CN108359633A - A kind of beaver rabbit dermis of skin hair papilla cell isolated culture method - Google Patents
A kind of beaver rabbit dermis of skin hair papilla cell isolated culture method Download PDFInfo
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Abstract
The present invention relates to technical field of cell biology, especially a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method.The beaver rabbit dermis of skin hair papilla cell isolated culture method includes:(1) beaver rabbit skin of back is taken, strip is cut into, is placed in II type separation enzyme solutions, is digested overnight in 4 DEG C, after next day digests in 37 DEG C of incubators, remove epidermis；(2) mud will be shredded into remaining dermis of skin part after step (1) removes epidermis processing, it is placed in D Collagenase Types and digests 4 6h, it is in a liquid state to digestion skin and has observed papilla under the microscope and dissociate, digestion is terminated with the DMEM containing fetal calf serum；(3) will be centrifuged through step (2) treated liquid skin, and collect cell, and discard supernatant liquid, cell culture medium be resuspended to get.Compared with the prior art, the isolated culture method science, reasonable, acellular mechanical damage in culture success ratio height and separation process.
Description
Technical field：
The present invention relates to technical field of cell biology, especially a kind of beaver rabbit dermis of skin hair papilla cell is separately cultured
Method.
Background technology：
Hair is attached to the outer surface of skin, not only has defence and defencive function to animal and people, also has and increase letter
Breath exchange and beautiful effect (Messenger, 1993).Hair is generated by hair follicle, hair follicle (Hair follicle) be epidermis to
The skin accessory organ that can control hair growth formed after corium recess, is mutual by embryonic period, embryonic phase neuroderm and mesenchyma
Effect, what final development was formed be made of multi-layer cellular, with the complicated dynamic for synthesizing a variety of special keratoprotein functions
Organ, the spy with unique structure and periodic regeneration (periodic cycle for undergoing growth period, decline phase and stand-down)
Property.Hair follicle formed with atomization in, refer at least to 20 kinds of different cell masses, mainly have papilla, hair matrix, interior
The cells such as sheath, external root sheath form.
Since hair follicle has the characteristic of cyclical growth, good model is provided for regeneration.The periodicity of hair follicle
Growth is mediated by hair follicle stem cells to be occurred, the tranquillization of hair follicle stem cells and activation regulated and controled by stem cell microenvironment (Paus etc.,
2004).Hair follicle stem cells are located at Hair Follicle Bulge area, but micro-loop of the people to influence hair follicle stem cells self-renewing and differentiation at present
Border understanding is simultaneously few, is more not thorough.Wherein, from stand-down to the conversion in growth period, that is, the startup in growth period is hair follicle life
The link of long most critical.When obstacle occurs for the periodic regeneration function of hair follicle, it just will appear the hair follicles disease such as alopecia, alopecia areata.
Jahoda etc. 2011 reports that micro-capsule, which is made, in hair papilla cell is implanted into rat ear skin, can induce ear hair follicle regeneration, and have
The hair papilla cell conditioned medium of people is used for the treatment of clinical alopecia areata.With the development of society, people to have protection with
The hair of beauty function is increasingly paid attention to, and research regenerated to hair follicle cycle is more and more important.
Papilla (Dermal papilla) is most important part in hair follicle dermal components, and hair follicle is controlled in hair follicle
Development, growth and reconstruction, and the size of hair follicle is determined to a certain extent.Hair papilla cell is that a group in corium is specifically changed
Mesenchyma source cell, play key effect in hair follicle cycle change procedure, be directly to participate in hair follicle in entire hair follicle
The key component of growth cycle regulation and control.Numerous studies show that in animal hair follicle cycle growth, hair papilla cell is dry thin with hair follicle
The signal communication of periodically regularity occurs for born of the same parents, the closest in hair follicle cycle startup (Millar, 2002；Aoi etc., 2012；
Hill etc., 2013), illustrate that hair papilla cell can provide microenvironment support for hair follicle stem cells, and regenerated in hair follicle cycle
Initial period plays a role.Papilla is the center of a secretion factor, can generate a large amount of signal factor, by activation or
Inhibition hair follicle cycle growth signals access performance adjustment effect (Peus etc., 1996；Roh etc., 2004；Dipo etc., 2005).
It is small and wrapped up by hair follicle however, since papilla is located at the basal part of hair follicle, therefore detach difficult.Mesh
Before, most common method is mainly microdissection in the separation process of papilla, and the method is to instrument and equipment and asepsis ring
Border is more demanding, and operator is needed to possess more skilled microseparation, and great work intensity is time-consuming longer, while obtaining
Hair papilla cell adherent rate is relatively low, and hair papilla cell is moved out difficulty.In addition, microdissection partition method can detach antenna hair follicle,
The larger hair follicles such as mouse skin hair follicle, goat skin hair follicle, and, individual small hair follicle more for picture beaver rabbit skin follicle quantity
The separation of papilla is simultaneously not suitable for, and separation is difficult；In addition mechanically decoupled that hair papilla cell is caused to damage, adherence rate is low, training
It is not high to support beaver rabbit corium hair papilla cell success rate.Therefore, obtaining beaver rabbit skin follicle hair papilla cell not only becomes research otter
The rabbit hair capsule cyclical growth variation, influence factor and regulatory mechanism crucial material, for improve beaver rabbit hide quality breeding change
It is good to lay the foundation, it also can also regenerate research for the human hair of medical domain and model is provided.
Invention content：
The present invention provides a kind of beaver rabbit dermis of skin hair papilla cell isolated culture methods, compared with the prior art, point
From cultural method science, reasonable, acellular machinery in culture beaver rabbit dermis of skin hair papilla cell success rate height and separation process
Damage, adherent rate are up to 99% up to 95-100%, successfully culture rate.The beaver rabbit skin obtained using the isolated culture method is true
Fur papillose cell can be established and improve being separately cultured suitable for the animal dermis hair papilla cell more than skin follicle quantity,
Not only research model material can be provided to illustrate beaver rabbit hair follicle growth law of development, influence factor and regulatory mechanism, to improve
The breeding improvement of beaver rabbit hide quality lays the foundation, and can regenerate research for the human hair of medical domain and provide material,
Solve problems of the prior art.
Used technical solution is the present invention to solve above-mentioned technical problem：
A kind of beaver rabbit dermis of skin hair papilla cell isolated culture method, including following operating procedure:
(1) take beaver rabbit skin of back, be cut into strip, be placed in II type separation enzyme solutions in, be digested overnight in 4 DEG C, next day in
After being digested in 37 DEG C of incubators, epidermis is removed；
(2) mud will be shredded into remaining dermis of skin part after step (1) removes epidermis processing, and will be placed in D Collagenase Types
Middle digestion 4-6h, until digestion skin is in a liquid state and has observed papilla under the microscope and dissociates, with the DMEM containing fetal calf serum
Terminate digestion；
(3) it will be centrifuged through step (2) treated liquid skin, and collect cell, discard supernatant liquid, cell culture base weight
It is outstanding to get.
Step (2) the digestion temperature is 37 DEG C, digestion time 4-6h.
2000r/min centrifuges 4-6min, is repeated 2 times；500r/min centrifuges 2-4min, is repeated 4 times；2000r/min is centrifuged
3-7min is repeated 2 times；500r/min centrifuges 2-4min, is repeated 4 times.
The concrete operation step of step (3) described centrifugation is followed successively by：2000r/min centrifuges 5min, is repeated 2 times；500r/
Min centrifuges 3min, is repeated 4 times；2000r/min centrifuges 5min, is repeated 2 times；500r/min centrifuges 3min, is repeated 4 times.
Step (1) the beaver rabbit skin of back is 25-35 age in days beaver rabbit skin of back.
Beneficial effects of the present invention：
(1) isolated culture method of the present invention detaches the scheme that enzyme and D Collagenase Types are combined using II type, successfully starts up
The original cuiture of beaver rabbit dermis of skin hair papilla cell, and substantially increase separative efficiency.Now it is directed to beaver rabbit dermis of skin hair breast
The culture of capitulum there has been no being combined the report for carrying out its corium hair papilla cell culture using above-mentioned enzyme, and is currently used for point
Low separation efficiency from corium hair papilla cell, complex steps are inconvenient.
(2) the beaver rabbit dermis of skin hair papilla cell system of isolated culture method structure of the present invention can stablize passage, and grow
Splitting status is good.
(3) the beaver rabbit dermis of skin hair papilla cell of isolated culture method structure of the present invention, in identification, papilla is special
Representation α-SMA expression is positive.
In addition, identifying that as a result display shows this hair by morphological observation, growth curve drafting and immunocytochemistry
The cell of bright isolated culture method acquisition is beaver rabbit dermis of skin source property hair papilla cell, can be to study beaver rabbit hair follicle growth and divide
The mechanism of change, various types of cells interaction mechanism provides good utility model especially in studying hair follicle.
Description of the drawings：
Fig. 1 is the beaver rabbit dermis of skin hair papilla cell (100X) that the present invention cultivates；
Fig. 2 is that the beaver rabbit dermis of skin hair papilla cell Giemsa that the present invention cultivates dyes (100X)；
Fig. 3 is the expression for the beaver rabbit dermis of skin hair papilla cell specific mark object α-SMA that the present invention cultivates；
Fig. 4 is the expression for the beaver rabbit dermis of skin hair papilla cell specific mark object Vimentin that the present invention cultivates；
Fig. 5 is the beaver rabbit dermis of skin hair papilla cell growth curve that the present invention cultivates；
Fig. 6 is recovery growing state after the beaver rabbit dermis of skin hair papilla cell that the present invention cultivates freezes.
Wherein：
Fig. 1 includes Fig. 1 a, Fig. 1 b, Fig. 1 c, Fig. 1 d, Fig. 1 e, Fig. 1 f,
Fig. 1 a are the hair papilla cell for cultivating 3-4 days separate outs, are triangle or short fusiformis；
Fig. 1 b are that culture enters exponential phase cell on the 7th day, form sheet of cell；
Fig. 1 c are when cultivating 9-10 days, and cell confluency reaches 80% or more, and starts dead cell occur；
Fig. 1 d are that the cell cultivated 14 days merges completely, form growth dense area.
Fig. 1 e are cell for 24 hours after passage, Acceleration of growth；
Fig. 1 f are secondary culture 5-6 days, and cell merges completely, form growth dense area, the growth of whirlpool sample；
Fig. 3 includes Fig. 3 a, Fig. 3 b, Fig. 3 c and Fig. 3 d,
Fig. 3 a are α-SMA immunohistochemistry (100X) positive findings；
Fig. 3 b are α-SMA immunohistochemistry (100X) negative control；
Fig. 3 c are α-SMA immunofluorescences (200X) positive findings；
Fig. 3 d are α-SMA immunofluorescences (200X) negative control；
Fig. 4 includes Fig. 4 a, Fig. 4 b, Fig. 4 c and Fig. 4 d,
Fig. 4 a are Vimentin immunohistochemistry (100X) positive findings；
Fig. 4 b are Vimentin immunohistochemistry (100X) negative control；
Fig. 4 c are Vimentin immunofluorescences (200X) positive findings；
Fig. 4 d are Vimentin immunofluorescences (200X) negative control；
Fig. 6 includes Fig. 6 a and Fig. 6 b,
Fig. 6 a are the cell cultivated after thawing for 24 hours, and cellular morphology is uniform, and cell growth is rapid；
Fig. 6 b are recovery about 5-6 days cells of culture, degrees of fusion up to 90%, cellular morphology with freeze it is preceding almost the same,
Whirlpool sample is grown.
Specific implementation mode：
In order to clarify the technical characteristics of the invention, below by specific implementation mode, and its attached drawing is combined, to this hair
It is bright to be described in detail.
(1) materials and methods
1. main agents
DMEM (Dulbecco ' s Modified Eagle Medium) basic (1X) culture medium is Gibco Products；
Fetal calf serum (Fetal bovine serum, FBS) is Gibco Products；II types detach enzyme (Dispase II) and D type glue
Protoenzyme (Collagenase D) is Sigma Products；Vimentin (VIM) Antibody, α smooth muscle actin
(a-SMA) Antibody SA1021-mouse IgG SABC immunohistochemical staining kits, SA1062-- mouse IgGs SABC-
FITC immunofluorescence dyeing kits, DAB developing solutions are Wuhan doctor's moral product；1 × PBS buffer solution, mycillin mixed liquor
(100 ×), trypsase-EDTA digestive juices (0.25%), Ji's nurse Sa dyeing liquor (Giemsa stain), DAPI solution (1mg/
), mL Triton X-100 are Suo Laibao products；The disposable tissue culture plate in 6 holes, steril cell culture dish are Ztel's product；
2. experimental animal and sample collection
30 age in days beaver rabbits are purchased from Taishan District Mount Taishan kind warren, and band to laboratory, Shearing shears cut off the back rabbit hair, by skin group
It knits and first uses iodine tincture disinfection, alcohol takes off iodine disinfection, with sterile scissors clip skin, places in the PBS containing mycillin；
3. the preparation of solution
(1) preparation of 0.25mg/mL Dispase II solution：0.25g Dispase II enzyme powders accurately are weighed, are added
It in 100mL 0.01M PBS, is dispensed after being completely dissolved, 4 DEG C of preservations；
(2) preparation of 0.1mg/mL Collagenase solution Ds:0.1g Collagenase D enzyme powders accurately are weighed,
It is added in 100mL 0.01M PBS, is dispensed after being completely dissolved, -20 DEG C of preservations；
(3) preparation of cell culture medium：10% fetal calf serum, mould mycin mixed liquor (100X) are added in DMEM culture mediums
1%；
(4) preparation of cells frozen storing liquid：Containing 10%DMSO, the DMEM of 30% fetal calf serum.
(2) embodiment
Beaver rabbit dermis of skin hair papilla cell isolated culture method of the present invention, includes the following steps：
1, after taking 30 age in days health beaver rabbits 1, cervical dislocation to put to death, skin of back cropping, alcohol swab disinfection, sterilize scissors
Clip skin is put into added in dual anti-PBS 50mL centrifuge tubes, in band to iuntercellular aseptic superclean bench, is clamped with tweezers
Skin, sterile PBS are rinsed repeatedly；
2, skin is cut into 2cm X 5cm strips with eye scissors, be placed in 0.25mg/mL Dispase II solution, in
4 DEG C are digested overnight 12-15h, and PBS is rinsed after next day digests 30min in 37 DEG C of incubators, throws off epidermis；
3, epidermis residue dermal partial will be thrown off and shred into mud, be placed in 0.1mg/mL Collagenase solution Ds 37 DEG C
5h is digested, observation is in a liquid state, and a large amount of DP separate outs are seen under microscope, is disappeared with the DMEM terminations containing 10% (volume) fetal calf serum
Change；
4, differential centrifugation collects cell, and concrete operations are：2000r/min centrifuges 5min, is repeated 2 times, and abandons supernatant, collects hair
The mixture of nipple hair fibr tissue and other cell components；By the papilla hair fiber tissue of collection and other are thin
The mixture of born of the same parents' ingredient continues 500r/min and centrifuges 3min, is repeated 4 times, abandons supernatant, removes other disseminated cells mixed；Continue
2000r/min centrifuges 5min, is repeated 2 times；500r/min centrifuges 3min, is repeated 4 times；It has centrifuged to abandon supernatant and use every time and has contained 10%
Serum DMEM culture mediums are resuspended, and the filtering of 0.75uM filters obtains purer DP；
5, filtrate is laid on the disposable tissue culture plate in six holes, 37 DEG C, 5%CO2Saturated humidity incubator culture.It changes within every 5 days
Liquid is primary.
Reference examples 1 detach beaver rabbit dermis of skin hair papilla cell using microscope separation method
Beaver rabbit dermis of skin hair papilla cell isolated culture method, including following operating procedure：
1, after taking 30 age in days health beaver rabbits 1, cervical dislocation to put to death, skin of back cropping, alcohol swab disinfection, sterilize scissors
Clip skin is put into added in dual anti-PBS 50mL centrifuge tubes, in band to iuntercellular aseptic superclean bench, is clamped with tweezers
Skin, sterile PBS are rinsed repeatedly；
2, it is cut off along corium subcutaneous tissue intersection in super-clean bench with sterile scissors, discards epidermis；Aseptic nipper is gently pressed
Subcutaneous fat part containing hair follicle middle and lower part squeezes out hair follicle incisxal edge tip, and incisxal edge tip then is clamped by hair in tweezers with another
It is completely extracted from subcutaneous fat capsule middle and lower part；The hair follicle of extraction is placed in culture dish, interior to contain sterile PBS；
3, under microscope, be clamped with tweezers above the nearly ball top of hair follicle, due to epithelial portion connect with corium portion it is closer,
It can only assemble to the migration of ball top portion compared with the hair matrix cell of evacuation and melanocyte etc., it is rich that this so that ball top portion forms an inside
Spherical shape containing pressure.1mL syringe needles are used to cut the dermal sheath of the nearly papilla rhizome portion of ball top at this time, due to releasing for pressure
It puts and completely releases papilla；Finally, papilla rhizome portion is completely cut through with 1mL syringe needles, Pasteur is used in combination to inhale
Pipe picks up free papilla；
4, inoculation contains 10% fetal calf serum, and six holes of the DMEM culture solutions of 1% mould mycin mixed liquor (100X) are disposable
Tissue culture plate, 37 DEG C, 5%CO2Saturated humidity incubator culture changes the liquid once for every 5 days.
Reference examples 2：Beaver rabbit skin dermal papilla cell is detached using D Collagenase Type separation methods
Beaver rabbit dermis of skin hair papilla cell isolated culture method, including following operating procedure：
1, after taking 30 age in days health beaver rabbits 1, cervical dislocation to put to death, skin of back cropping, alcohol swab disinfection, sterilize scissors
Clip skin is put into added in dual anti-PBS 50mL centrifuge tubes, in band to iuntercellular aseptic superclean bench, is clamped with tweezers
Skin, sterile PBS are rinsed repeatedly；
2, beaver rabbit skin is shredded into mud in super-clean bench with sterile scissors, it is molten is put in 0.1mg/mL Collagenase D
37 DEG C of 5 hours of digestion, observation are in a liquid state in liquid, and a large amount of DP separate outs are seen under microscope, whole with the DMEM containing fetal calf serum
Only digest；
3, differential centrifugation collects cell, and concrete operations are：2000r/min centrifuges 5min, is repeated 2 times, and abandons supernatant, collects hair
The mixture of nipple hair fibr tissue and other cell components；500r/min centrifuges 3min, is repeated 4 times, abandons supernatant, removes
Other disseminated cells mixed；2000r/min centrifuges 5min, is repeated 2 times；500r/min centrifuges 3min, is repeated 4 times；It uses every time
The culture mediums of DMEM containing serum are resuspended, the filtering of 0.75uM filters；
4, filtrate is laid on the disposable tissue culture plate in six holes, 37 DEG C, 5%CO2Saturated humidity incubator culture.It changes within every 5 days
Liquid is primary.
(3) identification method
1, morphological observation
The adherent beaver rabbit dermis of skin hair papilla cell being separately cultured and growing state and thin are observed under microscope normal light
Born of the same parents' form.After hair papilla cell growth converges completely, secondary culture is carried out.Old culture medium is discarded first, sterile PBS is rinsed,
Trypsase-EDTA the digestive juices of 1mL 0.25% are added per hole, digest 4min, microscopically observation to cell list under room temperature
When gap occurs in layer contraction protrusion, cell culture fluids of the 2mL containing 10% fetal calf serum is added and terminates digestion, blows and beats, 1000r/min
Cell is collected by centrifugation, by 104A/hole bed board.
2, Giemsa is dyed
Sterile cell climbing sheet is put into disposable 6 porocyte culture plates, 2mL cell suspensions is taken to be added in six orifice plates,
It is placed in CO2It is cultivated in incubator；After cell grows up to good cell monolayer, culture medium is discarded, PBS is added after rinsing without water beetle
Alcohol fixes 15min, takes out creep plate with tweezers, then rinse cell with new absolute methanol, is put into Ji's nurse Sa dyeing liquor and contaminates immediately
Color 5-10min, through gradient alcohol dehydration, dimethylbenzene is transparent, and last neutral gum mounting, microscopically observation is taken pictures.
3, immunohistochemistry is identified
Sterile cell climbing sheet is put into disposable 6 porocyte culture plates, 2mL cell suspensions is taken to be added in six orifice plates,
It is placed in CO2It is cultivated in incubator；After cell grows up to good cell monolayer, 30min, PBS punchings are fixed with 4% paraformaldehyde
It washes 3 times, each 3min, is carried out according to SA1021-mouse IgG SABC immunohistochemical staining kit specification steps
The detection of Vimentin and a-SMA protein expressions.
4, identified by immunofluorescence
Sterile cell climbing sheet is put into disposable 6 porocyte culture plates, 2mL cell suspensions is taken to be added in six orifice plates,
It is placed in CO2It is cultivated in incubator；After cell grows up to good cell monolayer, 30min, PBS punchings are fixed with 4% paraformaldehyde
It washes 3 times, each 3min, is carried out according to SA1062- mouse IgG SABC-FITC immunofluorescence dyeing kit specification steps
The detection of Vimentin and a-SMA protein expressions.
5, growth curve is drawn
Take the good cell of upgrowth situation with 104A/hole bed board, takes 3 holes daily, continuous statistics 14 days, cell counting board
Count daily cell density；Using incubation time as abscissa, using cell density as ordinate, cell growth curve is drawn.
6, cell cryopreservation and recovery
The cell for selecting logarithmic phase growth, is made cell suspension using the method for secondary culture, cell cryopreservation is then added
Liquid (contains 10%DMSO, the DMEM culture solutions of 20% fetal calf serum), with 106/ mL cell densities, are sub-packed in the cryopreservation tube of 2mL,
Sealed membrane seals, and has marked Cell Name and date etc..Cryopreservation tube is sequentially placed into 4 DEG C of 30min, -20 DEG C of 30min, -80 DEG C of mistakes
Night finally moves into liquid nitrogen and preserves for a long time.
When recovery cell, cryopreservation tube is taken out from liquid nitrogen, put into 40 DEG C of water-baths makes its thawing, the interior preheatings of 5min rapidly
Culture medium be diluted to 10 times of original volume or more.Low-speed centrifugal 10min goes supernatant that fresh culture is added and is cultivated.
(4) qualification result and analysis
1) isolated culture method effect compares
Table 1
It, can be with as it can be seen from table 1 using the method that is combined with D Collagenase Types of II types provided by the present invention separation enzyme
Obtain 1.0*108A papilla, up to ten million, and microscope separation method only 10,000, single clostridiopetidase A D separation is better than aobvious
Micro mirror separation method, but the method that separative efficiency is combined not as good as II types separation enzyme with D Collagenase Types.In addition, utilizing institute of the present invention
The papilla adherent rate of the method separation of offer is up to 95-100%, and culture success ratio is also above other two methods.Thus may be used
See, for the method for the present invention in the cell quantity of acquisition, adherent rate and culture success ratio etc. are significantly better than that existing method.
In addition, the separation enzyme and clostridiopetidase A with existing report are combined
2) morphological observation
It is rounded, oval that the present invention detaches the papilla obtained, does suspension culture, starts after about 8h adherent, and culture is for 24 hours
Most papillas can be securely adherent, and can see within 3-4 days has cell to be grown from papilla edge, and radioactivity is grown around,
Cell of moving out is triangle or short fusiformis (Fig. 1 a).At the 7th day or so, cell entered exponential phase, and endochylema is abundant, growth
Rapidly, radial growth around, at this time papilla gradually lose the shape of original, form sheet of cell (Fig. 1 b).
At 9-10 days or so, cell growth range no longer expanded, and fusion rate reaches 80% or more, and started dead cell (figure occur
1c), cell merges completely after about 14 days, forms growth dense area, and the cell after fusion is substantially arranged radially, and can carry out the
Primary passage (Fig. 1 d).By passing on for the first time, 12h cells can be adherent, and cellular morphology is uniform compared with primary cell for 24 hours, cell life
Long to accelerate (Fig. 1 e), in culture 5-6 days, cell merged completely, is formed and grows dense area, cell with spindle shape fiber-like multipole to
Arrangement, whirlpool sample growth, can be passed on (Fig. 1 f) again.
3) Giemsa is dyed
The cell culture that the present invention is separately cultured forms the cell climbing sheet of good single layer after Giemsa is dyed, it is seen that cell
Matter is in light blue, the visible nucleus with 1-2 kernel of the nearly centre of cell space, is in aubergine (Fig. 2).
4) specific mark object is expressed
Using immunocytochemical stain technology detect in vitro culture hair papilla cell specific marker object α-SMA and
Hair follicle corium source cell marker object Vimentin expressions.α-SMA Showed by immune group result is negative control with Fig. 3 b
It compares, the expression of α-SMA can be obviously observed in Fig. 3 a；Meanwhile immunofluorescence results are also shown, and negative control Fig. 3 d ratios,
Fig. 3 c cytoplasm sends out green fluorescence, that is, the cell express alpha-SMA albumen being separately cultured.Equally, the immunohistochemistry of Vimentin
Fig. 4 a and immunofluorescence Fig. 4 c are shown, are separately cultured cell expression Vimentin.The above results show that the present invention is separately cultured
Hair papilla cell really be hair follicle corium source property hair papilla cell.
5) growth curve is drawn
Cell growth curve observation is carried out to the cell that the present invention is separately cultured, first 3 days after cell is adherent, growth was slow
Slowly, quantity is without significant change；Enter exponential phase within 4-10 days, reach peak of proliferation within the 12nd day, decline phase, table are entered after 12 days
The hair papilla cell of bright separation is grown and splitting ability is vigorous (Fig. 5).
6) cell cryopreservation and recovery ability
After the logarithmic phase hair papilla cell recovery being separately cultured to the present invention for freezing 3 months, Trypan Blue counts system
Meter finds that hair papilla cell is averaged motility rate 74.5%.Cell after defrosting finds that cell is adherent still rapid through culture, and 12h is thin
Born of the same parents can be adherent, and cellular morphology is uniform for 24 hours, and cell growth accelerates (Fig. 6 a)；It is cultivated in Tissue Culture Dish about 5-6 days or so thin
Born of the same parents' degree of converging up to 90%, cellular morphology with freeze before it is almost the same, cell continues culture and finds after covering with, hair papilla cell with
There is layering growth again to arrangement, the growth of whirlpool sample, cell in spindle shape fiber-like multipole, and it is specific solidifying still to retain it
Collection property growth characteristics (Fig. 6 b).
Above-mentioned specific implementation mode cannot function as limiting the scope of the invention, for the technology people of the art
For member, any alternate modification or transformation made to embodiment of the present invention are all fallen in protection scope of the present invention.
Place is not described in detail by the present invention, is the known technology of those skilled in the art of the present technique.
Claims (5)
1. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method, it is characterised in that：Including following operating procedure:
(1) beaver rabbit skin of back is taken, strip is cut into, is placed in II type separation enzyme solutions, is digested overnight in 4 DEG C, next day is in 37 DEG C
After being digested in incubator, epidermis is removed；
(2) mud will be shredded into remaining dermis of skin part after step (1) removes epidermis processing, and will be placed in D Collagenase Types and disappears
Change 4-6h, until digestion skin is in a liquid state and has observed papilla under the microscope and dissociates, is terminated with the DMEM containing fetal calf serum
Digestion；
(3) it will be centrifuged through step (2) treated liquid skin, and collect cell, and discard supernatant liquid, cell culture medium is resuspended, i.e.,
.
2. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method according to claim 1, it is characterised in that：Step
Suddenly (2) described digestion temperature is 37 DEG C, digestion time 4-6h.
3. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method according to claim 1, it is characterised in that：Step
Suddenly the concrete operation step of (3) described centrifugation is followed successively by：2000r/min centrifuges 4-6min, is repeated 2 times；500r/min centrifuges 2-
4min is repeated 4 times；2000r/min centrifuges 3-7min, is repeated 2 times；500r/min centrifuges 2-4min, is repeated 4 times.
4. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method according to claim 3, it is characterised in that：Step
Suddenly the concrete operation step of (3) described centrifugation is followed successively by：2000r/min centrifuges 5min, is repeated 2 times；500r/min centrifuges 3min,
It is repeated 4 times；2000r/min centrifuges 5min, is repeated 2 times；500r/min centrifuges 3min, is repeated 4 times.
5. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method according to claim 1, it is characterised in that：Step
Suddenly (1) described beaver rabbit skin of back is 25-35 age in days beaver rabbit skin of back.
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