CN109321514B

CN109321514B - Humanization skin and preparation method thereof

Info

Publication number: CN109321514B
Application number: CN201811236546.1A
Authority: CN
Inventors: 孙理华; 张勇
Original assignee: Wuhan Aoxiang Biotechnology Co Ltd
Current assignee: WUHAN AOXIANG BIOTECHNOLOGY Co.,Ltd.
Priority date: 2018-06-11
Filing date: 2018-10-23
Publication date: 2019-06-21
Anticipated expiration: 2038-10-23
Also published as: CN109045357A; CN109321514A; CN109045357B

Abstract

The present invention provides a kind of humanization skins and preparation method thereof.The preparation method includes the following steps: that antigen animal cortex will be gone at least to be divided into 3 layers of cortex from deep layer to shallow-layer, successively plants the first groups of cells, the second groups of cells and third groups of cells from deep layer to shallow-layer on the cortex, cultivate to obtain the humanization skin；First groups of cells includes vascular endothelial cell and fibroblast, and second groups of cells includes perpendicular myocyte and keratinocyte, the third groups of cells include short columnar epithelial cell and melanocyte.Humanization skin of the invention solves the regeneration issues of dermatoplastic histocompatbility and skin, is used directly in patient and the beauty of fire victim and large-area burns sequelae, can realize the healing of the surface of a wound especially III degree burn wound quickly.Preparation method in the present invention makes its application on human skin that large-scale production may be implemented, to cope with the case for being badly in need of large area skin transplanting.

Description

Humanization skin and preparation method thereof

Technical field

The present invention relates to field of tissue engineering technology, more particularly, to a kind of humanization skin and preparation method thereof.

Background technique

Currently, the treatment to the skin burn wounded, the especially treatment to the extensive deep burn wounded, the best surface of a wound Covering is autologous skin.However, wounded's auto-skin grafting can not only generate the new surface of a wound of skin donor site, but also since corium damages Wound can also cause the formation of skin donor site stasis of blood trace and pigmentation, it is often more important that the large-area burns wounded itself can be used skin deficient. Therefore, it is badly in need of seeking a kind of ideal human skin.

In the prior art, usually using acellular dermal matrix matrix, Integra artificial skin etc..Wherein, acellular dermal matrix base Matter is needed after being applied to the surface of a wound with other dressing covering protections, expensive；Although and the Integra artificial skin through being commercialized The advantages of can using immediately, can voluntarily being decomposed after transplanting and induce host's epidermal growth, but the artificial skin is transplanting Easy infection afterwards, somewhat expensive.In order to overcome drawbacks described above, in existing research, also has and be made after handling Animal Skin manually Dermal scaffold, has document to provide a kind of preparation method of low-immunogenicity pig dermal support, and this method passes through series of steps The skin of domestic pig is handled, subcutaneous tissue and coupled dermal partial lamina reticularis are removed, retains 0.5~0.6mm thickness corium net Then shape layer removes de- cell and the high immunogenic components such as laminin, IV collagen type of degrading.This method is prepared Pigskin be used as growth of the defective tissue in repair process to depend on bracket when in use.

Existing method be only refer only to the reparation of defective tissue, but not targetedly, be not real yet Source of people skin substitutes, such as when being directed to III degree burn wound do not provide one and relatively effectively and are able to achieve and quickly control More skin graft, using the dermal scaffold of general defect of skin, healing time is longer, and repellency is larger.

Summary of the invention

The first object of the present invention is to provide a kind of preparation method of humanization skin, and this method comprises the following steps:

Antigen animal cortex will be gone at least to be divided into 3 layers of cortex from deep layer to shallow-layer, from deep layer to shallow-layer on the cortex The first groups of cells, the second groups of cells and third groups of cells are successively planted, is cultivated to obtain the humanization skin；

First groups of cells includes vascular endothelial cell and fibroblast, and second groups of cells includes perpendicular myocyte And keratinocyte, the third groups of cells include short columnar epithelial cell and melanocyte.

The humanization skin obtained using preparation method of the invention has well solved dermatoplastic histocompatbility And the regeneration issues of skin, it is used directly in patient and the beauty of fire victim and large-area burns sequelae, it can be with The healing of the surface of a wound especially III degree burn wound is realized quickly.

Wherein, in the present invention it is possible to cortex be divided into 4 layers, 5 layers, 6 layers, 7 layers even 8 layers etc. as needed, only need to protect Demonstrate,prove the plantation depth sequence between the first groups of cells, the second groups of cells and third groups of cells.These, it is preferred to first Groups of cells is vascular endothelial cell and fibroblast, and the second groups of cells is perpendicular myocyte and keratinocyte, third cell Group is short columnar epithelial cell and melanocyte.

In the present invention, it is preferred to be 6 layers, i.e., in a preferred embodiment of the invention, above-mentioned preparation method be can wrap Include following steps: antigen animal cortex will be gone to be divided into 6 layers of cortex from deep layer to shallow-layer, on the cortex from deep layer to shallow-layer according to Secondary plantation vascular endothelial cell, fibroblast, perpendicular myocyte, keratinocyte, short columnar epithelial cell and melanin are thin Born of the same parents cultivate to obtain the humanization skin.

In a preferred embodiment of the invention, the source for removing antigen germfree animal cortex be pig, ox, sheep or Rabbit, preferably pig.

Wherein, when for 3 layers of cortex, every layer of cortex with a thickness of 0.02~0.6mm, preferably 0.16~ 0.24mm.In a preferred embodiment of the invention, when for 6 layers of cortex, every layer of cortex with a thickness of 0.01~ 0.24mm, preferably 0.08~0.11mm.

In a preferred embodiment of the invention, the condition of the culture are as follows: 37 DEG C, saturated humidity, 5%CO₂Culture It is cultivated in case.It changes 2/3 culture solution within every 3-5 days, until cell 90% contacts, collects cell with pancreas enzyme -EDTA solution digestion, according to The corresponding culture medium diluting cells of 4~5 times of culture solutions.

In a preferred embodiment of the invention, sterilization steps are also typically included, sterilization steps are placed in repopulating cell Before.The specific steps of sterilizing are preferred are as follows: obtained cortex is successively handled 10~60min, sterile washing by hypochlorous acid respectively Agent, new gill go out 10~60min of processing, handle again through physiological saline after sterile water washing.Wherein, more preferably, it will obtain Cortex successively handle 30~40min, sterile rinsing agent by hypochlorous acid respectively, new gill goes out 30~40min of processing, sterile water It is handled again through physiological saline after washing.More preferably, obtained cortex is successively passed through into 0.7~0.9ppm hypochlorous acid disinfection 30 Minute, sterile rinsing agent 3 times, 0.2% new gill, which goes out, to be sterilized 30 minutes, and sterile washing 2 times, medical grade physiological saline is handled 2 times, Obtain antigen germfree animal cortex.

In a preferred embodiment of the invention, the culture medium of the plantation vascular endothelial cell is to contain VEGF's DMEM culture medium, the fibroblastic culture medium of the plantation are the DMEM culture medium containing FGF, described kind of tree planter myocyte's Culture medium is the DMEM culture medium containing MGF, and the culture medium of the plantation keratinocyte is the DMEM culture containing KGF The culture medium of base, the short columnar epithelial cell of plantation is the DMEM culture medium containing EGF, the training of the plantation melanocyte Support the DMEM culture medium that base is MGS.

The spent antigen defatted animal micromicro of the present invention is with oneself processing or commercial goods, and all cell provenances are all from Human body or stem cell, including Chinese Fetal Foreskin Fibroblasts ring cutting relic, donor, aborted fetus, the stem cell of separation and class stem cell.

In a preferred embodiments of the invention, seed cell preparation includes the following steps:

1) provenance comes from human body or stem cell, including Wrapping annulus cuts operation relic, donor, aborted fetus, separation it is dry Cell and class stem cell；

2) tissue in step 1) source is sufficiently cleaned through sterile saline, to be impregnated 5 minutes in 75% medicinal alcohol, Physiology salt wash 1 time after, sterilized 8 minutes in 0.2% new gill goes out, then washed 2 times with PBS solution, with enzyme (neutral proteinase, Trypsase etc.) or mechanical system separation different levels cell, be collected in different test tubes respectively, and indicate level；

3) by step 2 or 1), cell or stem cell are according to different cell types, and with DMEM+X, (X is the cell of different levels With the different factors, reagent) culture medium, at 37 DEG C, saturated humidity, 5%CO₂It is cultivated in incubator, changes 2/3 training within each 3-5 days Nutrient solution collects cell with pancreas enzyme -EDTA solution digestion until cell 90% is contacted, and is diluted according to 4~5 times with corresponding culture medium Cell.

In a preferred embodiment of the invention, the specific steps of the culture are as follows:

The cortex of repopulating cell is successively superimposed from deep layer to shallow-layer, adds DMEM culture medium to closing on top layer, culture 5~ 7d.Wherein, shallow-layer is top layer, i.e., the cortex containing melanocyte is top layer.

In a preferred embodiment of the invention, the preparation method of humanization skin specifically comprises the following steps:

Antigen animal cortex will be gone to be divided into 6 layers of cortex from from deep layer to shallow-layer, be successively labeled as cortex a, b, c, d, e, f, After sterilizing, vascular endothelial cell is planted on the cortex a, fibroblast is planted on cortex b, and cortex c Shang kind tree planter flesh is thin Born of the same parents plant keratinocyte on cortex d, short columnar epithelial cell are planted on cortex e, plants melanocyte on cortex f, when After cell realizes 50~70% contact on cortex, above-mentioned cortex is successively superimposed from deep layer to shallow-layer, upper layer is outer layer f, Add DMEM culture medium to top layer is closed on, cultivates 5~7d to obtain the humanization skin.

Wherein, superposition refers specifically to stack the cortex of repopulating cell according to a → b → c → d → e → f, and wherein top layer is f Layer, lowest level are a layers.

Wherein, it is described go antigen animal cortex be grease removal, removal of impurities, except DNA, except after RNA and de- antigen go antigen move Object cortex.Method commonly used in the art can be used to come grease removal, removal of impurities, remove DNA, except RNA and de- antigen.Wherein, more excellent Selection of land is by lauryl sodium sulfate, sodium hydroxide, peregal, bleeding agent, ammonium sulfate, neutral proteinase, lipase, DNA A series of processing such as enzyme, RNA enzyme, impulse electric field, deionized water with grease removal, removal of impurities, remove DNA, except RNA, de- antigen.It is preparing In process, Animal Skin must not be with the poisonous and harmful drug contact such as any heavy metal ion, dyestuff, benzene class.

Another object of the present invention is to provide the humanization skins obtained by above-mentioned preparation method.

A further object of the present invention is to provide above-mentioned preparation method or the humanization skins as made from above-mentioned preparation method The application in burn wound graft or beauty treatment skin is being prepared, especially in the graft for the treatment of III degree burn wound Using.

The present invention is layered according to structural order and plants by that will remove the sterile layering animal of antigen (pig, ox, sheep, rabbit etc.) skin Different types of cortex forms cell, and using compound criteria, the source of people skin for the bioactivity being prepared solves skin The histocompatbility of transplanting and the regeneration issues of skin, can make Wound healing quickly, be used directly for fire victim and In the patient of large-area burns sequelae and beauty, it can especially meet the needs of III degree burn wound patient well.This hair Preparation method in bright makes its application on human skin that large-scale production may be implemented, to cope with the case for being badly in need of large area skin transplanting.

Specific embodiment

With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for Illustrate the present invention, but is not intended to limit the scope of the invention.

Unless otherwise specified, the conventional means that technological means used is well known to those skilled in the art in embodiment, institute It is commercial goods with raw material.

Embodiment 1

Present embodiments provide a kind of humanization skin, it is specific the preparation method is as follows:

1, sterile to remove antigen degreasing pigskin layer

1) pigskin passes through lauryl sodium sulfate, sodium hydroxide, peregal, bleeding agent, ammonium sulfate, neutral proteinase, rouge A series of processing such as fat enzyme, DNA enzymatic, RNA enzyme, impulse electric field, deionized water, with grease removal, removal of impurities, except DNA, except RNA, de- anti- It is former；

2) step 1) is obtained into skin plane layer and is divided into 6 layers, every layer with a thickness of 0.1mm, from deep layer to shallow-layer number be respectively a, b,c,d,e,f；

3) cortex for obtaining step 2) successively sterilizes 30 minutes by 0.7-0.9ppm hypochlorous acid respectively, sterile rinsing agent 3 times, 0.2% new gill, which goes out, to be sterilized 30 minutes, and sterile washing 2 times, medical grade physiological saline is handled 2 times, and it is sterile to obtain antigen Animal cortex.

2, prepared by seed cell

1) provenance is from Chinese Fetal Foreskin Fibroblasts ring cutting operation relic；

2) tissue of step 1) is sufficiently cleaned through sterile saline, to be impregnated 5 minutes in 75% medicinal alcohol, physiology After salt is washed 1 time, sterilized 8 minutes in 0.2% new gill goes out, then washed 2 times with PBS solution, with enzyme (neutral proteinase, pancreas egg White enzyme etc.) or mechanical system separation different levels cell, be collected in different test tubes respectively, it is thin to respectively obtain blood vessel endothelium Born of the same parents, fibroblast, perpendicular myocyte, keratinocyte, short columnar epithelial cell, melanocyte, successively mark A, B, C, D, E,F；

3) by step 2) cell according to different type, using different culture medium DMEM+X, (X is used in different levels cell The factor, reagent) cultivated.The X for cultivating vascular endothelial cell is VEGF, the X of keratinocyte is KGF, is trained fibre The X of dimension cell is FGF, the X of the perpendicular myocyte of culture is MGF, the X of the short columnar epithelial cell of culture is EGF and culture melanin The X of cell is MGS.Condition of culture are as follows: 37 DEG C, saturated humidity, 5%CO₂It is cultivated in incubator, changes 2/3 culture solution within every 3 days, directly It is contacted to cell 70%, collects cell with pancreas enzyme -EDTA solution digestion, it is thin with corresponding culture medium dilution according to 4~5 times of culture solutions Born of the same parents.

3, humanization skin preparation method

1) go antigen germfree animal cortex unfolding in culture dish individual, according to 0.05-0.lml/cm²Add culture medium, Preculture 3 hours or more, suck culture medium, add corresponding level cell (a layers of corresponding A cell marking are a-A, and so on B-B, c-C, d-D, e-E, f-F) and culture medium, it cultivates and obtains cortex-cell；

2) cortex-cell for cultivating step 1), changes primary culture for 3-5 days, and cell reaches 50-70% contact, removes skin Layer-cell complexes are successively put in a new culture dish by a-A, b-B, c-C, d-D, e-E, f-F laminated structure, the most bottom a-A Layer adds DMEM culture medium to close to the top layer, cultivates 5-7 days, obtain composite layer i.e. source of people skin；

3) step 2) composite layer (source of people skin) is taken out, according to the size of patient wound, shape, cuts symbol composite layer, Source of people skin at this time can be used to transplant.

Embodiment 2

A kind of humanization skin is present embodiments provided, specific preparation method is same as Example 1, it is different only in that, In the sterile step 2) for removing antigen degreasing pigskin layer every layer with a thickness of 0.24mm；The culture in the step 3) of seed cell preparation The X of vascular endothelial cell is VEGF, the X of keratinocyte is KGF, and it is thin for FGF, the perpendicular flesh of culture to cultivate fibroblastic X It is EGF that the X of born of the same parents, which is EGF, the X of the short columnar epithelial cell of culture is EGF and cultivates the X of melanocyte.

Embodiment 3

A kind of humanization skin is present embodiments provided, specific preparation method is same as Example 1, it is different only in that, In the sterile step 2) for removing antigen degreasing pigskin layer every layer with a thickness of 0.01mm；Item is cultivated in the step 3) of seed cell preparation Until cell 90% contacts in part, cell is collected with pancreatin~EDTA solution digestion, according to the corresponding culture of 4~5 times of culture solutions Base diluting cells.

Embodiment 4

A kind of humanization skin is present embodiments provided, specific preparation method is same as Example 1, it is different only in that, this Embodiment is ox-hide.

Embodiment 5

A kind of humanization skin is present embodiments provided, specific preparation method is same as Example 1, it is different only in that, In the sterile step 2) for removing antigen degreasing pigskin layer every layer with a thickness of 0.24mm；It will step in the step 3) of seed cell preparation The rapid skin plane layer that 1) obtains is divided into 3 layers, and every layer is numbered respectively l, m, n with a thickness of 0.2mm, from deep layer to shallow-layer；

In humanization skin preparation method:

1) go antigen germfree animal cortex unfolding in culture dish individual, according to 0.05-0.lml/cm²Add culture medium, Preculture 3 hours or more, culture medium is sucked, adds the cell of corresponding level (l layers of corresponding first groups of cells labeled as l-L, with this Analogize m-M, n-N) and culture medium, it cultivates and obtains cortex-cell；Wherein, the first groups of cells is for vascular endothelial cell and at fiber Cell；Second groups of cells is perpendicular myocyte and keratinocyte, and third groups of cells is that short columnar epithelial cell and melanin are thin Born of the same parents；

2) cortex-cell for cultivating step 1), changes primary culture for 3-5 days, and cell reaches 50-70% contact, removes skin Layer-cell complexes are successively put in a new culture dish by l-L, m-M, n-N laminated structure, and the l-L bottom adds DMEM to train Base is supported to close to the top layer, cultivates 5-7 days, obtains composite layer i.e. source of people skin；

Experimental example

Comparative example 1 used in this experimental example is the artificial skin that is commercialized in this field, comparative example 2 be in embodiment 1 Raw material is identical with step, and difference is the cortex for removing subcutaneous tissue being divided into 2 layers, trains respectively with epidermal cell and dermal cell Support obtained artificial skin；

The preparation method of comparative example 3 is same as Example 5, unlike, the first groups of cells is vascular endothelial cell and erects Myocyte；Second groups of cells is fibroblast and short columnar epithelial cell, and third groups of cells is keratinocyte and black Plain cell.

Clinical report

1, experimental subjects

III degree burnt degree advanced stage granulation wound, and the age is no more than 60 years old research object.The research object is not suffered a shock The complex injuries such as phase, chemical poisoning do not merge the serious primary disease such as angiocarpy, the cerebrovascular, liver, kidney, hemopoietic system, do not have Allergic constitution person or autoimmune disease person.

2, experimental method

1) preoperative planning blood, routine urinalysis, Liver and kidney function.

2) transfer operation

By the dermatoplasty of cultured Examples 1 to 5 and comparative example 1~3 to III degree burnt degree advanced stage granulation wound On.By the composite skin graft being attached on petrolatum gauze on the surface of a wound, corium downwards (in Examples 1 to 3 a-A towards Under), epidermis faces upward (f-F faces upward in Examples 1 to 3), sutures composite skin with 3-0 silk thread, is fixed on the surface of a wound, stays length Line places salt water gauze with big mesh on petrolatum gauze, is packaged slightly pressure dressing.

3) postoperative with sensitive antibiotic 5-7 days.

After postoperative 7-10 days of granulation wound Composite skin, suture is cut off, soaks internal layer dressing with physiological saline, gently After removal, inspects transplanting Composite Skin and survived situation by area, observe color, secretion, the edge of wound situation of skin.With oil gauze, salt Water gauze and gauze continue slightly pressurized package, dressing 2 times weekly in art the latter moon.

3, efficacy assessment standard

It is effective: 75% or more composite skin wound healing of transplanting.

Effective: transplanting composite skin wound healing is in 30%-75%.

Invalid: transplanting composite skin wound healing is not and 30%.

4, experimental result

This experiment has carried out clinical research in certain research institute, is selected in 40 patients altogether, is divided into 8 groups at random, 8 groups make respectively With the humanization skin in Examples 1 to 5, comparative example 1~3, is observed through art the latter moon, it is compound for organizational project to evaluate it The improvement situation of deep burn and scar is repaired in dermatoplasty, and the curative effect of each MAIN OUTCOME MEASURES is as follows:

(1) it wound secretion: is oozed out without wound secretion.

(2) surface of a wound swelling: noninvasive edema of the face is swollen.

(3) edge of wound reacts: being negative reaction (reacting without edge of wound)

(4) wound healing percentage: using there is 4 20 days left sides after the transfer in 5 of the humanization skin of embodiment 1 It is right effective；Using have in 5 of the humanization skin of embodiment 24 after the transfer 25 days or so it is effective；Use embodiment 3 5 of humanization skin in have 4 after the transfer 25 days or so it is effective；Using in 5 of the humanization skin of embodiment 4 Have 4 after the transfer 25 days or so it is effective；Using having 4 after the transfer 23 days in 5 of the humanization naughty of embodiment 5 Left and right is effective.Using have in 5 of the artificial skin of comparative example 13 transplanting one month after it is effective, use the people of comparative example 2 Have in 5 of work skin 4 it is effective after transplanting one month, using thering are 3 transplanting in 5 of the artificial skin of comparative example 3 It is effective after one month.

Simultaneously using Examples 1 to 5 humanization skin patient's postoperation recovery effect compared with comparative example 1, comparative example 2 with And the effect in comparative example 3 is more preferable, can preferably merge with patient's autologous skin, rejection is less.

Finally, method of the invention is only preferable embodiment, it is not intended to limit the scope of the present invention.It is all Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in protection of the invention Within the scope of.

Claims

1. a kind of preparation method of humanization skin, which comprises the steps of:

Antigen animal cortex will be gone at least to be divided into 3 layers of cortex from deep layer to shallow-layer, every layer of cortex with a thickness of 0.01~ 0.24mm successively plants the first groups of cells, the second groups of cells and third groups of cells on the cortex from deep layer to shallow-layer, culture To obtain the humanization skin；

First groups of cells includes vascular endothelial cell and fibroblast, and second groups of cells includes perpendicular myocyte and angle Matter forms cell, and the third groups of cells includes short columnar epithelial cell and melanocyte.

2. preparation method according to claim 1, which is characterized in that the source for removing antigen germfree animal cortex is Pig, ox, sheep or rabbit.

3. preparation method according to claim 2, which is characterized in that the source for removing antigen germfree animal cortex is Pig.

4. preparation method according to claim 1, which is characterized in that it is described plantation vascular endothelial cell culture medium be containing There is the DMEM culture medium of VEGF, fibroblastic culture medium is the DMEM culture medium containing FGF, the perpendicular myocyte's Culture medium is the DMEM culture medium containing MGF, and the culture medium of the keratinocyte is described in the DMEM culture medium containing KGF The culture medium of short columnar epithelial cell is the DMEM culture medium containing EGF, and the culture medium of the melanocyte is the DMEM of MGS Culture medium.

5. preparation method according to claim 1, which is characterized in that the condition of the plantation are as follows: 37 DEG C, saturated humidity, 5%CO₂It is cultivated in incubator, changes 2/3 culture solution within every 3-5 days, until cell 90% contacts, received with pancreas enzyme -EDTA solution digestion Collect cell, according to the corresponding culture medium diluting cells of 4~5 times of culture solutions.

6. preparation method according to any one of claim 1 to 5, which is characterized in that the specific steps of the culture are as follows:

The cortex of repopulating cell is successively superimposed from deep layer to shallow-layer, adds DMEM culture medium to top layer is closed on, cultivates 5~7d.

7. preparation method according to any one of claim 1 to 5, which is characterized in that the preparation method includes as follows Step:

Antigen animal cortex will be gone to be divided into 6 layers of cortex from deep layer to shallow-layer, successively planted on the cortex from deep layer to shallow-layer Vascular endothelial cell, fibroblast, perpendicular myocyte, keratinocyte, short columnar epithelial cell and melanocyte, culture To obtain the humanization skin.

8. preparation method according to any one of claims 1 to 5, which is characterized in that the preparation method includes following step It is rapid:

Antigen animal cortex will be gone to be divided into 6 layers of cortex from deep layer to shallow-layer, be successively labeled as cortex a, b, c, d, e, f, after sterilizing, Vascular endothelial cell is planted on the cortex a, and fibroblast, cortex c Shang kind tree planter myocyte, cortex d are planted on cortex b Upper plantation keratinocyte plants short columnar epithelial cell on cortex e, melanocyte is planted on cortex f, when cell is in skin After realizing 50~70% contact on layer, above-mentioned cortex is successively superimposed from deep layer to shallow-layer, adds DMEM culture medium to closing on most 5~7d is cultivated to obtain the humanization skin in upper layer.

9. the humanization skin that preparation method described in any item of the claim 1 to 8 obtains.

10. preparation method described in any item of the claim 1 to 8 or humanization skin as claimed in claim 9 are controlled in preparation Treat the application in the graft of III degree burn wound.