CN107320781A

CN107320781A - Organization engineering skin containing living cells and preparation method thereof

Info

Publication number: CN107320781A
Application number: CN201710562163.2A
Authority: CN
Inventors: 刘少辉; 车七石
Original assignee: Guangzhou Rainhome Pharm and Tech Co Ltd
Current assignee: Guangzhou Rainhome Pharm and Tech Co Ltd
Priority date: 2017-07-11
Filing date: 2017-07-11
Publication date: 2017-11-07
Anticipated expiration: 2037-07-11
Also published as: CN107320781B

Abstract

The invention belongs to tissue engineering technique field, specifically related to a kind of organization engineering skin containing living cells and preparation method thereof, the organization engineering skin includes epidermis, emulation cortex and skin corium, described emulation cortex includes epithelial cells, fibroblast and biologic bracket material, and wherein epithelial cells and fibroblastic density ratio are 0.1 0.2:1；Described biologic bracket material is by gelatin, chitosan, glutamine peptide and hyaluronic acid in mass ratio 67:1.5‑2:0.5‑1:1 composition, artificial skin produced by the present invention has a good biocompatibility, good plasticity and mechanical strength, can preferably wound repairing.

Description

Organization engineering skin containing living cells and preparation method thereof

Technical field

The invention belongs to tissue engineering technique field, and in particular to a kind of organization engineering skin containing living cells and its system Preparation Method.

Background technology

Organizational engineering (tissueengineering) is one and is combined with cell biology, engineering science and materialogy, Tissue or organ substitute are built in vitro or in vivo, and one for repairing, maintaining or improving body form and function is emerging Cross discipline.Wherein organization engineering skin is to build artificial skin substitute using the principle of organizational project, is moved for skin Plant, can effectively solve clinically defect of skin reparation, for the problem that skin is not enough.

Skin tissue engineering is to develop earliest, most ripe field in organizational engineering, and the first ratifies listing through FDA Tissue engineering product is exactly organization engineering skin.Clinically the ulcer of skin ulcer including diabetes, veins intractable ulcer, Ulcer etc. caused by pyoderma gangraenosum ulcer and some other diseases, conventional treatment method tends not to obtain satisfaction Effect, and effect is notable after using artificial skin treatment.

At present, there are a variety of skin products to come out, and for clinical treatment, achieved certain curative effect, but deposit simultaneously In some defects, such as poor compatibility, rejection, cultivation cycle is long, and bubble is easily formed after transplanting, scar contracture easily occurs, Cause loss of moist etc. in skin, in order that organization engineering skin is more nearly human body skin, increasing research is entered to it Innovation of having gone is improved.

Notification number discloses a kind of organization engineering skin construction method for CN102462864B Chinese patent, and this method is adopted With human epidermal stem cell, dermal fibroblast as seed cell, with by the of the same race without thin of Human plactnta IV Collagen Type VIs modification Born of the same parents' dermal matrix is configured to a kind of active double-layer tissue engineering skin as support using gas-liquid interface isolated culture is compound Skin.The organization engineering skin that this method is built is closer to the structure of natural skin, and wherein dermal collagen supporting structure is complete, epidermis There is the cell of the different differentiation degree of multilayer in layer, the morphology requirement of organization engineering skin is reached, but the invention culture medium In add hyclone, easily occur rejection.

Publication No. CN104740689A Chinese patent application discloses a kind of composite tissue engineering skin containing living cells Skin and preparation method thereof.The composite tissue engineering skin both contains living cells, and degradable polymeric material protective layer is contained again, is made Obtain composite tissue engineering skin isolating power enhancing, cost reduction, extended shelf-life.Overcome in the prior art containing living thin The organization engineering skin of born of the same parents is with high costs, and the shelf-life is short, the defect of isolating power difference.But used in the invention it is degradable Polymeric material protective layer degradation time in human body is long, easily causes surface of a wound docile effect poor.

The content of the invention

In order to overcome the deficiencies in the prior art, the invention discloses a kind of organization engineering skin containing living cells and its preparation Method, obtained artificial skin has good biocompatibility, and good plasticity and mechanical strength can be repaired preferably The surface of a wound.

Concrete technical scheme of the present invention is as follows：

A kind of organization engineering skin containing living cells, including epidermis, emulation cortex and skin corium, described emulation cortex Including epithelial cells, fibroblast and biologic bracket material, wherein epithelial cells and fibroblastic density ratio are 0.1- 0.2:1；Described biologic bracket material is by gelatin, chitosan, glutamine peptide and hyaluronic acid 6-7 in mass ratio:1.5-2: 0.5-1:1 composition.

The present invention adds emulation cortex between epidermis and skin corium, epidermis is contacted with emulation cortex and more steps up Close, it is to avoid epidermis directly contacts the flaky shortcoming of appearance with skin corium, experiment proves that the angle protein contained in emulation cortex is thin Born of the same parents are unordered with fibroblastic growth, can be effectively increased the toughness of organization engineering skin, improve wound healing effect；And this Invent in the biologic bracket material used and add glutamine peptide, be conducive to it to decompose, and test discovery, add glutamine peptide The toughness and elasticity of skin can be improved, apply be conducive in biologic bracket material improve its mechanical strength, glutamine peptide with Human body has preferable compatibility, is conducive to organization engineering skin to have when using with the surface of a wound and carrys out good affinity, in addition, paddy acyl Amine peptide can also supply nutrients for follow-up cell growth.

Further, the preparation method of described emulation cortex is：Under the conditions of 4 DEG C, by biologic bracket material and DMEM Culture medium by volume 1:After 1 mixing, epithelical cell growth factor and Basic Fibroblast Growth Factor are added, fiber finer is inoculated with into Born of the same parents and the mixed liquor of epithelial cells, two kinds of cell inoculation gross densities are 2 × 10⁶/ ml, wherein epithelial cells with it is fibroblastic Density ratio is 0.1-0.2:1,37 DEG C, 5%CO₂Cultivated 4-5 days in incubator, be subsequently placed in culture dish and solidified, form imitative Skin corium；

The amount ratio of described epithelical cell growth factor, Basic Fibroblast Growth Factor and DMEM culture mediums is 2-3ng: 12-15ng:1ml。

Further, described biologic bracket material by gelatin, chitosan, glutamine peptide and hyaluronic acid in mass ratio 6.5:1.75:0.75:1 composition.

Further, the preparation method of described biologic bracket material is：Under the conditions of 50 DEG C, gelatin is configured to quality Fraction is 10% aqueous gelatin solution, and chitosan is dissolved in to be configured to mass fraction in the acetum that mass fraction is 2% be 2% The acetum of chitosan, two kinds of solution are mixed, and add glutamine peptide and hyaluronic acid, are stirred, and stand 24-36 small When, obtained mixed liquor is then prolonged into film forming for stream on 20 DEG C of horizontal polyvinyl chloride panel in surface temperature, is using pH finally 7.2-7.4 sodium hydroxide solutions soak 12 hours, dry, produce.

Further, present invention also offers the preparation method of the organization engineering skin containing living cells, step is：

(1) epithelial cells and fibroblast are obtained

(2) emulation cortex is prepared

Under the conditions of 4 DEG C, by biologic bracket material and DMEM culture mediums by volume 1:After 1 mixing, epidermal cell is added The mixed liquor of growth factor and Basic Fibroblast Growth Factor, inoculation fibroblast and epithelial cells, two kinds of cell inoculations are total Density is 2 × 10⁶/ ml, wherein epithelial cells are 0.1-0.2 with fibroblastic density ratio:1,37 DEG C, 5%CO₂In incubator Culture 4-5 days, is subsequently placed in culture dish and is solidified, and forms emulation cortex；Described epithelical cell growth factor, alkalescence into The amount ratio of fibroblast growth factor and DMEM culture mediums is 2-3ng:12-15ng:1ml；

(3) skin corium is prepared

Fibroblast is inoculated into the emulation cortical surface that step (2) is obtained, fibroblast culture medium, inoculation is added Density is 1 × 10⁶/ ml, 37 DEG C, 5%CO₂Cultivated 1-3 days in incubator, obtain corium-emulation cortex；

(4) epidermis is prepared

Epithelial cells is inoculated into the emulation cortical surface for corium-emulation cortex that step (3) is obtained, epidermal cell is added Culture medium, inoculum density is 1 × 10⁶/ ml, 37 DEG C, 5%CO₂Cultivated 6-8 days in incubator, obtain corium-emulation cortex-epidermis Layer；

(5) organization engineering skin is built

The corium that step (4) is obtained-emulation cortex-epidermis is laid in the bottom of culture dish, adds and contains 1 × 10^- ⁴In the DMEM culture mediums of mol/L calcium chloride, 37 DEG C, 5%CO₂Cultivate 6-8 days, produce in incubator.

Further, present invention also offers obtain epithelial cells in step (1) to be with fibroblastic method：

Transcription factor OCT4, SOX2, KLF4 and C-MYC are imported into human urine cell, then using cell density as 1 × 10⁶ It is seeded in multipotential stem cell inducing culture, 37 DEG C, 5%CO₂Cultivated in incubator, liquid is changed daily, continuous culture 25-30 days, Obtain induced multi-potent stem cell；By induced multi-potent stem cell using cell density as 1 × 10⁶It is seeded in mescenchymal stem cell induction training Support in base, 37 DEG C, 5%CO₂Cultivated in incubator, change liquid within every 2 days, continuous culture 14-16 days obtains mescenchymal stem cell；

The acquisition amplification of epithelial cells：By mescenchymal stem cell with density 1 × 10⁶It is seeded in epithelial cells inducing culture Middle differentiation obtains epithelial cells, when epithelial cells length is to 80%, removes epithelial cells inducing culture, is cultivated using DMEM/F12 Liquid is cleaned, plus to obtain angle protein unicellular by DPBS digestion 5min, the 400r/min centrifugations 5min of the EDTA containing 0.5mM, then by angle protein Unicellular to be inoculated in cultured epidermal cell base, inoculum density is 1 × 10⁶/ ml, 37 DEG C, 5%CO₂Cultivated in incubator, you can；

It is fibroblastic to obtain amplification：By mescenchymal stem cell with density 1 × 10⁶It is seeded in fibroblast induction training Support differentiation in base and obtain fibroblast, when fibroblast length is to 80%, removes fibroblast inducing culture, use DMEM/F12 nutrient solutions are cleaned, plus DPBS digestion 5min, the 400r/min centrifugations 5min of the EDTA containing 0.5mM obtains into fiber list Cell, then will be inoculated in fibroblast culture medium into fiber is unicellular, and inoculum density is 1 × 10⁶/ ml, 37 DEG C, 5% CO₂Cultivated in incubator, you can.

Further, described cultured epidermal cell base is：The culture medium based on DMEM/F12, additive and consumption For：The μ g/mL of bovine brain pituitary extract 50, hydrocortisone 5ng/mL, cholera toxin 1 × 10^-10Mol/L, insulin 0.05ng/ ML, adenine 1.8 × 10^-4Mol/L, penicillin 100IU/mL, the μ g/mL of transferrins 10, the μ g/mL of glutamic acid 5, epidermal cell life The long μ g/mL of the factor 10, monoethanolamine 1 × 10^-5mol/L；

Described fibroblast culture medium is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain is hung down The μ g/mL of body extract 50, insulin 5ng/mL, the μ g/ml of hydrocortisone 300, the μ g/mL of adenine 15, the μ g/ of vitamin C 120 ML, the μ g/mL of Basic Fibroblast Growth Factor 8, the μ g/mL of transferrins 10；

Described epithelial cells inducing culture is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain The μ g/mL of pituitary extract 50, penicillin 100IU/mL, the μ g/mL of streptomysin 100, epithelical cell growth factor 20ng/mL, pancreas islet 10 μ g/mL of element, the μ g/mL of vitamin A acid 8, the μ g/mL of calcium chloride 6；

Described fibroblast inducing culture is：The culture medium based on DMEM/F12, additive and consumption are：Ox The μ g/mL of pituitary extract 50, penicillin 100IU/mL, the μ g/mL of streptomysin 100, transforming growth factor β l15ng/mL, alkalescence FGF2 0ng/mL, the μ g/mL of pancreas islet plain sheet selenium transferrin 10,0.1 μm of ol/L of dexamethasone；

Described multipotential stem cell inducing culture is：The culture medium based on fibroblast culture medium, additive and Consumption is：L-GLUTAMINE 2mmol/L, nonessential amino acid 0.02mmol/mL；

Described mescenchymal stem cell inducing culture is：The culture medium based on DMEM/F12, additive and consumption are： The μ g/mL of bovine brain pituitary extract 50, l-GLUTAMINE 2mmol/L.

Described DMEM/F12 weight ratio is 2：1.

The epidermal cell culture base and corium culture medium used in the present invention is changed using the mixing of commercial DMEM and F12 culture mediums Enter culture medium, F12 culture mediums contain various trace elements, and DMEM medium nutrient contents concentration is high, be allowed to be blended in addition ox Pituitary extract, instead of traditional blood serum medium, reduces the content of antigen, reduces the infection of virus.

A kind of organization engineering skin structure containing living cells obtained by the present invention is corium-emulation cortex-epidermis, its The random growth layer containing fibroblast and epithelial cells, epidermis is cultivated in emulation cortex both sides respectively in middle emulation cortex With skin corium, the existing layer structure of organization engineering skin for obtaining culture is difficult layering and peeled off, improves organization engineering skin again Mechanical strength, and bovine brain pituitary extract present in fibroblast after transplanting in skin corium and culture medium can promote Enter wound healing, the various growth factors of secretion can accelerate surface of a wound cell and fibroblastic intergrowth, with good Compatibility, wound healing time is short, in addition, the biologic bracket material that the present invention is used can not only protect the surface of a wound not by the external world Pollution, and with good biodegradability.

Compared with prior art：Instant invention overcomes the skin corium of individual layer living cell tissue engineering skin in the prior art appearance Easily by extraneous bacterium infection, and the shortcoming of water holding capacity difference, it is with high costs also to compensate for double-deck living cell tissue's engineering skin, Poor compatibility, biologic bracket material is difficult the shortcoming absorbed, and makes skin mechanical performance the invention belongs to three layers of skin texture It is stronger.

Embodiment

The present invention is further detailed below by embodiment.The composition that the present invention is used belongs to conventional production Product, wherein, chitosan：CAS.148411-57-8, is purchased from big magnificent great achievement medication chemistry (group) Co., Ltd in Wuhan；DMEM、 F12：Upper Asteriidae bio tech ltd；Bovine brain pituitary extract：BPE brands：ScienCell, article No. 0713；Pancreas islet Element：CAS：11061-68-0, Hubei Xin Kang medication chemistries Co., Ltd.

The kinds of culture medium of embodiment 1 and composition

A kind of cultured epidermal cell base is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain hypophysis is carried Take the μ g/mL of thing 50, hydrocortisone 5ng/mL, cholera toxin 1 × 10^-10Mol/L, insulin 0.05ng/mL, adenine 1.8 × 10^-4Mol/L, penicillin 100IU/mL, the μ g/mL of transferrins 10, the μ g/mL of glutamic acid 5, the μ g/mL of epithelical cell growth factor 10, Monoethanolamine 1 × 10^-5mol/L；

A kind of fibroblast culture medium is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain hypophysis The μ g/mL of extract 50, insulin 5ng/mL, the μ g/ml of hydrocortisone 300, the μ g/mL of adenine 15, the μ g/mL of vitamin C 120, The μ g/mL of Basic Fibroblast Growth Factor 8, the μ g/mL of transferrins 10；

A kind of epithelial cells inducing culture is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain is hung down The μ g/mL of body extract 50, penicillin 100IU/mL, the μ g/mL of streptomysin 100, epithelical cell growth factor 20ng/mL, insulin 10 μ g/mL, the μ g/mL of vitamin A acid 8, the μ g/mL of calcium chloride 6；

A kind of fibroblast inducing culture is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain The μ g/mL of pituitary extract 50, penicillin 100IU/mL, the μ g/mL of streptomysin 100, transforming growth factor β l15ng/mL, alkalescence into Fibroblast growth factor 20ng/mL, the μ g/mL of pancreas islet plain sheet selenium transferrin 10,0.1 μm of ol/L of dexamethasone；

A kind of multipotential stem cell inducing culture is：The culture medium based on fibroblast culture medium, additive and use Measure and be：L-GLUTAMINE 2mmol/L, nonessential amino acid 0.02mmol/mL；

A kind of mescenchymal stem cell inducing culture is：The culture medium based on DMEM/F12, additive and consumption are：Ox The μ g/mL of pituitary extract 50, l-GLUTAMINE 2mmol/L；

Described DMEM/F12 weight ratio is 2：1.

A kind of organization engineering skin containing living cells of embodiment 2

The described organization engineering skin containing living cells includes epidermis, emulation cortex and skin corium, described artificial leather Layer includes epithelial cells, fibroblast and biologic bracket material, and wherein epithelial cells is with fibroblastic density ratio 0.15:1；Described biologic bracket material is by gelatin, chitosan, glutamine peptide and hyaluronic acid in mass ratio 6.5:1.75: 0.75:1 constitutes, and its preparation method is：Under the conditions of 50 DEG C, gelatin is configured to mass fraction for 10% aqueous gelatin solution, will Chitosan is dissolved in the acetum for being configured to that mass fraction is 2% chitosan in the acetum that mass fraction is 2%, by two kinds Solution is mixed, and adds glutamine peptide and hyaluronic acid, is stirred, and 30 hours is stood, then by obtained mixed liquor in table Face temperature prolongs film forming for 20 DEG C of horizontal polyvinyl chloride panel upstream, the use of pH is finally that 7.3 sodium hydroxide solutions soak 12 hours, Dry, produce；

The preparation method of the described organization engineering skin containing living cells,

(1) epithelial cells and fibroblast are obtained

Concretely comprise the following steps：Transcription factor OCT4, SOX2, KLF4 and C-MYC are imported into human urine cell, it is then close with cell Spend for 1 × 10⁶It is seeded in multipotential stem cell inducing culture, 37 DEG C, 5%CO₂Cultivated in incubator, liquid is changed daily, continuous culture 27 days, obtain induced multi-potent stem cell；By induced multi-potent stem cell using cell density as 1 × 10⁶It is seeded in mescenchymal stem cell In inducing culture, 37 DEG C, 5%CO₂Cultivated in incubator, change liquid within every 2 days, continuous culture 15 days obtains mescenchymal stem cell；

(2) emulation cortex is prepared

Under the conditions of 4 DEG C, by biologic bracket material and DMEM culture mediums by volume 1:After 1 mixing, epidermal cell is added The mixed liquor of growth factor and Basic Fibroblast Growth Factor, inoculation fibroblast and epithelial cells, two kinds of cell inoculations are total Density is 2 × 10⁶/ ml, wherein epithelial cells are 0.15 with fibroblastic density ratio:1,37 DEG C, 5%CO₂Cultivated in incubator 4.5 days, it is subsequently placed in culture dish and is solidified, forms emulation cortex；Described epithelical cell growth factor, basic fibroblast The amount ratio of growth factor and DMEM culture mediums is 2.5ng:13.5ng:1ml；

(3) skin corium is prepared

Fibroblast is inoculated into the emulation cortical surface that step (2) is obtained, fibroblast culture medium, inoculation is added Density is 1 × 10⁶/ ml, 37 DEG C, 5%CO₂Cultivated 2 days in incubator, obtain corium-emulation cortex；(4) epidermis is prepared

Epithelial cells is inoculated into the emulation cortical surface for corium-emulation cortex that step (3) is obtained, epidermal cell is added Culture medium, inoculum density is 1 × 10⁶/ ml, 37 DEG C, 5%CO₂Cultivated 7 days in incubator, obtain corium-emulation cortex-epidermis；

(5) organization engineering skin is built

The corium that step (4) is obtained-emulation cortex-epidermis is laid in the bottom of culture dish, adds and contains 1 × 10^- ⁴In the DMEM culture mediums of mol/L calcium chloride, 37 DEG C, 5%CO₂Cultivate 7 days, produce in incubator.

The culture medium that embodiment 2 is related to uses the formula that embodiment 1 is provided

A kind of organization engineering skin containing living cells of embodiment 3

Difference with embodiment 2 is that described biologic bracket material is by gelatin, chitosan, glutamine peptide and hyaluronic acid In mass ratio 6:2:1:1 composition, other be the same as Examples 2.

A kind of organization engineering skin containing living cells of embodiment 4

Difference with embodiment 2 is that described biologic bracket material is by gelatin, chitosan, glutamine peptide and hyaluronic acid In mass ratio 7:1.5:0.5:1 composition, other be the same as Examples 2.

A kind of organization engineering skin containing living cells of comparative example 1

Difference with embodiment 2 is, glutamine peptide is replaced using fibrin, described biologic bracket material by gelatin, Chitosan, fibrin and hyaluronic acid in mass ratio 6.5:1.75:0.75:1 composition, other be the same as Examples 2.

A kind of organization engineering skin containing living cells of comparative example 2

Difference with embodiment 2 is, without the step of emulating cortex is prepared in preparation method, to use biologic bracket material Instead of emulating cortex, obtained organization engineering skin is corium-biologic bracket material-epidermis, other be the same as Examples 2.

The assay of the living cells of test example 1

Using mtt assay to the living cells content in 2-4 of the embodiment of the present invention and organization engineering skin made from comparative example 1-2 It is measured, obtained organization engineering skin is preserved after 15 days and 40 days respectively, living cells content is determined respectively.

Test method：By 1mm²The skin of area is cut into small pieces, and is put into nutrient solution, and addition MTT (concentration 5mg/ml, often 10⁶Cell 200ul) be incubated 4 hours under the conditions of 37 DEG C, discard nutrient solution, the skin after the completion of incubation shredded, add DMSO in Lower 100 revs/min of normal temperature shakes 20 minutes, the supernatant extraction after concussion is put its OD value is determined under ELIASA；

Same method determines the OD values with contained living cells in homalographic；

Living cells content=skin OD values/living cells OD value × 100%, it is specific as shown in table 1.

The activity of living cells result of table 1

	Embodiment 2	Embodiment 3	Embodiment 4	Comparative example 1	Comparative example 2
						After 20 days	85%	83%	82%	72%	68
After 40 days	61%	58%	56%	45%	43%

From table 1 it follows that organization engineering skin made from embodiment 2-4 is after preserving 20 days, living cells content is protected Hold more than 82%, after preserving 40 days, living cells content is maintained at more than 56%, and organizational project skin made from comparative example 1-2 The content that skin preserves living cells after same time is greatly reduced, and illustrates the organization engineering skin storage life of the invention provided more Long, glutamine peptide influences the cell of organization engineering skin in the preparation of further clear artificial leather layer and biologic bracket material Activity.

The animal implant tests textured of test example 2

Test material：Male Balb/c-nu mouse (nude mice, the western pul-Bi Kai experimental animals Co., Ltd in Shanghai provides), 20 ± 1 grams, 30 of body weight.

Test method：Pretreatment：Nude mice shaves skin of back hair, in mouse after chloraldurate intraperitoneal injection of anesthesia In the median line of back, the circular skin holostrome of an a diameter of 6mm is removed with operating scissors, lesion thickness is uniform；

(1) transplant：2-4 of the embodiment of the present invention and organization engineering skin made from comparative example 1-2 are transplanted in the surface of a wound, set Positive controls：Normal healing, is fixed organization engineering skin, Sterilized application covers after transplanting using intradermal suturing skill Lid；

(2) post surgery treatment：All mouse carry out single cage nursing under identical condition, and surface of a wound situation is observed after transplanting.Tool Body result is as shown in table 2：

The healing time of table 2

	Embodiment 2	Embodiment 3	Embodiment 4	Comparative example 1	Comparative example 2	Control group
							Heal area (1 week)	69%	65%	67%	49%	46%	32%
Heal area (2 weeks)	100%	99%	99%	91%	90%	80%
							Complete healing time/day	14	14.5	15	18	19	23

From Table 2, it can be seen that embodiment 1-3 compares comparative example 1-2 and control group, healing rate faster, and passes through Transplant experiment observation is found：Control group：Surface of a wound crust is relatively thin, and color is dark red, and healing rate is slow, easily occurs breakage, is cured completely After conjunction, crust is to lower recess, and skin is tight, and gently touching neoplastic skin does not have any elasticity.

Comparative example 1-2：The surface of a wound and organization engineering skin stick that effect is good, and blister is produced, and crust is slightly elastic, gently touches Touch neoplastic skin slightly more tight than normal skin.

Embodiment 2-4：The surface of a wound and organization engineering skin stick that effect is good, and blister is produced, and surface of a wound skin becomes flexible, Gently touch neoplastic skin tight, it is identical with normal skin sense of touch.

Transplant after 4 weeks, sections observation discovery is carried out to surface of a wound skin, work is organized using made from 2-4 of the embodiment of the present invention Epidermis and dermis physically well develop after journey dermatoplasty, and form uses comparative example 1 is obtained to organize close to normal skin Healing rate is slow after engineering skin transplanting, and skin holostrome structure is relatively thin relative to normal skin, is organized using comparative example 2 is obtained Skin holostrome structure is thicker relative to normal skin after engineering skin transplanting, illustrates that organizational project can be caused by lacking emulation cortex Skin Cell hyperplasia.

The elasticity of test example 3 and toughness test

Subjects：The surface of a wound skin obtained after being transplanted 4 weeks using the specimen method described in test example 2；

Specimen method：The square surface of a wound skins of 5 × 5mm are taken, skin both sides is forced in, calculates the elasticity and toughness of skin, Toughness：The power that skin applies when broken；Elasticity：Deformation (length change) size when skin is broken；Concrete outcome is as shown in table 3.

The skin elasticity of table 3 and toughness data result

From table 3 it is observed that the organization engineering skin that embodiment 2-4 is prepared has bigger elasticity and toughness, And comparative example 1-2 toughness and elasticity are slightly worse, control group is normal healing, and its internal hetero-organization or angiogenic growth speed are slow, and And because healing time is long, atrophy directly occurs for the surface of a wound, cause surface of a wound skin degradation.

The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.It is any ripe Know the personage of this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as Into all equivalent modifications or change, should by the present invention claim be covered.

Claims

1. a kind of organization engineering skin containing living cells, it is characterised in that described including epidermis, emulation cortex and skin corium Emulation cortex include epithelial cells, fibroblast and biologic bracket material, wherein epithelial cells with it is fibroblastic close Degree is than being 0.1-0.2:1；Described biologic bracket material is by gelatin, chitosan, glutamine peptide and hyaluronic acid 6- in mass ratio 7:1.5-2:0.5-1:1 composition.

2. the organization engineering skin as claimed in claim 1 containing living cells, it is characterised in that the preparation of described emulation cortex Method is：Under the conditions of 4 DEG C, by biologic bracket material and DMEM culture mediums by volume 1:After 1 mixing, epidermal cell life is added The mixed liquor of the long factor and Basic Fibroblast Growth Factor, inoculation fibroblast and epithelial cells, two kinds of cell inoculations are total close Spend for 2 × 10⁶/ ml, wherein epithelial cells are 0.1-0.2 with fibroblastic density ratio:1,37 DEG C, 5%CO₂Trained in incubator Support 4-5 days, be subsequently placed in culture dish and solidified, form emulation cortex；Described epithelical cell growth factor, alkalescence are into fibre The amount ratio for tieing up growth factor and DMEM culture mediums is 2-3ng:12-15ng:1ml.

3. the organization engineering skin containing living cells as claimed in claim 1, it is characterised in that described biologic bracket material by Gelatin, chitosan, glutamine peptide and hyaluronic acid in mass ratio 6.5:1.75:0.75:1 composition.

4. the organization engineering skin containing living cells as described in claim 1 or 3, it is characterised in that described biological support material The preparation method of material is：Under the conditions of 50 DEG C, gelatin is configured to mass fraction for 10% aqueous gelatin solution, chitosan is dissolved in The acetum that mass fraction is 2% chitosan is configured in the acetum that mass fraction is 2%, two kinds of solution are mixed, And glutamine peptide and hyaluronic acid are added, stir, 24-36 hours are stood, then by obtained mixed liquor in surface temperature Prolong film forming for 20 DEG C of horizontal polyvinyl chloride panel upstream, the use of pH is finally that 7.2-7.4 sodium hydroxide solutions soak 12 hours, dries in the air It is dry, produce.

5. the preparation method of the organization engineering skin containing living cells as described in claim any one of 1-4, it is characterised in that step Suddenly it is：

(1) epithelial cells and fibroblast are obtained

(2) emulation cortex is prepared

Under the conditions of 4 DEG C, by biologic bracket material and DMEM culture mediums by volume 1:After 1 mixing, epidermal growth is added The mixed liquor of the factor and Basic Fibroblast Growth Factor, inoculation fibroblast and epithelial cells, two kinds of cells are inoculated with gross density For 2 × 10⁶/ ml, wherein epithelial cells are 0.1-0.2 with fibroblastic density ratio:1,37 DEG C, 5%CO₂Cultivated in incubator 4-5 days, it is subsequently placed in culture dish and is solidified, forms emulation cortex；Described epithelical cell growth factor, basic fibroblast The amount ratio of growth factor and DMEM culture mediums is 2-3ng:12-15ng:1ml；

(3) skin corium is prepared

Fibroblast is inoculated into the emulation cortical surface that step (2) is obtained, fibroblast culture medium, inoculum density is added For 1 × 10⁶/ ml, 37 DEG C, 5%CO₂Cultivated 1-3 days in incubator, obtain corium-emulation cortex；

(4) epidermis is prepared

Epithelial cells is inoculated into the emulation cortical surface for corium-emulation cortex that step (3) is obtained, cultured epidermal cell is added Base, inoculum density is 1 × 10⁶/ ml, 37 DEG C, 5%CO₂Cultivated 6-8 days in incubator, obtain corium-emulation cortex-epidermis；

(5) organization engineering skin is built

The corium that step (4) is obtained-emulation cortex-epidermis is laid in the bottom of culture dish, adds and contains 1 × 10^-4Mol/L chlorine In the DMEM culture mediums for changing calcium, 37 DEG C, 5%CO₂Cultivate 6-8 days, produce in incubator.

6. the preparation method of the organization engineering skin as claimed in claim 5 containing living cells, it is characterised in that described step (1) epithelial cells is obtained in is with fibroblastic method：

Transcription factor OCT4, SOX2, KLF4 and C-MYC are imported into human urine cell, then using cell density as 1 × 10⁶It is seeded in In multipotential stem cell inducing culture, 37 DEG C, 5%CO₂Cultivated in incubator, liquid is changed daily, continuous culture 25-30 days is lured Lead multipotential stem cell；By induced multi-potent stem cell using cell density as 1 × 10⁶It is seeded in mescenchymal stem cell inducing culture In, 37 DEG C, 5%CO₂Cultivated in incubator, change liquid within every 2 days, continuous culture 14-16 days obtains mescenchymal stem cell；

The acquisition amplification of epithelial cells：By mescenchymal stem cell with density 1 × 10⁶It is seeded in epithelial cells inducing culture and divides Change obtains epithelial cells, when epithelial cells length is to 80%, removes epithelial cells inducing culture, clear using DMEM/F12 nutrient solutions Wash, plus to obtain angle protein unicellular by DPBS digestion 5min, the 400r/min centrifugations 5min of the EDTA containing 0.5mM, it is then that angle protein is slender Born of the same parents are inoculated in cultured epidermal cell base, and inoculum density is 1 × 10⁶/ ml, 37 DEG C, 5%CO₂Cultivated in incubator, you can；

It is fibroblastic to obtain amplification：By mescenchymal stem cell with density 1 × 10⁶It is seeded in fibroblast inducing culture Middle differentiation obtains fibroblast, when fibroblast length is to 80%, removes fibroblast inducing culture, uses DMEM/ F12 nutrient solutions are cleaned, plus to obtain into fiber unicellular by DPBS digestion 5min, the 400r/min centrifugations 5min of the EDTA containing 0.5mM, Then it will be inoculated in into fiber is unicellular in fibroblast culture medium, inoculum density is 1 × 10⁶/ ml, 37 DEG C, 5%CO₂Incubator Middle culture, you can.

7. the preparation method of the organization engineering skin containing living cells as described in claim 5 or 6, it is characterised in that

Described cultured epidermal cell base is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain hypophysis is extracted The μ g/mL of thing 50, hydrocortisone 5ng/mL, cholera toxin 1 × 10^-10Mol/L, insulin 0.05ng/mL, adenine 1.8 × 10^-4Mol/L, penicillin 100IU/mL, the μ g/mL of transferrins 10, the μ g/mL of glutamic acid 5, the μ g/mL of epithelical cell growth factor 10, Monoethanolamine 1 × 10^-5mol/L；

Described fibroblast culture medium is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain hypophysis is carried Take the μ g/mL of thing 50, insulin 5ng/mL, the μ g/ml of hydrocortisone 300, the μ g/mL of adenine 15, the μ g/mL of vitamin C 120, alkali Property fibroblast growth factor 8 μ g/mL, the μ g/mL of transferrins 10；

Described epithelial cells inducing culture is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain hypophysis The μ g/mL of extract 50, penicillin 100IU/mL, the μ g/mL of streptomysin 100, epithelical cell growth factor 20ng/mL, the μ of insulin 10 G/mL, the μ g/mL of vitamin A acid 8, the μ g/mL of calcium chloride 6；

Described fibroblast inducing culture is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain is hung down The μ g/mL of body extract 50, penicillin 100IU/mL, the μ g/mL of streptomysin 100, transforming growth factor β l15ng/mL, alkalescence are into fibre Tie up Porcine HGF 20ng/mL, the μ g/mL of pancreas islet plain sheet selenium transferrin 10,0.1 μm of ol/L of dexamethasone；

Described multipotential stem cell inducing culture is：The culture medium based on fibroblast culture medium, additive and consumption For：L-GLUTAMINE 2mmol/L, nonessential amino acid 0.02mmol/mL；

Described mescenchymal stem cell inducing culture is：The culture medium based on DMEM/F12, additive and consumption are：Bovine brain The μ g/mL of pituitary extract 50, l-GLUTAMINE 2mmol/L；

Described DMEM/F12 weight ratio is 2：1.