A kind of skin model and application
Technical field
The present invention relates to a kind of skin model, and this model is in the research to application on human skin disease, the application of the screening of dermal drug and the clinical prods aspect of exploitation treatment wound.Belong to histocytology and tissue engineering field.
Background technology
For a long time, dermatologist and skin surgery doctor are striving for the skin that can bear complete function clinically more always, are used for the treatment of the wound of skin and eliminate skin scar.There is a lot of reasons can cause the infringement of skin: the wound that such as genetic diseases, burn and scald, chronic trauma cause as diabetes.A lot of skin lesion self can not recover later, the large area burn and scald of the such as degree of depth, very easily causes and infect and cause death after widespread skin damage.At present, prevailing Therapeutic Method is that the skin that do not damage with other positions of operation transplantation self is to burn and scald injury.But this method has very large limitation, if large-area burns on the one hand, the limited source of self normal skin; On the other hand, the skin survival rate after transplanting is low is also the clinical significant challenge faced at present.So need In vitro culture to obtain technology carry out amplifying cells for transplanting.The succedaneum that the current Cell binding framework albumen cultivating amplification comes together to be formed application on human skin as collagen protein replaces complete skin transplantation to play very large using value clinically, but this skin substitutes just forms very thin epidermis and corium, the accessory organ that can not form subcutaneous tissue and skin, as hair, sweat gland etc., is the skin products not having normal function.The foot ulcers that diabetes patient is common for another example, the microenvironment due to diabetes patient causes this skin ulcer to be difficult to healing, method treatment not good at present.
Tissue engineering hair growth contains cutify tissue or cell to target area to induce hair follicle to be formed.The induction of hair follicle and the interaction constantly between the active epithelial cell of growth needs and Interstitial cell.But not allly transplant from hair follicle cortex the generation that the cell of gained all has the ability to induce new hair follicle.Research in the past shows, the hair follicle separated from human body scalp can be transplanted to the mouse back of immunodeficiency, successfully returns to regular regeneration circulation afterwards.By transplanting the human skin succedaneum of In vitro culture saltant type (TSCII gene deletion) fibroblast formation, successful regeneration goes out hair.But the hair follicle of this regeneration can not form normal hair shaft, and to form normal hair shaft be one of the key element judging whether to be formed hair follicles maturity.Other researchs show, the Graftskin be made up of epidermis cell and Interstitial cell can be used for transplanting to promote that hair follicle is formed.The United States Patent (USP) 2012/0095445 applied for by people such as Zheng, describe from the epidermis be separated and hypodermal cell, generate hair follicle in vitro method and reagent, these cells are single culture, go down to posterity, and are then combined to form hair follicle implanted thereafter.But because hair follicle is implanted after must being formed in vitro again, this process is restricted.
The United States Patent (USP) (patent No. 2011/0321180) of Lee etc. reports and becomes muroid hair follicle from the mouse skin stem cell regenerating of newborn mice fresh separated.Because the quantity of fresh separated cell is limited, regeneration skin just needs to use the cell cultivating amplification.Unfortunately, multipotential cell often can cause cell differentiation to cause the loss of required cell type ability in cultivation.This patent of invention does not report the skin of people and the regeneration of hair in addition.What this research was is the mouse cell of fresh separated, instead of people's cell of In vitro culture.
In a word, at present, also do not have in the world can bear the complete functional and skin of people with ripe hair of having again with the cell of cultured and amplified in vitro.
Summary of the invention
We have invented separation and the cultural method of the multi-functional cell of human skin, and transplant this cultured cells bears complete meritorious energy human skin with it again technology to mice.This technology comprises the separation method by obtaining multi-functional epidermis and hypodermal cell, multi-functional epidermis and hypodermal cell in-vitro multiplication and maintains polyfunctional cultural method.The human skin regenerated by above-mentioned technical method is contained with normal epidermis, corium and a complete hypodermis layer.The skin of this regeneration comprises hair, sebaceous gland, sweat gland, nerve and blood vessel containing all skin accessories.And the hair of regeneration has normal pegmentation and the human hair follicle of the maturation of energy loop cycle growth.The area that the skin regenerated covers on mice is at 0.1 ~ 2cm
2in region, every 1cm
2the hair follicle of 50-2000 maturation can be produced.
Technical scheme of the present invention is: a kind of method of cell regeneration mankind intact skin tissue of In vitro culture, is characterized in that, comprise the following steps:
(1) tissue samples containing multi-functional epidermis and hypodermal cell of scalp being derived from fetal scalp, neonatal foreskin or adult is gathered; In addition from the Interstitial cell of the SOX2 gene expression of any tissue with still can have the ability after having the ability to form monoclonal epidermis cell and freezing thawing from any tissue samples to form the cell of application on human skin;
(2) the fresh tissue samples tissue digestion enzyme (Dispase proteolytic enzyme) gathered is spent the night in low temperature 4 DEG C digest, within second day, separate epidermis and dermal partial by Mechanical Method;
(3) skin portion is shredded rear cultivation at trypsin typsin) in 37 DEG C of water baths be separated epidermis cell with 40 order bore filter nets after 30 minutes; Dermal partial is shredded rear cultivation 37 DEG C of water baths in Collagenase and isolate multi-functional true chrotoplast with 40 order bore filter nets after 30 minutes;
(4) separately cultivate from the epidermis cell after separate tissue and hypodermal cell, epidermis cell and hypodermal cell can with any culture medium culturings for cell self-sow, comprise MEM, DMEM, RPMI, F-12, CNT07 etc. in conjunction with different somatomedin as fibroblast growth factor (FGF), epithelical cell growth factor (EGF), B27 (neuronal cell cultures) Summing Factor PC-1 (primitive cell culture) replenisher etc.; Cell culture medium is recommended to be the DMEM/F-12 culture medium containing glutamine; The cultivation of epidermis cell adds 2-10 μM of Rock kinases inhibitor in the medium in addition if Y-27632 is to maintain the multifunctionality of epidermis cell; The culture environment of epidermis cell and hypodermal cell should close to human physiological environment, and culture medium pH value is 6-8, and preferably 7.4; The cultivation temperature of cell is 30-40 DEG C, is preferably 35 DEG C-37 DEG C; Can cultivate in conjunction with hypoxia the multifunctionality that method that (2%-10%) or cell aggregation cultivate strengthens cell in addition, and increase the expression of hypodermal cell SOX2 gene.
(5) hypodermal cell is cultivated and is changed a culture fluid in every 5 days, and epidermis cell changes a culture fluid for every 2 days; Just go down to posterity in culture vessel 80-90% coverage rate when Growth of Cells reaches; Hypodermal cell goes down to posterity in 1:3-6 ratio, can pass 2-6 generation; Epidermis cell goes down to posterity in the ratio of 1:2-4, at least can pass 2-4 generation;
(6) the multifunctional meter chrotoplast after cultivating propagation and hypodermal cell digest formation individual cells respectively, then with epidermis cell: hypodermal cell is that the number ratio of 1-10:1 is mixed and made into cell mixing suspension in DMEM/F12 (1:1) culture fluid, and cumulative volume is at 100-200 microlitre; Cell mixing suspension can add its functional cell as melanocyte simultaneously, endotheliocyte etc.; Then cell mixing suspension is transferred to on the semipermeable polymer (as polyethylene terephthalate film) in 3.0 μm of ventilative apertures or pellosil substrate, and in 37 DEG C of environment, hatch 1-2 hour, make the complete coherent film of cell also to be formed simultaneously cell aggregation group to maintain the multifunctionality of cell;
(7) transplanted cells is to receptor: the wound of the intact skin organization formation first cutting away one piece of 0.5-2.0 square centimeter size on receptor without skin histology but no infringement underlying muscle tissue; Then cover on whole wound by the substrate being attached with hypodermal cell and epidermis cell mixing liquid, cell face contact wound, substrate faces up; And by substrate and skin closure, wrapping, or the regeneration site cell mixing suspension containing passage cell directly can being injected skin or hair needs.Prove by experiment by cell mixture as wound, the cell of transplanting can autologous tissue also stimulate and induction mutually, impels the formation comprising the human body such as hair follicle and relevant body of gland intact skin structure.
Use TGFβ_1 antibody and β 2 antibody (TGF beta 1/beta 2), the local immunosuppression agent such as thyroliberin (ACTH), melanotropin Alpha antibodies (alpha MSH), Ciclosporin A (Cyclosporine A) or rapamycin (Rapamycin) coordinate the transplanting of cell, can reduce inflammation and graft-rejection like this.These local immunosuppression agent can before or after transplanted cells, and by shallow injection, microneedle therapy or local treatment, immunosuppressant can be transferred in receptor.
This technology can be applied to set up people skin model to do drug test and screening; The model of test skin corrosion; The skin setting up regenerating human to evaluate new treatment technology, as laser therapy, cold therapy art, dermal abrasion art etc.
The skin that this technology may be used for setting up regenerating human carrys out drugs method for dermal drug delivery; Research ultraviolet and illumination are to the infringement of skin; The medicine of research treatment dermatosis; The medicine of research treatment hair diseases; The medicine of research treatment Chinese gland disease; The medicine of research treatment disease of sebaceous glands; Research skin anti-aging; Research skin disappears fat medicine; The cirsoid disease of research skin; The drug screening of the treatment of research individual character and individual character.
What this technology may be used for setting up people sets up application on human skin wound and bum model; Set up the model of diabetes patient's skin ulcer; Set up the animal model of hair growth; Set up specific skin disease model: as psoriasis, skin carcinoma, eczema, alopecia areata, male alopecia, epidermis blister is sick, acne, albinism, skin aging etc.
This technology may be used for exploitation cell products for clinical treatment regenerated hairs and sebaceous gland; Treat various alopecia diseases; Promote wound healing; Treat chronic recovery to close; The skin ulcer for the treatment of diabetes; The healing for the treatment of scar tissue; Treatment scar tissue also forms normally functioning sweat gland, subcutaneous tissue and sebaceous gland; For the wound healing without cicatrix; For ear, the reparation of nose and eyelid; Remove the skin healing after tatooing; Treatment psoriasis and skin albinism; In treatment skin, skin aging and crease-resistant is treated by neural infringement and regenerated bark undertissue.
Useful achievement of the present invention is, in the following examples, we successfully show with the hypodermal cell from embryo's scalp of In vitro culture and regenerate the intact skin of people from the nude mice back being transplanted to immunodeficiency after neonatal epidermal cells of prepuce mixing and comprise full hypodermis layer and ripe hair follicle.Skin and the hair follicle of regeneration have normal pigmentation.The skin of regeneration can will maintain the form of its human skin and performance at least more than 6 months with it Mus.The skin that we also show regeneration in addition has the function of wound healing.
Accompanying drawing explanation
Fig. 1 is nude mice model regenerates skin and hair photo after 6 months; Wherein right figure is the amplification picture of left figure; The skin that this figure shows regeneration be have Pigmented
Fig. 2 is the tissue slice figure of 6 months regeneration skin of Fig. 1.The structure that this figure shows regeneration skin is similar with the scalp structure of normal person containing epidermal area, skin corium, the hair follicle of hypodermis layer and maturation and sebaceous gland.
Detailed description of the invention
Above-mentioned innovation can be understood further with reference to following instance:
Example: regenerate the functional skin of people and hair follicle with the cell of cultured and amplified in vitro on nude mice
Materials and methods:
Animal: the nude mice (or homozygote nu/nu sudden change Mus) of immunodeficiency or non-diabetic obesity mice/severe combined immunodeficiency Mus are albino rats, these two kinds of Mus all produce rejection because immunodeficiency makes it reduce to allosome tissue etc., are therefore suitable for transplanting and tumour transplatation as various heteroplasm.
Cell and cell culture medium: the epidermis cell dissociated takes from human neonatal foreskin.Hypodermal cell is from the scalp of 15 weeks fetuses.Cell, after separate tissue, is cultivated in hypodermal cell culture medium DMEM/F12 (1:1) (Gibco, Cat.#11039).And epidermis cell middle cultivation in the CNT07 culture medium containing protein kinase (ROCK) inhibitor Y27632.Hypodermal cell and epidermis cell need cultivate the period 3, the third generation respectively.Then 150 μ l cell suspending liquids DMEM/F12 (1:1) (Gibco, Cat.#11039) are mixed and made into 1:1 ratio.Then cell suspending liquid is transferred on pellosil (Invitrogen), and be placed in 37 DEG C of cell culture incubators and cultivate 1-1.5 hour, make cell adhere to pellosil completely.
The transplanting of cell: cut full thickness skin from there being the nude mice back of immunodeficiency and form open wound, be covered on the wound of Mus by the pellosil with cell, cell face directly contacts with wound, above pellosil covers.Every nude mice can be cooked 1 to 2 transplanting with it, and pellosil and nude mice skin is sewed up, and will scribble the antiseptic gauze of ointment as on wound, and with sterilization bandage nude mice.Nude mice after transplanting is observed weekly and Taking Pictures recording, observable 12 weeks to a year.
Histologic analysis: observing the new skin that can regenerate Mus with dissection light microscopy latter stage with it, and the sample of regeneration skin is collected in excision, sample freezing processing Hou Zuo10μm tissue physiology cuts into slices, and haematoxylin-eosin stains method dyes.In brief, tissue physiology's section first soaks setting in the formalin of 10%, rinses, dye three minutes, then use distilled water flushing, then use eosin stains 30 seconds with haematoxylin with 1xPBS.Again respectively with alcohol flushing and dehydration that concentration is 70%, 90% and 100%.After dehydration, section dimethylbenzene rinses, and coverslip covers, and by xylyl setting agent solidifying and setting, and regenerates the organizational structure of skin by optics microscopic examination.
Alkali phosphatase (AP) is painted: carry out AP according to the workbook (Roche NBT/BCIP stock solution, Cat#11681451001) of the product graft to 6 weeks and 12 weeks painted.By the section of 10 μm of freezing tissue physiologies through PBS rinsing, the acetone refreezed fixes 10 minutes.With 1XPBS solution rinsing twice, each 5 minutes, be placed in AP buffer (0.1M Tris-HCL, pH9.5,0.1M NaCl, 0.05M MgCl2) in 5 minutes, with dyeing liquor (200 μ l NBT/BCIP stock solutions are dissolved in 10mlAP buffer) the fixation 5-30 minute of fresh configuration.Positive AP reaction can produce navy blue coloring effect, every microscopic examination in 5 minutes once, to determine incubation time.After navy blue coloring effect occurs, remove dyeing liquor, rinse section.Then redye 30 seconds, ethanol dehydration with Yihong, coverslip covers, and diphenyl solid agent is fixed.
Utilize cultured cell that green fluorescent protein (GFP) is expressed to regenerate skin: to be produce GFP-express cell, the horn cell of the retroviral transmission foreskin of expressing with GFP or neonate scalp hypodermal cell, every T75 Tissue Culture Flask consumption is the virus liquid of 6 milliliters.Infect three little after replace with normal growth medium.Next day, collect the cell of infection and mix with the foreskin horn cell do not infected or hypodermal cell, the cell suspending liquid of mixing carries out being transplanted to as the above-mentioned method described with it nude mice, monitor its change, and collected the skin regenerated afterwards with 12 weeks, and whether form green regeneration skin with fluorescence microscopy.
Immunofluorescence is analyzed: immunofluorescence should operate by standard.Do 10 μm of sections after freezing, fix 10 minutes with 4% paraformaldehyde/PBS, PBS rinses, and under room temperature, Block buffer hatches (PBS of 10% donkey serum+2% bovine serum albumin) 1 hour.Primary antibodie is hatched: (conjugation mouse-anti bovine serum albumin (CD49) α 6-closes element (stem cell to basement membrane labelling primary antibodie, cat.10111)), epidermis cell or bottom cellular layer labelling (anti-human keratin (BD,) or corium (leaf) cell marking (the anti-Vimentin of rabbit (cellular signal transduction, cat.3932)) cat.550951).Section is placed in above antibody-solutions 4 DEG C of overnight incubation.Next day, section is placed in PBS solution and rinses, two anti-hatch 1 hour.By DAPI (Vector Laboratory) solid containing solid agent, checked the expression of each albumen by Laser Scanning Confocal Microscope analysis.
Result:
Post operation observes weekly the skin formational situation of mouse transplanting place.After transplanting 4 weeks, the wound surface of transplanting place heals completely, and is formed with the new skin of pigment, does not have cicatrization, the wound healing that may be used for without cicatrix is described.By the 12nd week, skin surface produced the clear and legible hair with pigment of naked eyes.Because the nude mice of immunodeficiency does not have melanocyte can not form skin and the hair of pigment, illustrate that newborn skin and hair are the cultured cells from transplanting.Longer hair can sustainable growth, within 6 months, can reach 3cm long (as shown in Figure 1).This prove further by graft area or surrounding skin injection or and Skin Cell mixing, and with the use of immunosuppressant, allochthonous melanocyte can be applied in the skin of regeneration and hair follicle and reach the colour of skin accepted in appearance.
Tissue section strain (haematoxylin-eosin stains method) analysis display (as shown in Figure 2) in 6 months: the structure of regeneration skin is closely similar with the skin texture of adult's scalp, containing epidermal area, skin corium and hypodermis layer.And its ripe hair shaft of hair of regeneration is connected to sebaceous gland and dermal papilla.The hair follicle that we observe different growing stages appears on same tissue slice, the hair follicle of this suggestion regeneration be have a function carry out cycling deposition.The ability of hair follicle cycling deposition weighs the major criterion of hair follicles maturity.In order to further monitor the function of hair cycle, hair is cut short to observe the hair cycle cycle.After cutting one month, find that the hair of regeneration can regrow out, this proves that hair of regeneration has function further.It should be noted that the formation also having sweat gland at some regeneration skin area.Before the regeneration of sweat gland, nobody reported.
In order to analyze the structure of regenerated hairs further, we utilize alkaline phosphatase staining to identify the dermal papilla (dermal papilla) of restructuring hair follicle.Alkali phosphatase is the mark of Division identification dermal papilla and other dermal fibroblasts.In order to further labelling regenerated hairs and skin, the hair follicle of alkaline phosphatase staining mark regeneration was carried out to the 6th week and the 12nd week regeneration skin.The mastoid process that the hair of rear 6th week commitment is transplanted in dyeing display is alkaline phosphatase positive, and the hair follicle of the hair of the 12nd week maturation and periphery thereof are alkaline phosphatase positive.This result shows that the hair follicle regenerated is normal mature.
In order to show that the skin of regeneration is from the cultured cell transplanted, the foreskin horn cell of incubation uses green fluorescent protein-labelling retroviral infection before transplantation, so that transplanted cells is traceable under fluorescence microscope.12 weeks after the transfer, regeneration skin was separated from transplant, analyzes under fluorescence microscope.The epidermis cell part of all regeneration skin is comprising epidermal area, and hair and sebaceous gland all present fluorescence green, shows that they derive from the foreskin horn cell of the cultivation of green fluorescent protein-labelling.In contrast, all dermal partial, comprise the mastoid process of hair follicle, be then all negative for green fluorescent protein.This experiment also advises that we externally can carry out genetic modification to set up the model of specific skin disease to cultured cells.
For the skin and the hair follicle that prove regeneration are not further formed by the mouse cell of receptor, but from the Skin Cell of people of the In vitro culture transplanted, we analyze skin and the hair follicle of regeneration with the immunofluorescence staining of the cell protein antibody only identifying people, analyzed by immunofluorescence microscopy, reaffirm that the skin of regeneration derives from human body cell.First, we use Cytokeratin (Pan-cytokeratin) antibody, this antibody identifiable design all people body epithelial tissue cells.The composition of the epidermis cell that keratoprotein dyeing display regeneration skin is all, comprises epidermal area, hair follicle and sebaceous gland, all show positive, and the epidermal tissue of Mus is negative.Then, we identify with anti-human vimentin antibodies (vimentin) hypodermal cell that human body is all.The dermal tissue of this dyeing display regeneration comprises mastoid process and the aobvious positive of subcutaneus adipose tissue of corium, but the dermal tissue of Mus is negative.Above result shows that the skin regenerated is tissue completely.
In addition, we find can to cultivate with hypoxia the expression that method that (2%-10%) or cell aggregation cultivate increases hypodermal cell SOX2 gene, and transplant the hair follicle formation efficiency that the high cell of SOX2 gene expression can promote and improve people.
Finally, for proving that the skin regenerated is functional skin further, we have 12 weeks after the transfer, a diameter and the degree of depth wound at 2mm is opened in the middle of the regeneration skin that nude mice is formed, simultaneously also open an onesize wound with it in contrast nude mice, whether the skin observing regeneration has ability to heal.We find that skin about the 9 days wounds regenerated heal completely, and agglutination is consistent with the skin of people, but longer than the skin healing time of Mus (just healing in Mus 5-7 days).Organizing of immunostaining display healing is also human skin tissue, illustrates and regenerates the healing ability that skin has self.This result shows that regeneration skin is functional human body skin further, may be used for the research of various disease as wound healing.
In a word, above entity result can find out separation and Culture with us and implantation technique, we can successfully regenerate the complete skin and the hair that have function of people with it nude mice, this skin and hair model have to be applied such as the research to application on human skin and hair diseases very widely, and wound and bald clinical prods are treated in screening and the exploitation of dermal drug.