CN106962274A - A kind of organization engineering skin repairs the experiment research of nude mice full thickness dermal - Google Patents
A kind of organization engineering skin repairs the experiment research of nude mice full thickness dermal Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Materials For Medical Uses (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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
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.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN113455433A (en) * | 2021-08-03 | 2021-10-01 | 浙江省海洋水产研究所 | Crab biology measuring device |
CN115418386A (en) * | 2022-11-03 | 2022-12-02 | 成都诺医德医学检验实验室有限公司 | Damage healing evaluation method based on skin organoid, model and application |
-
2017
- 2017-03-30 CN CN201710201874.7A patent/CN106962274A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN113455433A (en) * | 2021-08-03 | 2021-10-01 | 浙江省海洋水产研究所 | Crab biology measuring device |
CN113455433B (en) * | 2021-08-03 | 2022-05-10 | 浙江省海洋水产研究所 | Crab biology measuring device |
CN115418386A (en) * | 2022-11-03 | 2022-12-02 | 成都诺医德医学检验实验室有限公司 | Damage healing evaluation method based on skin organoid, model and application |
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