chitosan；
Sweat gland cells Fiber differentiation：Contain hEGF, cholera toxin, trilute and chlorination second
The DMEM culture mediums of phatidylcholine；
Sweat gland cells culture medium：DMEM culture mediums containing EGF, ox pituitary extract, penicillin and streptomysin.
Further, the epidermal stem cells culture medium is the DMEM cultures containing the following components in terms of ultimate density
Base：0.1-2ng/mL hydrocortisones, 0.01-1ng/mL insulin, 1-5 × 10-4Mol/L adenines, 50-200IU/mL moulds
Element, 5-50ng/mL hEGFs, 2-50 μ g/mL transferrins, 1-10 μ g/mL glutamic acid, 1-20 μM of Y-27632 and
0.01-1mg/mL carboxymethyl chitosans.
Further, the sweat gland cells inducing culture is the DMEM cultures containing the following components in terms of ultimate density
Base：25-100ng/mL hEGFs, 0.1-2 × 10-10Mol/L cholera toxins, 0.5-5 × 10-7Mol/L triiodo first shapes
Gland original ammonia acid, 2-8 × 10-5Mol/L acecolines；
The sweat gland cells culture medium is the DMEM culture mediums containing the following components in terms of ultimate density：10-100ng/
ML EGF, 10-50mg/mL ox pituitary extracts, 50-200U/mL penicillin and 50-200 μ g/mL streptomysins.
Compared with prior art, the beneficial effects of the present invention are：
1) present invention use human body in vitro tissue sample as sampling sample in a creative way, effective to avoid blood sampling, bone
The unnecessary pain such as marrow sample, while ethics misgivings are avoided, it has long-range meaning；
2) method provided by the invention, the acquisition of epidermal stem cells is more convenient quick, and it is lower and used that prototype is immunized
Culture medium is free serum culture, avoids the pollution of pathogen, ensure that safe reliability；
3) present invention, which has prepared, is beneficial to the homogeneous growth of epidermal stem cells, differential period consistent culture medium group, is applied to
The different phase of separation, induction differentiation culture and Multiplying culture, from identified by immunofluorescence collection of illustrative plates, the form of epidermal stem cells
With preferable uniformity；
4) culture medium group provided by the invention can preferably control growth, directed differentiation and the propagation of cell, and it is as phase
Supporting culture medium group, there is preferable application prospect.
Brief description of the drawings
Fig. 1 is the identified by immunofluorescence figure of the embodiment of the present invention 1；
Fig. 2 is the identified by immunofluorescence figure of the embodiment of the present invention 2；
Fig. 3 is the identified by immunofluorescence figure of the embodiment of the present invention 3；
Fig. 4 is 1-3 of embodiment of the present invention flow cytomery figure.
Embodiment
Below, with reference to accompanying drawing and embodiment, the present invention is described further, it is necessary to which explanation is, not
Under the premise of afoul, new implementation can be formed between various embodiments described below or between each technical characteristic in any combination
Example.
The present invention provides the method that differentiation sweat gland cells are induced by epidermal stem cells, using human body in vitro tissue as raw material
Source, sampling and preparation process are disputed in the absence of ethics.
In detailed description below, such as reagent or instrument used by non-specified otherwise, can by commercially available mode or
Conventional laboratory facilities obtain.
The present invention provides a kind of method that differentiation sweat gland cells are induced by epidermal stem cells, comprises the following steps：
1) epidermal stem cells are separated from isolated skin tissue, Secondary Culture is carried out in epidermal stem cells culture medium；
The epidermal stem cells culture medium is to contain hydrocortisone, insulin, adenine, penicillin, human epidermal growth
The factor, transferrins, glutamic acid, the DMEM culture mediums of Y-27632 and carboxymethyl chitosan；
Serum is not contained in the epidermal stem cells culture medium, carboxymethyl chitosan is a kind of derivative of chitosan, in table
Skin stem cell media has preferable compatibility with epidermal stem cells, help epidermal stem cells can be made homogeneous as carrier
Ground, form controllably grow；
2) epidermal stem cells for obtaining step 1) carry out Secondary Culture, when its degrees of fusion reaches 80%, discard culture
Base, cleaned with DMEM/F12, add sweat gland cells inducing culture and carry out induction differentiation；
The sweat gland cells Fiber differentiation be containing hEGF, cholera toxin, trilute and
The DMEM culture mediums of acecoline；The sweat gland cells Fiber differentiation homogeneous can induce epidermal stem cells to orient to sweat gland cells
Differentiation；
3) after cultivating some days, treat occur polygon, the as cell of paving stone sample, sweat gland cells in culture medium, will train
Foster base changes sweat gland cells culture medium into, after cultivating some days, is passed on and is bred；
The sweat gland cells culture medium is the DMEM culture mediums containing EGF, ox pituitary extract, penicillin and streptomysin；
The sweat gland cells culture medium contribute to sweat gland cells equably, form controllably passes on and breeds.
Embodiment 1：
1) preparation of culture medium：
DMEM culture mediums are taken, preparation is obtained containing 0.5ng/mL hydrocortisones, 0.05ng/mL insulin, 1.8 × 10- 4Mol/L adenines, 100IU/mL penicillin, 15ng/mL hEGFs, 10 μ g/mL transferrins, 5 μ g/mL paddy ammonia
The DMEM culture mediums of acid, 5 μM of Y-27632 and 0.1mg/mL carboxymethyl chitosans；
Sweat gland cells inducing culture：Take DMEM culture mediums, preparation obtain containing 50ng/mL hEGFs, 1 ×
10-10Mol/L cholera toxins, 1 × 10-7Mol/L trilutes, 5 × 10-5The DMEM of mol/L acecolines
Culture medium；
Sweat gland cells culture medium：DMEM culture mediums are taken, preparation obtains extracting containing 50ng/mL EGF, 25mg/mL Niu Chuiti
The DMEM culture mediums of thing, 100U/mL penicillin and 100 μ g/mL streptomysins.
2) acquisition and culture of epidermal stem cells
Epidermal stem cells concrete operations are separated from isolated skin tissue is：After taking 3-6 year healthy infants foreskin stereotomy
The foreskin abandoned, under aseptic condition, chloramphenicol cleaning skin histology, add dispase to act on 18-24h in 4 DEG C, separate epidermis group
Knit；Sterile PBS epidermal tissue, 0.25% pancreas enzyme -EDTA digestion 4h, adds epidermal stem cells culture medium and terminates, 1000r/
Min, 5min is centrifuged, collect epidermal cell, sterile PBS three times, add epidermal stem cells culture medium and be resuspended, as
The coated culture plates of Matrigel are cultivated, and obtain epidermal stem cells；
3) induction and culture of sweat gland cells
The epidermal stem cells that step 2) is prepared carry out Secondary Culture, when its degrees of fusion reaches 80%, discard culture
Base, DMEM/F12 are washed once, are added sweat gland cells Fiber differentiation and are carried out induction differentiation；
Liquid, culture 7d or so are changed daily, treat occur polygon, the as cell of paving stone sample, sweat gland cells in culture medium,
Change culture medium into sweat gland cells culture medium, after cultivating 3 days, passed on and bred.
Embodiment 2：
Embodiment 2 as different from Example 1,
1) preparation of culture medium：
Epidermal stem cells culture medium：DMEM culture mediums are taken, preparation is obtained containing 0.1ng/mL hydrocortisones, 0.01ng/
ML insulin, 1 × 10-4Mol/L adenines, 50IU/mL penicillin, 5ng/mL hEGFs, 2 μ g/mL transferrins,
The DMEM culture mediums of 1 μ g/mL glutamic acid, 1 μM of Y-27632 and 0.01mg/mL carboxymethyl chitosan；
Sweat gland cells inducing culture：DMEM culture mediums are taken, preparation is obtained containing 25ng/mL hEGFs, 0.1
×10-10Mol/L cholera toxins, 0.5 × 10-7Mol/L trilutes, 2 × 10-5Mol/L acecolines
DMEM culture mediums；
Sweat gland cells culture medium：DMEM culture mediums are taken, preparation obtains extracting containing 10ng/mL EGF, 10mg/mL Niu Chuiti
The DMEM culture mediums of thing, 50U/mL penicillin and 50 μ g/mL streptomysins.
Embodiment 3：
Embodiment 3 as different from Example 1,1) preparation of culture medium：
Epidermal stem cells culture medium：DMEM culture mediums are taken, preparation is obtained containing 2ng/mL hydrocortisones, 1ng/mL pancreas islet
Element, 5 × 10-4Mol/L adenines, 200IU/mL penicillin, 50ng/mL hEGFs, 50 μ g/mL transferrins, 10 μ
The DMEM culture mediums of g/mL glutamic acid, 20 μM of Y-27632 and 1mg/mL carboxymethyl chitosans；
Sweat gland cells inducing culture：DMEM culture mediums are taken, preparation is obtained containing 100ng/mL hEGFs, 2
×10-10Mol/L cholera toxins, 5 × 10-7Mol/L trilutes, 8 × 10-5Mol/L acecolines
DMEM culture mediums；
Sweat gland cells culture medium：DMEM culture mediums are taken, preparation obtains carrying containing 100ng/mL EGF, 50mg/mL Niu Chuiti
Take the DMEM culture mediums of thing, 200U/mL penicillin and 200 μ g/mL streptomysins.
Comparative example 1：
As different from Example 1, sweat gland cells culture medium is the KSFM culture mediums containing 10vt%FBS to comparative example 1.
Performance detection and effect assessment：
1.MTT methods detect cell viability
The epidermal stem cells for taking each embodiment to be obtained with comparative example, are detected, its vigor is as shown in the table using mtt assay：
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example 1 | |
| Vigor [%] | 96.57 | 83.78 | 80.38 | 97.11 |
The vigor of cell is significantly higher than embodiment 2-3 (p wherein made from embodiment 1<0.05) it is and poor compared with comparative example 1
Different unobvious (P>0.05), illustrate that free serum culture has reached identical effect with commercially available containing serum free culture system.
2. identified by immunofluorescence sweat gland cells surface marker
The cell made from 0.25% pancreatin digestion embodiment 1, with 1 × 105Inoculum concentration be inoculated in 12 orifice plates, treat it
Adherent, 4% paraformaldehyde fixes 2h, and PBS is washed three times；
200 μ L primary antibody dilution normal temperature is added to close 1-2h；The primary antibody dilution be containing 10vt% serum,
0.3vt%TritonX-100 PBS；
Discard primary antibody dilution, add 200 mono- antiantibody (dilution factor 1 of μ L mouse source CK19, CK15:100), 4 DEG C of incubations
Overnight, PBS is flushed three times 3 times, each 5min；
200 μ L FITC are added to mark anti-mouse secondary antibody (1：400), reacting at normal temperature without light 1h, PBS are rinsed；PI is used before upper machine testing
Contaminate core.
Observation result is visible under fluorescence inverted microscope, and positive indication's thing CEA, CK14, CK18, CK8 of sweat gland cells exist
Positive expression in most sweat gland cells.Fluorescein PE labelled antibodies are combined with CEA, CK14, CK18, CK8 monoclonal antibody,
Red fluorescence is shown as in endochylema, the karyon of DAPI reagents dye sweat gland tissue derived cell is blueness.
Wherein, testing result such as Fig. 1-3 of sweat gland cells is made in embodiment 1-3.From Fig. 1-3 as can be seen that CEA, CK14,
CK18, CK8 albumen are the specific marker proteins of sweat gland cells, and high CEA, CK14, CK18, CK8 expression quantity is positive table
Reach, the cell for illustrating to induce is sweat gland cells.And each protein positive rate of embodiment 1 is apparently higher than embodiment 2-3 (p<
0.05)。
3. flow cytomery sweat gland cells surface marker
Epidermal stem cells are digested with 0.25% pancreatin and collect cell, and cell number is 2 × 105It is individual；
Cell born of the same parents are resuspended in 1mLDPBS and washed 2 times, 200g, centrifuge 5min, and obtained cell precipitation adds 1mL70%
Fixed 2h is resuspended in the alcohol of precooling；
Centrifugation discards fixer, adds 200 mono- antiantibody (dilution factor 1 of μ L mouse source CK19, CK15, CK10:100) normal temperature
Lower incubation 30min；
1mL PBS are washed, and mark secondary antibody (dilution factor 1 with FITC respectively:200) normal temperature lucifuge is incubated 1h；
After PBS is washed twice, PBS is discarded, adding appropriate PBS according to cell concentration is resuspended cell, is examined using flow cytometer
Survey.
Wherein, testing result such as Fig. 4 of sweat gland cells embodiment 1-3 is made.
By flow cytometry analysis showed, the positive expression amount of the CK14 albumen in sweat gland cells of embodiment 1 is 95%
More than, the positive expression amount of CEA albumen is more than 75%, and the negative expression quantity of MSX-1 albumen is below 2%；Embodiment 2 exists
The positive expression amount of CK14 albumen is that the positive expression amount of more than 82%, CEA albumen is more than 65% in sweat gland cells, and MSX-
The negative expression quantity of 1 albumen is below 4%；The positive expression amount of the CK14 albumen in sweat gland cells of embodiment 3 is more than 80%,
The positive expression amount of CEA albumen is more than 62%, and the negative expression quantity of MSX-1 albumen meets sweat gland cells below 5%
The expression of results of surface marker.And each protein expression of embodiment 1 is substantially better than embodiment 2-3 (p<0.05).
Above-mentioned embodiment is only the preferred embodiment of the present invention, it is impossible to the scope of protection of the invention is limited with this,
The change and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed scope.
Claims (10)
1. a kind of method that differentiation sweat gland cells are induced by epidermal stem cells, comprises the following steps：
1) epidermal stem cells are separated from isolated skin tissue, Secondary Culture is carried out in epidermal stem cells culture medium；
The epidermal stem cells culture medium be containing hydrocortisone, insulin, adenine, penicillin, hEGF,
Transferrins, glutamic acid, the DMEM culture mediums of Y-27632 and carboxymethyl chitosan；
2) epidermal stem cells for obtaining step 1) carry out Secondary Culture, when its degrees of fusion reaches 80%, discard culture medium, use
DMEM/F12 is cleaned, and is added sweat gland cells inducing culture and is carried out induction differentiation；
The sweat gland cells Fiber differentiation is to contain hEGF, cholera toxin, trilute and chlorination
The DMEM culture mediums of acetylcholine；
3) after cultivating some days, treat occur polygon, the as cell of paving stone sample, sweat gland cells in culture medium, by culture medium
Change sweat gland cells culture medium into, after cultivating some days, passed on and bred；
The sweat gland cells culture medium is the DMEM culture mediums containing EGF, ox pituitary extract, penicillin and streptomysin.
2. the method as described in claim 1, it is characterised in that in step 1), the isolated skin is organized as healthy infants bag
The foreskin abandoned after skin stereotomy.
3. the method as described in claim 1, it is characterised in that in step 1), it is thin that epidermal stem is separated from isolated skin tissue
Born of the same parents' concrete operations are：Take the foreskin abandoned after 3-6 year healthy infants foreskin stereotomy, under aseptic condition, chloramphenicol cleaning skin
Tissue, add dispase to act on 18-24h in 4 DEG C, separate epidermal tissue；Sterile PBS epidermal tissue, 0.25% pancreas enzyme -EDTA
4h is digested, epidermal stem cells culture medium is added and terminates, with 1000r/min centrifugation 5min, collects epidermal cell, sterile PBS
Cleaning three times, add epidermal stem cells culture medium and be resuspended, cultivated as the coated culture plates of Matrigel, obtain epidermal stem
Cell.
4. the method as described in claim 1, it is characterised in that in step 2), when induction is broken up, change sweat gland cells daily and lure
Lead culture medium.
5. the method as described in claim any one of 1-4, it is characterised in that the epidermal stem cells culture medium is containing with most
The DMEM culture mediums of the following components of final concentration：0.1-2ng/mL hydrocortisones, 0.01-1ng/mL insulin, 1-5 ×
10-4Mol/L adenines, 50-200IU/mL penicillin, 5-50ng/mL hEGFs, 2-50 μ g/mL transferrins, 1-
10 μ g/mL glutamic acid, 1-20 μM of Y-27632 and 0.01-1mg/mL carboxymethyl chitosan.
6. the method as described in claim any one of 1-4, it is characterised in that the sweat gland cells inducing culture be containing with
The DMEM culture mediums of the following components of ultimate density meter：25-100ng/mL hEGFs, 0.1-2 × 10-10Mol/L is suddenly
Random toxin, 0.5-5 × 10-7Mol/L trilutes, 2-8 × 10-5Mol/L acecolines.
7. the method as described in claim any one of 1-4, it is characterised in that the sweat gland cells culture medium is containing with final
The DMEM culture mediums of the following components of densimeter：10-100ng/mL EGF, 10-50mg/mL ox pituitary extracts, 50-200U/
ML penicillin and 50-200 μ g/mL streptomysins.
8. a kind of culture medium group that differentiation sweat gland cells are induced by epidermal stem cells, including following culture medium：
Epidermal stem cells culture medium：Containing hydrocortisone, insulin, adenine, penicillin, hEGF, turn iron
Albumen, glutamic acid, the DMEM culture mediums of Y-27632 and carboxymethyl chitosan；
Sweat gland cells Fiber differentiation：Contain hEGF, cholera toxin, trilute and acetyl chloride courage
The DMEM culture mediums of alkali；
Sweat gland cells culture medium：DMEM culture mediums containing EGF, ox pituitary extract, penicillin and streptomysin.
9. culture medium group as claimed in claim 8, it is characterised in that
The epidermal stem cells culture medium is the DMEM culture mediums containing the following components in terms of ultimate density：0.1-2ng/mL hydrogen
Change cortisone, 0.01-1ng/mL insulin, 1-5 × 10-4Mol/L adenines, 50-200IU/mL penicillin, 5-50ng/mL people
EGF, 2-50 μ g/mL transferrins, 1-10 μ g/mL glutamic acid, 1-20 μM of Y-27632 and 0.01-1mg/mL carboxylic
Methyl chitosan.
10. culture medium group as claimed in claim 8, it is characterised in that
The sweat gland cells inducing culture is the DMEM culture mediums containing the following components in terms of ultimate density：25-100ng/
ML hEGFs, 0.1-2 × 10-10Mol/L cholera toxins, 0.5-5 × 10-7Mol/L trilutes, 2-8
×10-5Mol/L acecolines；
The sweat gland cells culture medium is the DMEM culture mediums containing the following components in terms of ultimate density：10-100ng/mL
EGF, 10-50mg/mL ox pituitary extract, 50-200U/mL penicillin and 50-200 μ g/mL streptomysins.
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