CN106962274A - A kind of organization engineering skin repairs the experiment research of nude mice full thickness dermal - Google Patents

Abstract

The invention discloses the experiment research that a kind of organization engineering skin repairs nude mice full thickness dermal, comprise the following steps：1) healthy male nude mouse is taken to be divided into 2 groups, respectively organizational project group and blank control group, every group of 10 nude mices；2) treat that nude mice anesthesia back part center is cut into the round defect that diameter is about 1cm；3) the hAECs DED organization engineering skin flap coverages built are used at organizational project group nude mice defect, blank control group is covered without anything；4) observation of healing postoperative wound surface situation and histology are carried out；5) drawn materials in postoperative surrounding in nude mice back art area, histotomy, row HE dyeing；6) statistical analysis.The present invention transplants the hAECs DED organization engineering skins of structure in the nude mice surface of a wound, the effect that organization engineering skin product is used to repair nude mice full thickness dermal is detected and understood in terms of Wound healing rate, wound healing time, histopathology, and experimental basis and technical support are provided for the clinical practice in future.

Description

A kind of organization engineering skin repairs the experiment research of nude mice full thickness dermal

Technical field

The present invention relates to tissue engineering technique field, it is specifically related to a kind of organization engineering skin and repairs nude mice full thickness skin The experiment research of defect.

Background technology

The final purpose of organization engineering skin is will to form artificial skin product, transplant in the skin for needing to repair, rebuild At defect, the purpose for repairing human body defect skin histology is reached.Recent domestic expert is continuously attempted to various types of groups The application of weaver's journey skin products is clinically.Because stem cell enough causes the continuous self-renewing of organization engineering skin and propagation, energy Good repairing effect is achieved, therefore existing many scholars build group using stem cell as tissue engineering skin seed cells Knit engineering skin.But the application of these stem cells all has certain limitation, such as its source, quantity, materials wound all Govern its development.We successfully build hAECs-DED organization engineering skins by tissue engineering technique.HAECs is on DED The preferable stratified epithelium tissue of angling is formed, not only institutional framework and cellular morphology are similar to normal human skin, and newborn epithelium Propagation and the feature of differentiation are also similar to normal human skin.

Skin graft is to supplement skin and temporary closure wound as skin-covering agent earliest, and promotes healing.Tissue Engineering skin transplanting is to be combined the functioning cell of in vitro culture with appropriate extracellular matrix, is transplanted to disease damage portion Position, during extracellular matrix is progressively degraded, the cell of plantation is formd with its inherent function by Proliferation, Differentiation Active mass.It is one of defect of skin disease most efficient methods such as current treatment intractable ulcer, large-area burns.But Many organization engineering skin products are still immature at present, exist that skin wears no resistance, autologous seed cell source lacks, allosome The shortcomings of seed cell induces immunological rejection, time-consuming for culture, medical expense is expensive.The organization engineering skin of structure according to Contained layer of structure difference can be divided into three types:Tissue engineering epidermis, tissue engineered dermal equivalent and all layers of skin used by tissue engineering.Group Weaver's journey epidermis exist it is not wear-resisting, easy shrink, it is easy to foaming the shortcomings of, and scar proliferation, anti-sense still occur after wound healing Ability is contaminated, elasticity is not good enough, the problems such as pliability is not enough, therefore this method is greatly limited in clinical practice.Tissue Skin elasticity, flexibility and mechanical endurance of the engineering corium after skin reconstruction process can increase wound healing, reduce scar Hyperplasia, and active fibroblast present in tissue engineered dermal equivalent can promote epidermal growth to break up, induction basilar memebrane is formed.But It is merely able to play a part of corium reconstruction after transplanting.Preferable organization engineering skin should repair epidermis and dermal tissue simultaneously, Because both institutional frameworks not only cutaneous function and profile, and with interactional mechanism, can promote each other Differentiation.Therefore, the direction of skin tissue engineering research from now on is mainly the new method explored and build all layers of skin used by tissue engineering. At present, scholar both domestic and external did the experiment and the research of clinical practice of more all layers of skin used by tissue engineering transplanting, such as Pellegrini etc. is made by screening full cloned stem cell colony as the epidermis seed cell for building artificial skin of Fibrin Support inoculation fibroblast builds skin corium, and made artificial composite skin is used for the treatment of Patients with Big Area Burn.

The progress of existing organization engineering skin transplanting is slow, and one of main cause is exactly to transplant the clinic in human body Research is by many restrictions such as ethics.Nude mice causes T cell dyspoiesis, therefore shortage immune response due to athymia, It is immunodeficient animals, therefore easily implantation heterogenous cell and tissue, have multiple experiments and use nude mice to exempt from for study on the carrier transplanting Epidemic disease achieves good effect.Lack effective immune response in nude mouse, be difficult to repel the fell skin tissue of implantation, can be by people Dermatoplasty is in structure application on human skin-nude mice model after nude mice.Fibroblast, epidermal stem cells are planted in glue by Berthod etc. Nude mice back is migrated on olynthus support, after in vitro culture.Blood vessel from host gradually gos deep into collagen sponge scaffold and reached Under epidermis, and the synthesis of various kinds of cell epimatrix, and energy can be promoted after complete rete vasculosum, the reconstruction skin maturation can be formed Elastin laminin is produced, skin function can be promoted to recover and the formation of scar after skin-grafting is prevented.Nude mice blood vessel can gradually invade fell The corium of skin, mutually being coincide with its dermal microvascular, there is provided the normal configuration that suitable internal microenvironment maintains application on human skin.Therefore with Nude mice is the animal model of defect of skin, available for the reasonable structure after tissues observed engineering skin transplanting survival, and is planted The situation of daughter cell Proliferation, Differentiation in vivo.

Can whether the organization engineering skin for checking external structure have normal function and play its epidermis after the transfer The effect of reconstruction, the present invention transplants the hAECs-DED organization engineering skins of structure in the nude mice surface of a wound, from Wound healing rate, wound The effect of organization engineering skin product is detected and understood in terms of face healing time, histopathology, is the clinical practice in future Experimental basis and technical support are provided.

The content of the invention

Present invention solves the technical problem that being：The experiment that a kind of organization engineering skin repairs nude mice full thickness dermal is provided Research method.

The technical scheme is that：

A kind of organization engineering skin repairs the experiment research of nude mice full thickness dermal, comprises the following steps：

1) take the healthy male nude mouse of 3-4 week old, 20,2 groups be randomly divided into according to lottery, respectively organizational project group and Blank control group, every group of 10 nude mices；It is injected intraperitoneally with 5% chloraldurate 0.01ml/g, nude mice enters after 3-5 minutes Narcosis；

2) it is fixed after nude mice is anaesthetized, routine alcohol and PVP-I sterilization nude mice skin of back spread hole towel；With Point centered on the center of nude mice back, the round defect that diameter is about 1cm is cut into eye scissors, removes full thickness skin at this, after Cleaned a wound with mycillin mixed liquor, to prevent infection；

3) it is described with the hAECs-DED organization engineering skin flap coverages built at organizational project group nude mice defect HAECs-DED organization engineering skins are the corium groups for removing epidermis with people using human amnion membrane (hAECs) as seed cell (De-epidermized dermis, DED) is knitted as timbering material, seed cell is planted in built-up on timbering material HAECs-DED organization engineering skins；Blank control group without anything cover, after with petrolatum oil gauze cover create Face, slightly the packing suture surface of a wound and pressure dressing；Every sub-cage rearing；Remove within postoperative 1 week gauze bag and throw off oily yarn, routinely change Medicine, Continuous Observation 4 weeks；

4) observation of healing postoperative wound surface situation and histology：The healing state of the postoperative Continuous Observation surface of a wound, respectively at One week, two weeks, three weeks, surrounding contrast wound healing situation recorded two groups of wound healing times according to visually observing；Use thin transparent Embrane method calculates two groups of Wound healing rate, i.e. Wound healing rate=(original surface of a wound area-, and do not heal surface of a wound area)/original the surface of a wound Area；

5) draw materials, fixed with 10% formalin in nude mice back art area in postoperative surrounding, conventional dehydration, FFPE, Histotomy, row HE dyeing；

6) statistical analysis：Using SPSS19.0 statistical softwares, measurement data uses independent samples t-test, and enumeration data is used Chi-square Test；P ＜ 0.05 are that difference is statistically significant.

Further, in such scheme, the construction method of the hAECs-DED organization engineering skins is：Using low dense Degree trypsase multistep digestion partition method handles HIGH DENSITY LIPOPROTEIN IN HEALTHY CHILDREN foreskin to extract hAEC with two step enzyme digestions, obtains into fibre Tie up cell suspension, Secondary Culture；The hAEC of the fibroblast of amplification in vitro culture to 3-5 generations and 2nd generation is inoculated with respectively In DED corium face and substrate film surface, vitro in organ's culture builds organization engineering skin.

Further, in such scheme, the specific construction method of the hAECs-DED organization engineering skins is：

1) hAECs be separately cultured and hAECs cell suspensions preparation：

Fritter in small, broken bits is cut into after amnion tissue is rinsed repeatedly with phosphate buffer (PBS), addition contains The ethylenediamine tetra-acetic acid of 0.05% trypsase -0.02% (EDTA) solution, 37 DEG C of digestion 10min, abandons digestive juice and (mainly goes removal of impurities Cell and cell fragment)；Above method digestive epithelium cell, 37 DEG C of digestion 30-40min, 200 mesh stainless (steel) wire mistakes are repeated afterwards Filter；Single cell suspension is collected, plus the culture medium containing 10% hyclone terminates digestion；Fragment of tissue same method weight after filtering Digest 1 time again；The cell being collected by centrifugation addition 10ng/ml EGF DMEM complete medium cultures, are placed in 37 DEG C, 5%CO₂ Cultivated in incubator；24h changes liquid first, and rear every 3 days full doses change liquid once, and cell can pass on training according to a conventional method after about 10 days Support；Fixed after second generation hAEC cell climbing sheets with 4% paraformaldehyde, Oct-3/4, SSEA-4 Immunofluorescence test are carried out respectively； Inverted microscope observes hAECs growth conditions, takes 1-2 for exponential phase hAECs；Culture medium is sucked, two are cleaned with PBS liquid Time；0.25% trypsase is added, jog blake bottle digests attached cell；Microscopic observation cell is rounded by oval, iuntercellular Gap increases, when a small amount of cell is started shedding off, and adds the DMEM culture mediums containing 10%FBS and terminates digestion；It is thin in piping and druming culture bottle wall Born of the same parents, are collected by centrifugation cell (1000r/min, 5min)；Supernatant is abandoned, is resuspended in hAECs complete mediums and cell suspension is made, Count, adjustment cell concentration to 5x10⁶/ ml, it is stand-by；

2) fibroblast (FB) be separately cultured and Fb cell suspensions preparation：

By in epidermal tissue's sample immersion D-Hanks solution, 4 DEG C save backup, and are used in 6 hours；By foreskin in nothing Rinsed 5 times with PBS (penicillin containing 100U/ml and 100U/ml streptomysins) under the conditions of bacterium, hypodermis is cut off with eye scissors, will Skin histology is cut into size about 0.5cm × 1cm elongate strip, is put into 4 DEG C of 16~18h of digestion in 2.5mg/ml Dispase enzymes； Next day is taken out, and epidermis is separated with corium；The dermal tissue isolated is put into 10ml sterile penicillin bottle, added 0.2% type Ⅳ collagen enzyme solutions 3ml, 37 DEG C of 2~3h of digestion, dermal tissue is shredded, add into bottle after digestion with eye scissors Enter the DMEM 3ml containing 10%FBS and terminate digestion, pasteur pipet piping and druming is tens of secondary, and low-speed centrifugal (1000r/min) 5min is abandoned Clear liquid, is resuspended with the DMEM culture mediums 1ml containing 10%FBS, and tally is calculated, and adjustment cell density is 2 × 10⁶/ ml, is inoculated in In batch cultur bottle；Put 5%CO₂, cultivate in 37 DEG C of incubators, liquid is changed after 24h first and removes not adherent cell, later 2~3 It changes liquid 1 time；When culture cell reaches 70%~80% Fusion Strain after 7~10 days, passage purifying culture can obtain into fiber Cell (FB)；Inverted microscope observes Fb growth conditions, takes the 3rd generation exponential phase Fb；Culture medium is sucked, is cleaned with PBS liquid Twice；0.25% trypsase is added, jog blake bottle digests attached cell, adds the DMEM culture mediums containing 10%FBS and terminates Digestion；Cell in piping and druming culture bottle wall, is collected by centrifugation cell (1000r/min, 5min)；Supernatant is abandoned, is resuspended in containing 10%FBS DMEM culture mediums in cell suspension is made, count, adjustment cell concentration to 5x105/ml, it is stand-by；

3) DED preparation and pretreatment：

Adult skin sample routine disinfection is taken, appropriate PBS is added, is placed in standby in 4 DEG C of refrigerators；PBS is used before preparation Sample for several times, routine disinfection；Subcutaneus adipose tissue layer is carefully removed with eye scissors, lcm × 1cm size skin grafts are made；It is dipped in 56 30min in DEG C PBS；Epidermis is removed with ophthalmic tweezers, people is gone to the dermal tissue (De-epidermized dermis, DED) of epidermis It is placed in Epp pipes, room temperature after about 5min, taking-up is inserted in liquid nitrogen rapidly and places 30min, is then inserted again in liquid nitrogen, continuous 10 Individual circulation；After be placed in -20 DEG C of refrigerators and save backup；DED is placed in 12h in 4 DEG C of refrigerators, hAECs is then dipped in and cultivates completely In base；5%CO₂, soak 48h in 37 DEG C of environment, it is standby；To promote hAECs preferably to attach before growth, inoculation, palpus will DED is pre-processed；

4) structure of hAECs-DED organization engineering skins：

Pretreated DED is placed in six orifice plates, DED basilar memebranes downwards, add hAEC complete mediums, 1ml/ The μ l of FB cell suspensions 150 got ready, are inoculated on the DED in 1-5 holes by hole with pipettor, add 150 μ lPBS to make on DED in the 6th hole For blank control；It is placed in 5%CO₂, be incubated overnight in 37 DEG C of environment, to promote FB to be attached at DED reticular corium face；Incubate After educating overnight, DED is overturn, the μ l of hAEC cell suspensions 150 got ready up, are inoculated in 1-5 holes by the basilar memebrane for making DED On DED substrate film surfaces, 150 μ lPBS are added in the 6th hole as blank control, it is rear that hAEC complete mediums are added per hole, it is allowed to soak No DED, be placed in 5%CO₂, cultivate in 37 DEG C of environment；Under liquid cultivate 3 days so that hAEC reaches fusion, after DED is placed in support On frame, the amount of culture medium is adjusted, hAEC is cultivated growth on gas-liquid interface；5%CO₂, continue the training of solution-air face in 37 DEG C of environment Support 2 weeks.

Further, in such scheme, adenyl cyclase activator is also added into described DMEM culture mediums And neurotrophic factor -3, concentration of volume percent of the adenyl cyclase activator in DMEM culture mediums is 0.01- 0.03%, concentration of volume percent of the neurotrophic factor -3 in DMEM culture mediums is 0.02-0.05%.

Further, in such scheme, the ethylenediamine tetra-acetic acid of 0.05% trypsase -0.02% (EDTA) solution Compound method be：Trypsase powder 0.05g, EDTA 0.02g is weighed to be dissolved in a little D-Hanks liquid；It is sufficiently stirred for having made Fully dissolved, 100ml is settled to D-Hanks；4 DEG C overnight；0.22 μm of miillpore filter positive press filtration is degerming, is sub-packed in sterile vials In, 10ml/ bottles；- 20 DEG C save backup.

Further, in such scheme, the formula of the hAECs complete mediums is：Basic DMEM culture mediums, 10% Hyclone, 10ng/mLEGF, penicillin and streptomycin each 30U/ml, 0.5~1.5ng/ml of EGF, DKK1 protein 10s~ 18ng/ml, 12~18ug/ml of transferrins, testosterone propionate 1 × 10^-9~1.2 × 10^-7mol/L。

The beneficial effects of the invention are as follows：The organization engineering skin of the present invention repairs the experimental study of nude mice full thickness dermal Method, by the hAECs-DED organization engineering skins of structure transplant in the artificial skin transplanted after the nude mice surface of a wound, 2 weeks i.e. with surrounding The basic fusion growth of rat tissue, after 3 weeks with the mouse skin appearance of surrounding without significant difference.The tissue work of histology display transplanting Journey skin is similar to regular skin structure, including epidermis and corium.Epidermal structure is clear, and cuticula is obvious.True epidermis boundary is clear Clear, corium is made up of the fiber of homogeneous.The present invention is detected in terms of Wound healing rate, wound healing time, histopathology Effect with understanding organization engineering skin product for repairing nude mice full thickness dermal, experiment is provided for the clinical practice in future Foundation and technical support.

Brief description of the drawings

On the day of Fig. 1 is organization engineering skin group, control group dermatoplasty, the 14th day, the 21st day wound healing situation.

Fig. 2 is organization engineering skin group, the postoperative 28 days histological observations of control group, wherein, Fig. 2-A are organizational project group, Fig. 2-B are blank control group.

Fig. 3 is two groups of Wound healing rate statistical charts.

Fig. 4 is two groups of healing time comparison diagrams.

Embodiment

Embodiment 1：

A kind of organization engineering skin repairs the experiment research of nude mice full thickness dermal, comprises the following steps：

1) the healthy male nude mouse of 3 week old is taken, 20,2 groups, respectively organizational project group and sky are randomly divided into according to lottery White control group, every group of 10 nude mices；It is injected intraperitoneally with 5% chloraldurate 0.01ml/g, nude mice enters anesthesia after 3 minutes State；

2) it is fixed after nude mice is anaesthetized, routine alcohol and PVP-I sterilization nude mice skin of back spread hole towel；With Point centered on the center of nude mice back, the round defect that diameter is about 1cm is cut into eye scissors, removes full thickness skin at this, after Cleaned a wound with mycillin mixed liquor, to prevent infection；

3) it is described with the hAECs-DED organization engineering skin flap coverages built at organizational project group nude mice defect HAECs-DED organization engineering skins are the corium groups for removing epidermis with people using human amnion membrane (hAECs) as seed cell (De-epidermized dermis, DED) is knitted as timbering material, seed cell is planted in built-up on timbering material HAECs-DED organization engineering skins；Blank control group without anything cover, after with petrolatum oil gauze cover create Face, slightly the packing suture surface of a wound and pressure dressing；Every sub-cage rearing；Remove within postoperative 1 week gauze bag and throw off oily yarn, routinely change Medicine, Continuous Observation 4 weeks；

4) observation of healing postoperative wound surface situation and histology：The healing state of the postoperative Continuous Observation surface of a wound, respectively at One week, two weeks, three weeks, surrounding contrast wound healing situation recorded two groups of wound healing times according to visually observing；Use thin transparent Embrane method calculates two groups of Wound healing rate, i.e. Wound healing rate=(original surface of a wound area-, and do not heal surface of a wound area)/original the surface of a wound Area；

5) draw materials, fixed with 10% formalin in nude mice back art area in postoperative surrounding, conventional dehydration, FFPE, Histotomy, row HE dyeing；

6) statistical analysis：Using SPSS19.0 statistical softwares, measurement data uses independent samples t-test, and enumeration data is used Chi-square Test；P ＜ 0.05 are that difference is statistically significant.

Wherein, the construction method of hAECs-DED organization engineering skins is：

1) hAECs be separately cultured and hAECs cell suspensions preparation：

Fritter in small, broken bits is cut into after amnion tissue is rinsed repeatedly with phosphate buffer (PBS), addition contains The ethylenediamine tetra-acetic acid of 0.05% trypsase -0.02% (EDTA) solution, 37 DEG C of digestion 10min, abandons digestive juice and (mainly goes removal of impurities Cell and cell fragment), the compound method of the ethylenediamine tetra-acetic acid of 0.05% trypsase -0.02% (EDTA) solution is：Weigh Trypsase powder 0.05g, EDTA 0.02g are dissolved in a little D-Hanks liquid, are sufficiently stirred for making to be completely dissolved, are used D-Hanks 100ml is settled to, 4 DEG C overnight, and 0.22 μm of miillpore filter positive press filtration is degerming, is sub-packed in sterile vials, 10ml/ bottles, -20 DEG C Preserve；；Above method digestive epithelium cell, 37 DEG C of digestion 30-40min, the filtering of 200 mesh stainless (steel) wires are repeated afterwards；Collect slender Born of the same parents' suspension, plus the culture medium containing 10% hyclone terminate digestion；Fragment of tissue repeats digestion 1 time with same method after filtering； The cell being collected by centrifugation addition 10ng/ml EGF DMEM complete medium cultures, are placed in 37 DEG C, 5%CO₂Trained in incubator Support；24h changes liquid first, and rear every 3 days full doses change liquid once, and cell can Secondary Culture according to a conventional method after 10 days；Second generation hAEC Fixed after cell climbing sheet with 4% paraformaldehyde, Oct-3/4, SSEA-4 Immunofluorescence test are carried out respectively；Inverted microscope is observed HAECs growth conditions, take 1st generation exponential phase hAECs；Culture medium is sucked, is cleaned twice with PBS liquid；Add 0.25% pancreas Protease, jog blake bottle digests attached cell；Microscopic observation cell is rounded by oval, space between cells increase, a small amount of cell When starting shedding off, add the DMEM culture mediums containing 10%FBS and terminate digestion；Cell in piping and druming culture bottle wall, is collected by centrifugation cell (1000r/min, 5min)；Supernatant is abandoned, is resuspended in hAECs complete mediums and cell suspension is made, is counted, adjustment cell is dense Spend to 5x10⁶/ ml, it is stand-by；

2) fibroblast (FB) be separately cultured and Fb cell suspensions preparation：

By in epidermal tissue's sample immersion D-Hanks solution, 4 DEG C save backup, and are used in 6 hours；By foreskin in nothing Rinsed 5 times with PBS (penicillin containing 100U/ml and 100U/ml streptomysins) under the conditions of bacterium, hypodermis is cut off with eye scissors, will Skin histology is cut into size about 0.5cm × 1cm elongate strip, is put into 4 DEG C of 16~18h of digestion in 2.5mg/ml Dispase enzymes； Next day is taken out, and epidermis is separated with corium；The dermal tissue isolated is put into 10ml sterile penicillin bottle, added 0.2% type Ⅳ collagen enzyme solutions 3ml, 37 DEG C of digestion 2h, dermal tissue is shredded, add and contain into bottle after digestion with eye scissors 10%FBS DMEM 3ml terminate digestion, and pasteur pipet piping and druming is tens of secondary, and low-speed centrifugal (1000r/min) 5min abandons supernatant Liquid, is resuspended with the DMEM culture mediums 1ml containing 10%FBS, and tally is calculated, and adjustment cell density is 2 × 10⁶/ ml, is inoculated in one In secondary property blake bottle；Put 5%CO₂, cultivate in 37 DEG C of incubators, liquid is changed after 24h first and removes not adherent cell, is changed within later 2 days Liquid 1 time；When culture cell reaches 70% Fusion Strain after 7 days, passage purifying culture can obtain fibroblast (FB)；It is inverted Micro- sem observation Fb growth conditions, take the 3rd generation exponential phase Fb；Culture medium is sucked, is cleaned twice with PBS liquid；Add 0.25% trypsase, jog blake bottle digests attached cell, adds the DMEM culture mediums containing 10%FBS and terminates digestion；Piping and druming Cell in bottle wall is cultivated, cell (1000r/min, 5min) is collected by centrifugation；Supernatant is abandoned, the DMEM trainings containing 10%FBS are resuspended in Support in base and cell suspension is made, count, adjustment cell concentration is stand-by to 5x105/ml；

3) DED preparation and pretreatment：

Adult skin sample routine disinfection is taken, appropriate PBS is added, is placed in standby in 4 DEG C of refrigerators；PBS is used before preparation Sample for several times, routine disinfection；Subcutaneus adipose tissue layer is carefully removed with eye scissors, lcm × 1cm size skin grafts are made；It is dipped in 56 30min in DEG C PBS；Epidermis is removed with ophthalmic tweezers, people is gone to the dermal tissue (De-epidermized dermis, DED) of epidermis It is placed in Epp pipes, room temperature after about 5min, taking-up is inserted in liquid nitrogen rapidly and places 30min, is then inserted again in liquid nitrogen, continuous 10 Individual circulation；After be placed in -20 DEG C of refrigerators and save backup；DED is placed in 12h in 4 DEG C of refrigerators, hAECs is then dipped in and cultivates completely In base；5%CO₂, soak 48h in 37 DEG C of environment, it is standby；To promote hAECs preferably to attach before growth, inoculation, palpus will DED is pre-processed；

4) structure of hAECs-DED organization engineering skins：

Pretreated DED is placed in six orifice plates, DED basilar memebranes downwards, add hAEC complete mediums, 1ml/ The μ l of FB cell suspensions 150 got ready, are inoculated on the DED in 1-5 holes by hole with pipettor, add 150 μ lPBS to make on DED in the 6th hole For blank control；It is placed in 5%CO₂, be incubated overnight in 37 DEG C of environment, to promote FB to be attached at DED reticular corium face；Incubate After educating overnight, DED is overturn, the μ l of hAEC cell suspensions 150 got ready up, are inoculated in 1-5 holes by the basilar memebrane for making DED On DED substrate film surfaces, 150 μ lPBS are added in the 6th hole as blank control, it is rear that hAEC complete mediums are added per hole, it is allowed to soak No DED, be placed in 5%CO₂, cultivate in 37 DEG C of environment；Under liquid cultivate 3 days so that hAEC reaches fusion, after DED is placed in support On frame, the amount of culture medium is adjusted, hAEC is cultivated growth on gas-liquid interface；5%CO₂, continue the training of solution-air face in 37 DEG C of environment Support 2 weeks.

Wherein, adenyl cyclase activator and neurotrophic factor -3, adenylate are also added into DMEM culture mediums Concentration of volume percent of the cyclase activators in DMEM culture mediums is 0.01%, and neurotrophic factor -3 is in DMEM culture mediums In concentration of volume percent be 0.02%.The formula of hAECs complete mediums is：Basic DMEM culture mediums, 10% tire ox blood Clearly, 10ng/mLEGF, each 30U/ml of penicillin and streptomycin, EGF 0.5ng/ml, DKK1 protein 10 ng/ml, transferrins 12ug/ml, testosterone propionate 1 × 10^-9mol/L。

Embodiment 2：

A kind of organization engineering skin repairs the experiment research of nude mice full thickness dermal, comprises the following steps：

1) the healthy male nude mouse of 4 week old is taken, 20,2 groups, respectively organizational project group and sky are randomly divided into according to lottery White control group, every group of 10 nude mices；It is injected intraperitoneally with 5% chloraldurate 0.01ml/g, nude mice enters anesthesia after 5 minutes State；

2) it is fixed after nude mice is anaesthetized, routine alcohol and PVP-I sterilization nude mice skin of back spread hole towel；With Point centered on the center of nude mice back, the round defect that diameter is about 1cm is cut into eye scissors, removes full thickness skin at this, after Cleaned a wound with mycillin mixed liquor, to prevent infection；

3) it is described with the hAECs-DED organization engineering skin flap coverages built at organizational project group nude mice defect HAECs-DED organization engineering skins are the corium groups for removing epidermis with people using human amnion membrane (hAECs) as seed cell (De-epidermized dermis, DED) is knitted as timbering material, seed cell is planted in built-up on timbering material HAECs-DED organization engineering skins；Blank control group without anything cover, after with petrolatum oil gauze cover create Face, slightly the packing suture surface of a wound and pressure dressing；Every sub-cage rearing；Remove within postoperative 1 week gauze bag and throw off oily yarn, routinely change Medicine, Continuous Observation 4 weeks；

4) observation of healing postoperative wound surface situation and histology：The healing state of the postoperative Continuous Observation surface of a wound, respectively at One week, two weeks, three weeks, surrounding contrast wound healing situation recorded two groups of wound healing times according to visually observing；Use thin transparent Embrane method calculates two groups of Wound healing rate, i.e. Wound healing rate=(original surface of a wound area-, and do not heal surface of a wound area)/original the surface of a wound Area；

5) draw materials, fixed with 10% formalin in nude mice back art area in postoperative surrounding, conventional dehydration, FFPE, Histotomy, row HE dyeing；

6) statistical analysis：Using SPSS19.0 statistical softwares, measurement data uses independent samples t-test, and enumeration data is used Chi-square Test；P ＜ 0.05 are that difference is statistically significant.

Wherein, the construction method of hAECs-DED organization engineering skins is：

1) hAECs be separately cultured and hAECs cell suspensions preparation：

Fritter in small, broken bits is cut into after amnion tissue is rinsed repeatedly with phosphate buffer (PBS), addition contains The ethylenediamine tetra-acetic acid of 0.05% trypsase -0.02% (EDTA) solution, 37 DEG C of digestion 10min, abandons digestive juice and (mainly goes removal of impurities Cell and cell fragment), the compound method of the ethylenediamine tetra-acetic acid of 0.05% trypsase -0.02% (EDTA) solution is：Weigh Trypsase powder 0.05g, EDTA 0.02g are dissolved in a little D-Hanks liquid, are sufficiently stirred for making to be completely dissolved, are used D-Hanks 100ml is settled to, 4 DEG C overnight, and 0.22 μm of miillpore filter positive press filtration is degerming, is sub-packed in sterile vials, 10ml/ bottles, -20 DEG C Preserve；；Above method digestive epithelium cell, 37 DEG C of digestion 40min, the filtering of 200 mesh stainless (steel) wires are repeated afterwards；Collect unicellular outstanding Liquid, plus the culture medium containing 10% hyclone terminate digestion；Fragment of tissue repeats digestion 1 time with same method after filtering；Centrifugation The addition 10ng/ml EGF DMEM complete medium cultures of the cell of collection, are placed in 37 DEG C, 5%CO₂Cultivated in incubator； 24h changes liquid first, and rear every 3 days full doses change liquid once, and cell can Secondary Culture according to a conventional method after 10 days；Second generation hAEC cells Fixed after creep plate with 4% paraformaldehyde, Oct-3/4, SSEA-4 Immunofluorescence test are carried out respectively；Inverted microscope is observed HAECs growth conditions, take 1-2 for exponential phase hAECs；Culture medium is sucked, is cleaned twice with PBS liquid；Add 0.25% Trypsase, jog blake bottle digests attached cell；Microscopic observation cell is rounded by oval, space between cells increase, Shao Liangxi When born of the same parents start shedding off, add the DMEM culture mediums containing 10%FBS and terminate digestion；Cell in piping and druming culture bottle wall, is collected by centrifugation thin Born of the same parents (1000r/min, 5min)；Supernatant is abandoned, is resuspended in hAECs complete mediums and cell suspension is made, is counted, cell is adjusted Concentration is to 5x10⁶/ ml, it is stand-by；

2) fibroblast (FB) be separately cultured and Fb cell suspensions preparation：

By in epidermal tissue's sample immersion D-Hanks solution, 4 DEG C save backup, and are used in 6 hours；By foreskin in nothing Rinsed 5 times with PBS (penicillin containing 100U/ml and 100U/ml streptomysins) under the conditions of bacterium, hypodermis is cut off with eye scissors, will Skin histology is cut into size about 0.5cm × 1cm elongate strip, is put into 4 DEG C of digestion 18h in 2.5mg/ml Dispase enzymes；Next day Take out, epidermis is separated with corium；The dermal tissue isolated is put into 10ml sterile penicillin bottle, 0.2% IV is added Collagenase Type solution 3ml, 37 DEG C of digestion 3h, dermal tissue is shredded, added into bottle and contain 10%FBS after digestion with eye scissors DMEM3ml terminate digestion, pasteur pipet piping and druming is tens of time, and low-speed centrifugal (1000r/min) 5min abandons supernatant, with containing 10%FBS DMEM culture mediums 1ml is resuspended, and tally is calculated, and adjustment cell density is 2 × 10⁶/ ml, is inoculated in disposable training Support in bottle；Put 5%CO₂, cultivate in 37 DEG C of incubators, liquid is changed after 24h first and removes not adherent cell, liquid is changed 1 time within later 3 days； When culture cell reaches 70%~80% Fusion Strain after 10 days, passage purifying culture can obtain fibroblast (FB)；It is inverted Micro- sem observation Fb growth conditions, take the 3rd generation exponential phase Fb；Culture medium is sucked, is cleaned twice with PBS liquid；Add 0.25% trypsase, jog blake bottle digests attached cell, adds the DMEM culture mediums containing 10%FBS and terminates digestion；Piping and druming Cell in bottle wall is cultivated, cell (1000r/min, 5min) is collected by centrifugation；Supernatant is abandoned, the DMEM trainings containing 10%FBS are resuspended in Support in base and cell suspension is made, count, adjustment cell concentration is stand-by to 5x105/ml；

3) DED preparation and pretreatment：

Adult skin sample routine disinfection is taken, appropriate PBS is added, is placed in standby in 4 DEG C of refrigerators；PBS is used before preparation Sample for several times, routine disinfection；Subcutaneus adipose tissue layer is carefully removed with eye scissors, lcm × 1cm size skin grafts are made；It is dipped in 56 30min in DEG C PBS；Epidermis is removed with ophthalmic tweezers, people is gone to the dermal tissue (De-epidermized dermis, DED) of epidermis It is placed in Epp pipes, room temperature after about 5min, taking-up is inserted in liquid nitrogen rapidly and places 30min, is then inserted again in liquid nitrogen, continuous 10 Individual circulation；After be placed in -20 DEG C of refrigerators and save backup；DED is placed in 12h in 4 DEG C of refrigerators, hAECs is then dipped in and cultivates completely In base；5%CO₂, soak 48h in 37 DEG C of environment, it is standby；To promote hAECs preferably to attach before growth, inoculation, palpus will DED is pre-processed；

4) structure of hAECs-DED organization engineering skins：

Pretreated DED is placed in six orifice plates, DED basilar memebranes downwards, add hAEC complete mediums, 1ml/ The μ l of FB cell suspensions 150 got ready, are inoculated on the DED in 1-5 holes by hole with pipettor, add 150 μ lPBS to make on DED in the 6th hole For blank control；It is placed in 5%CO₂, be incubated overnight in 37 DEG C of environment, to promote FB to be attached at DED reticular corium face；Incubate After educating overnight, DED is overturn, the μ l of hAEC cell suspensions 150 got ready up, are inoculated in 1-5 holes by the basilar memebrane for making DED On DED substrate film surfaces, 150 μ lPBS are added in the 6th hole as blank control, it is rear that hAEC complete mediums are added per hole, it is allowed to soak No DED, be placed in 5%CO₂, cultivate in 37 DEG C of environment；Under liquid cultivate 3 days so that hAEC reaches fusion, after DED is placed in support On frame, the amount of culture medium is adjusted, hAEC is cultivated growth on gas-liquid interface；5%CO₂, continue the training of solution-air face in 37 DEG C of environment Support 2 weeks.

Wherein, adenyl cyclase activator and neurotrophic factor -3, adenylate are also added into DMEM culture mediums Concentration of volume percent of the cyclase activators in DMEM culture mediums is 0.03%, and neurotrophic factor -3 is in DMEM culture mediums In concentration of volume percent be 0.05%.The formula of hAECs complete mediums is：Basic DMEM culture mediums, 10% tire ox blood Clearly, 10ng/mLEGF, each 30U/ml of penicillin and streptomycin, EGF 1.5ng/ml, DKK1 protein 18 ng/ml, transferrins 18ug/ml, testosterone propionate 1.2 × 10^-7mol/L。

The organization engineering skin group animal surface of a wound of above-described embodiment 1 is observed, healing result is as follows：

1 week after the transfer, build organization engineering skin group and preferably merged with autologous skin, it can be seen that certain surface of a wound Shrink, organizational project group Wound healing rate is 57.49% ± 6.11%, control group is 22.93% ± 4.26% (n=10).

2 weeks after transplanting, visually observe organizational project group wound healing and be substantially better than control group, graft color and autologous skin Skin color is close to (as shown in Figure 1).Organizational project group Wound healing rate be 92.80% ± 3.10%, control group be 54.57% ± 7.94%.

Transplant after 3 weeks, the organizational project group surface of a wound heals substantially, control group has the wound (as shown in Figure 1) that part is not healed, Organizational project group Wound healing rate is 99.83% ± 5.25%, and control group is 91.16% ± 4.79%.

Transplant after 4 weeks, two groups of naked eyes, which are seen, reaches complete healing.Transplant materials after 4 weeks and carry out histological observation, organize work Journey group epithelium clear layer, substantially, skin corium cell growth is good (as shown in fig. 2-b) for angling.On blank control group graft area Skin is relatively thin and has segmental defect, and skin corium is in disorder, there is inflammatory cell infiltration (as shown in Fig. 2-A).

Therefore observed within 3 weeks, organization engineering skin group Wound healing rate has apparently higher than control group at 1 week, 2 weeks respectively Significant difference (p<0.001) (as shown in Figure 3).

Statistical calculations go out two groups of wound healing times：Organizational project 17.51 ± 1.51d of group, control group be 22.80 ± 1.14d, therefore organizational project group wound healing time is significantly shorter than control group, and both have significant difference (p<0.001) (as schemed Shown in 4).

Claims (6)

1. a kind of organization engineering skin repairs the experiment research of nude mice full thickness dermal, it is characterised in that including following Step：

1) the healthy male nude mouse of 3-4 week old is taken, 20,2 groups, respectively organizational project group and blank are randomly divided into according to lottery Control group, every group of 10 nude mices；It is injected intraperitoneally with 5% chloraldurate 0.01ml/g, nude mice enters anesthesia after 3-5 minutes State；

2) it is fixed after nude mice is anaesthetized, routine alcohol and PVP-I sterilization nude mice skin of back spread hole towel；With nude mice Point centered on the center of back, the round defect that diameter is about 1cm is cut into eye scissors, removes full thickness skin at this, rear with blue or green Streptomysin mixed liquor cleans a wound, to prevent infection；

3) with the hAECs-DED organization engineering skin flap coverages built, described hAECs- at organizational project group nude mice defect DED organization engineering skins are the dermal tissue (De- for removing epidermis with people using human amnion membrane (hAECs) as seed cell Epidermized dermis, DED) as timbering material, seed cell is planted in built-up on timbering material HAECs-DED organization engineering skins；Blank control group without anything cover, after use petrolatum oil gauze flap coverage, The packing suture surface of a wound and slightly pressure dressing；Every sub-cage rearing；Remove gauze bag within postoperative 1 week and throw off oily yarn, route dressing change, even Continuous observation 4 weeks；

4) observation of healing postoperative wound surface situation and histology：The healing state of the postoperative Continuous Observation surface of a wound, respectively at one week, Two weeks, three weeks, surrounding contrast wound healing situation recorded two groups of wound healing times according to visually observing；Use thin transparent embrane method Calculating two groups of Wound healing rate, i.e. Wound healing rate ,=(original surface of a wound area-do not heal surface of a wound area)/original surface of a wound face Product；

5) draw materials, fixed with 10% formalin in nude mice back art area in postoperative surrounding, conventional dehydration, FFPE, tissue Section, carries out HE dyeing；

6) statistical analysis：Using SPSS19.0 statistical softwares, measurement data uses independent samples t-test, and enumeration data uses card side Examine；P ＜ 0.05 are that difference is statistically significant.

2. a kind of organization engineering skin as claimed in claim 1 repairs the experiment research of nude mice full thickness dermal, its It is characterised by, the construction method of the hAECs-DED organization engineering skins is：Digested and separated using low concentration trypsase multistep Method handles HIGH DENSITY LIPOPROTEIN IN HEALTHY CHILDREN foreskin to extract hAEC with two step enzyme digestions, obtains fibroblast suspension, Secondary Culture；Will Amplification in vitro culture to the fibroblast in 3-5 generations and the hAEC of 2nd generation is seeded in DED corium face and substrate film surface respectively, Vitro in organ's culture builds organization engineering skin.

3. a kind of organization engineering skin as claimed in claim 2 repairs the experiment research of nude mice full thickness dermal, its It is characterised by, the specific construction method of the hAECs-DED organization engineering skins is：

1) hAECs be separately cultured and hAECs cell suspensions preparation：

Fritter in small, broken bits is cut into after amnion tissue is rinsed repeatedly with phosphate buffer (PBS), adds and contains 0.05% pancreas The ethylenediamine tetra-acetic acid of protease -0.02% (EDTA) solution, 37 DEG C of digestion 10min, abandons digestive juice；Above method digestion is repeated afterwards Epithelial cell, 37 DEG C of digestion 30-40min, the filtering of 200 mesh stainless (steel) wires；Single cell suspension is collected, plus containing 10% hyclone Culture medium terminates digestion；Fragment of tissue repeats digestion 1 time with same method after filtering；The cell being collected by centrifugation addition 10ng/ Ml EGF DMEM complete medium cultures, are placed in 37 DEG C, 5%CO₂Cultivated in incubator；24h changes liquid first, and latter every 3 days complete Amount changes liquid once, and cell can Secondary Culture according to a conventional method after about 10 days；4% paraformaldehyde is used after second generation hAEC cell climbing sheets It is fixed, Oct-3/4, SSEA-4 Immunofluorescence test are carried out respectively；Inverted microscope observes hAECs growth conditions, takes 1-2 generations Exponential phase hAECs；Culture medium is sucked, is cleaned twice with PBS liquid；Add 0.25% trypsase, jog blake bottle, digestion Attached cell；Microscopic observation cell is rounded by oval, space between cells increase, when a small amount of cell is started shedding off, is added and is contained 10% FBS DMEM culture mediums terminate digestion；Cell in piping and druming culture bottle wall, is collected by centrifugation cell；Supernatant is abandoned, hAECs is resuspended in Cell suspension is made in complete medium, counts, adjustment cell concentration to 5x10⁶/ ml, it is stand-by；

2) fibroblast (FB) be separately cultured and Fb cell suspensions preparation：

By in epidermal tissue's sample immersion D-Hanks solution, 4 DEG C save backup, and are used in 6 hours；By foreskin in sterile bar Rinsed 5 times with PBS under part, cut off hypodermis with eye scissors, skin histology is cut into size about 0.5cm × 1cm elongate strip, It is put into 4 DEG C of 16~18h of digestion in 2.5mg/ml Dispase enzymes；Next day is taken out, and epidermis is separated with corium；It is true by what is isolated Skin tissue is put into 10ml sterile penicillin bottle, adds 0.2% type Ⅳ collagen enzyme solutions 3ml, 37 DEG C of 2~3h of digestion, digestion Dermal tissue is shredded with eye scissors afterwards, the DMEM 3ml containing 10%FBS are added into bottle and terminate digestion, pasteur pipet piping and druming Tens of times, low-speed centrifugal abandons supernatant, is resuspended with the DMEM culture mediums 1ml containing 10%FBS, and tally is calculated, and adjustment cell is close Spend for 2 × 10⁶/ ml, is inoculated in batch cultur bottle；Put 5%CO₂, cultivate in 37 DEG C of incubators, liquid is changed after 24h first and is removed not Adherent cell, changes liquid 1 time in later 2~3 days；When culture cell reaches 70%~80% Fusion Strain after 7~10 days, pass on pure Change culture, fibroblast (FB) can be obtained；Inverted microscope observes Fb growth conditions, takes the 3rd generation exponential phase Fb；Suck Culture medium, is cleaned twice with PBS liquid；0.25% trypsase is added, jog blake bottle digests attached cell, adds and contain 10% FBS DMEM culture mediums terminate digestion；Cell in piping and druming culture bottle wall, is collected by centrifugation cell；Supernatant is abandoned, is resuspended in containing 10% Cell suspension is made in FBS DMEM culture mediums, counts, adjustment cell concentration is stand-by to 5x105/ml；

3) DED preparation and pretreatment：

Adult skin sample routine disinfection is taken, appropriate PBS is added, is placed in standby in 4 DEG C of refrigerators；PBS sample is used before preparing For several times, routine disinfection；Subcutaneus adipose tissue layer is carefully removed with eye scissors, lcm × 1cm size skin grafts are made；It is dipped in 56 DEG C of PBS Middle 30min；Epidermis is removed with ophthalmic tweezers, the dermal tissue (De-epidermized dermis, DED) that people removes epidermis is placed in In Epp pipes, room temperature after about 5min, taking-up is inserted in liquid nitrogen rapidly and places 30min, is then inserted again in liquid nitrogen, continuous 10 are followed Ring；After be placed in -20 DEG C of refrigerators and save backup；DED is placed in 12h in 4 DEG C of refrigerators, is then dipped in hAECs complete mediums； 5%CO₂, soak 48h in 37 DEG C of environment, it is standby；

4) structure of hAECs-DED organization engineering skins：

Pretreated DED is placed in six orifice plates, DED basilar memebranes downwards, add hAEC complete mediums, 1ml/ holes are used The μ l of FB cell suspensions 150 got ready are inoculated on the DED in 1-5 holes by pipettor, add 150 μ lPBS to be used as sky on DED in the 6th hole White control；It is placed in 5%CO₂, be incubated overnight in 37 DEG C of environment, to promote FB to be attached at DED reticular corium face；It was incubated After night, DED is overturn, the basilar memebrane for making DED up, the μ l of hAEC cell suspensions 150 got ready is inoculated in the DED in 1-5 holes On substrate film surface, 150 μ lPBS are added in the 6th hole as blank control, it is rear that hAEC complete mediums are added per hole, it is allowed to submerge DED, is placed in 5%CO₂, cultivate in 37 DEG C of environment；Under liquid cultivate 3 days so that hAEC reaches fusion, after DED is placed in support frame On, the amount of culture medium is adjusted, hAEC is cultivated growth on gas-liquid interface；5%CO₂, continue the culture of solution-air face in 37 DEG C of environment 2 weeks.

4. a kind of organization engineering skin as claimed in claim 3 repairs the experiment research of nude mice full thickness dermal, its It is characterised by, adenyl cyclase activator and neurotrophic factor -3, adenosine is also added into described DMEM culture mediums Concentration of volume percent of the cyclase of acid activator in DMEM culture mediums is 0.01-0.03%, and neurotrophic factor -3 exists Concentration of volume percent in DMEM culture mediums is 0.02-0.05%.

5. a kind of organization engineering skin as claimed in claim 3 repairs the experiment research of nude mice full thickness dermal, its It is characterised by, the compound method of the ethylenediamine tetra-acetic acid of 0.05% trypsase -0.02% (EDTA) solution is：Weigh pancreas egg White enzyme powder 0.05g, EDTA 0.02g is dissolved in a little D-Hanks liquid；It is sufficiently stirred for making to be completely dissolved, uses D-Hanks constant volumes To 100ml；4 DEG C overnight；0.22 μm of miillpore filter positive press filtration is degerming, is sub-packed in sterile vials, 10ml/ bottles；- 20 DEG C of preservations It is standby.

6. a kind of organization engineering skin as described in claim 1-4 repairs the experiment research of nude mice full thickness dermal, Characterized in that, the compound method of the ethylenediamine tetra-acetic acid of 0.05% trypsase -0.02% (EDTA) solution is：Weigh pancreas Protease powders 0.05g, EDTA 0.02g are dissolved in a little D-Hanks liquid；It is sufficiently stirred for making to be completely dissolved, it is fixed with D-Hanks Hold to 100ml；4 DEG C overnight；0.22 μm of miillpore filter positive press filtration is degerming, is sub-packed in sterile vials, 10ml/ bottles；- 20 DEG C of guarantors Deposit standby.