CN110339060A - A kind of medicine box and preparation method thereof for wound repair - Google Patents
A kind of medicine box and preparation method thereof for wound repair Download PDFInfo
- Publication number
- CN110339060A CN110339060A CN201910636265.3A CN201910636265A CN110339060A CN 110339060 A CN110339060 A CN 110339060A CN 201910636265 A CN201910636265 A CN 201910636265A CN 110339060 A CN110339060 A CN 110339060A
- Authority
- CN
- China
- Prior art keywords
- wound
- medicine box
- cell suspension
- wound repair
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
- A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Dermatology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention discloses a kind of medicine box for wound repair, comprising: the self-existent fibronectin external preparation made of fibronectin and pharmaceutically acceptable auxiliary material and epidermal basal cell suspension.During carrying out wound repair, use the medicine box provided by the present invention for wound repair, first fibronectin is sprayed on the fresh granulation wound after debridement or the decellularized vascular matrix of transplanting, epidermal basal cell is sprayed again, cell can be made effectively to be adhered on the surface of a wound or the decellularized vascular matrix of transplanting, cell is prevented to be lost, so as to effectively facilitate wound healing and improve wound healing quality.The present invention can be used for the Plastic renovation of the surface of a wound of formation such as burn, the excision of scald, diabetic ulcer, scar excision, mole, subcutaneous Tumor resection for the medicine box of wound repair, significant effect, wound healing can not only be accelerated, but also healing quality can be significantly improved.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to a kind of for the medicine box of wound repair and its preparation side
Method.
Background technique
The surface of a wound is body since inherent or external factor causes skin integrity forfeiture to be formed by, repair always with
Being the urgent problem in clinic.The surface of a wound clinically can be temporally divided into acute wound and chronic wound.It is acute
The surface of a wound includes being formed after the operations such as major diseases or scar, Skin pigment abnormalities, innocent and malignant tumour such as burn, wound are cut off
Defect of skin, chronic wound include traumatic ulcer, diabetic ulcer, vascular ulcers, pressure ulcer, radioactivity burst
The defect of skin that ulcer, neoplastic ulcer, cicatricial ulcer etc. are formed.
Operation repair be the current treatment surface of a wound main method, still, either traditional grafts, skin flap transplantation,
Stamp skin grafting dermepenthesis, wear debris emission, netted skin grafting dermepenthesis, Composite skin etc. or emerging organization engineering skin, due to existing
Neoplastic skin wears no resistance, is easy the problems such as ulceration repeatedly after self skin scarcity, wound healing, not can effectively solve yet at present
The problem of wound repair.And often scar proliferation, sweat gland missing, skin lose original function after major disease fire victim's rehabilitation
Can, quality of life is low；And often protracted course of disease, less serious case greatly consume medical resource, severe one accompanying infection, septicopyemia to chronic wound
Mass formed by blood stasis etc. seriously endangers life.
Epidermal cell is the main cell for constituting skin epidermis, and epidermis normal configuration and physiology are maintained in proliferation, differentiation
Function, while being also the important seed cell in wound, it may be directly applied to wound repair.Currently, epidermal cell
Suspension transplantation method mainly has one-step method and two-step method.One-step method is by cell suspension directly transplanting to the surface of a wound.The hair such as Wu Changyan
Table paper " Effect study [J] China aesthetic medicine of the Autologous epidermis cell suspension in repairing skin tissue's Wound Defect,
2012,21 (12): 2201-2203 " is open, and epidermal cell suspension can promote skin in the skin grafing and mending skin blemish surface of a wound
The healing rate of Wound Defect mitigates the shrinking percentage of the surface of a wound, prevents cicatrization, mitigates pigmentation after wound, but due to cell
The reasons such as suspension itself flowing, after cell suspension is sprayed on the surface of a wound, cell is easy to be lost, and cannot effectively be adhered on the surface of a wound,
Higher epidermal cell Transplanting den- sity is not only needed as a result, and strong influence also is caused to therapeutic effect.
Two-step method refers to first transplants dermal matrix or cytoskeleton on the surface of a wound, such as acellular dermal matrix, collagen sponge
Deng then carrying out the transplanting of cell suspension again.Paper " the in vitro cultured epidermal stem cells composite high-molecular branch that Lu Kingdom's loyalty etc. is delivered
The damage of research [J] China and reparation magazine (electronic edition) of frame in-situ immobilization deep burn wound, 2011,6 (1): 20-32 " is public
It has opened and has been implanted into the surface of a wound at fiber-silk fibroin nano-fiber active scaffold for the Autologous epidermis of in vitro culture is stem cell combined,
As the method that tissue-engineering graft constructed repairs the III degree surface of a wound, the above method is with fibroblast-silk fibroin nano-fiber branch
Frame and amplification in vitro skin bit is digested by neutral proteinase and two step digestion method of trypsase-EDTA after utilize IV Collagen Type VI egg
The culture bottle of white modification quickly sticks epidermal stem cells that separation and Extraction obtains as tissue-engineering graft constructed；When wound repairing,
Fibroblast-silk fibroin nano-fiber bracket is first laid in the surface of a wound, then instillation concentration about 1 × 10 on bracket7/ml
Epidermal stem cells suspension, then with sterilizing pig acellular dermal cover bracket, finally suture, cover gauze.In this method, silk
Fibroin provides similar with tissue mainly for the preparation of cytoskeleton, simulation extra-cellular matrix structure for the growth and metabolism of cell
Growing environment, and this method mainly passes through fibroblast-fibroin albumen Nanowire in the epidermal stem cells suspension that instiled
Covering sterilizing pig acellular dermal prevents the losses of epidermal stem cells on dimensional scaffold, prevents the bacterial invasion surface of a wound, and epidermal stem
Cell can be proliferated under the protection of sterilizing pig acellular dermal, expand and finally differentiation generates epidermis prevention flap coverage.Though
So repairing the III degree surface of a wound by the above method can be improved the healing rate and healing quality of the surface of a wound, but its experiment condition requires height,
The wholistic therapy time is long, and operation is complicated, takes time and effort, is difficult to promote and apply in basic hospital；On the other hand, acellular dermal base
Matter is expensive, and treatment cost is high.
Summary of the invention
One of them of the invention is designed to provide a kind of medicine box for wound repair, to solve the above technical problems
At least one of.
It is above-mentioned to solve it is another object of the present invention to provide the preparation method of the above-mentioned medicine box for wound repair
At least one of technical problem.
According to an aspect of the invention, there is provided a kind of medicine box for wound repair, comprising: self-existent by fibre
Connect fibronectin external preparation and epidermal basal cell suspension made of albumen and pharmaceutically acceptable auxiliary material.
Present inventor has found that, compared to transplanting epidermal cell, transplanting epidermal basal is thin in the clinical research of the past
Born of the same parents (Epidermal Basal Cells, EBCs, mainly comprising keratinocyte, epidermal stem cells, fibroblast and black
Chromatophore etc.) Promote Chronic Ischemic Wound Healing can be more efficiently facilitated, and wherein play main function is epidermal stem cells
(Epidermal Stem Cells, ESCs).Fibronectin (fibronectin, FN) is that one kind is widely present in blood, body fluid
And the macromolecule glycoprotein in various tissues, molecular mass 450ku pass through chain by the subunit that two molecular masses are 220ku
Between disulfide bond be formed by connecting.In the course of the research, inventor has been surprisingly found that, when carrying out wound repair, after debridement
Fresh granulation wound or transplanting decellularized vascular matrix on, first spray fibronectin, then spray cell suspension, fibronectin
The ESCs in EBCs suspension can be quickly and effectively adsorbed, cell is enable effectively to be adhered to the decellularized vascular matrix of the surface of a wound or transplanting
On, prevent EBCs to be lost, so as to improve the therapeutic efficiency of EBCs, wound healing simultaneously improves healing quality.In addition, fine
Even albumen is commercialized product, is widely used to clinic, and has proven to the risks such as drug residue free, DNA residual, allergy and secondary work
With applied to wound repair, securely and reliably.
In the present invention, epidermal basal cell can be the cell for being derived from the cell of skin graft or manually cultivating, wherein skin graft
Can be self skin graft or artificial skin graft, (ideal artificial skin graft includes epidermis and corium double-layer structure and connects between the two
It connects closely, with flexibility and certain mechanical strength, is transplanted to after the surface of a wound and is well attached with the surface of a wound quickly, there is peace
Entirely, the advantage of virus is not carried, and epidermis and dermal components can complete own amplification, differentiation and function maturation, formation as early as possible and more connect
It is bordering on physiological permanent Graftskin etc.)；The cell manually cultivated can be the cell of cell bank preservation, can also be with
It is commercially available cell.
In some embodiments, epidermal basal cell suspension can be made by following methods:
(1) skin graft is digested with TrypLE Select digestive juice；
(2) it is rinsed skin graft with Lactated Ringer'S Solution and is terminated and digested, separated epidermis and corium, then scraped from basal layer of epidermis
Epidermal basal cell is taken, is diluted after collecting cell with Lactated Ringer'S Solution, obtains cell suspension；
(3) cell suspension is purified to get epidermal basal cell suspension.
Epidermal basal cell suspension is prepared by the above method, experiment condition requirement is low, can be completed in operating room, about
Time-consuming 30min, compared with the past organization engineering skin need to undergo in laboratory conditions cell secondary culture etc., epidermis of the present invention
The preparation method of basal cell has the advantages of quick, disposable completion, and in cell suspension obtained, and cell concentration is high, cell
It is active strong, average every 1cm2Cell number made from skin graft is about 106A, activity reaches 85% or more, epidermal basal cell obtained
It is transplanted simultaneously with fibronectin for wound repairing, average every 1cm2Cell portable is in 40~80cm acquired by skin graft2Wound
Face can also reduce the area of skin graft as a result,.
In some embodiments, in step (1), skin graft can be used to concentration under the conditions of 37 DEG C is 1 × TrypLE
The TrypLE Select digestive juice of 5~15 times of Select digestive juice concentration digests 10~20min.Pass through TrypLE Select
When digestive juice digests skin graft, it is ensured that the working concentration and TrypLE Select digestive juice of TrypLE Select digestive juice are to skin
The time of the digestion of piece can be only achieved the short operating time and epidermis and corium is easily isolated, and the cell quantity of acquisition is more, active ratio
The high purpose of example, that is, achieve the purpose that be quickly obtained epidermal basal cell suspension.
In some embodiments, in step (3), the step of purifying to cell suspension may include: with 100 μm thin
Born of the same parents' strainer filtering.Thus, it is possible to improve the purity of ESCs.
In some embodiments, in step (1), skin graft can be Razor thin skin.It is few that Razor thin skin corium contains band, and is easy to cut
It takes, skin donor site is unrestricted, and healing is rapid.
In some embodiments, the medicine box provided by the invention for being same as wound repair can be used for burning, scald, glycosuria
The Plastic renovation of the surface of a wound of formation such as characteristic of disease ulcer, scar excision, mole excision, subcutaneous Tumor resection, significant effect, not only
It can accelerate Wound healing rate, and healing quality can be significantly improved.
According to another aspect of the present invention, the preparation method of the above-mentioned medicine box for wound repair is provided, it will be by fibre
Connect fibronectin external preparation made of albumen and pharmaceutically acceptable auxiliary material and epidermal basal cell suspension is distributed into medicine box；
Wherein the preparation method of epidermal basal cell suspension includes the following steps:
(1) skin graft is digested with TrypLE Select digestive juice；
(2) it is rinsed skin graft with Lactated Ringer'S Solution and is terminated and digested, separated epidermis and corium, then scraped from basal layer of epidermis
Epidermal basal cell is taken, is diluted after collecting cell with Lactated Ringer'S Solution, obtains cell suspension；
(3) cell suspension is purified to get epidermal basal cell suspension.
In some embodiments, in step (1), it is 1 × TrypLE Select that skin graft is used to concentration under the conditions of 37 DEG C
The TrypLE Select digestive juice of 5~15 times of digestive juice concentration digests 10~20min.
In some embodiments, in step (3), the step of purifying to cell suspension includes: to be filtered with 100 μm of cells
Net filtration.
Compared with prior art, the beneficial effects of the present invention are:
(1) when medicine box provided by the invention is used for wound repairing, cell can be enable effectively to be adhered to the surface of a wound or transplanting
On decellularized vascular matrix, EBCs is prevented to be lost, so as to improve the therapeutic efficiency of EBCs, wound healing and improving is cured
Close quality；
(2) medicine box of the present invention is at low cost, and preparation method is simple and fast, and when being used for wound repairing, surgical procedure is simple, is easy to
It is promoted and applied in basic hospital；
(3) present invention is used for wound repairing for the medicine box of wound repair, can effectively facilitate angiogenesis, improve ESCs
Proliferative capacity and promote ESCs proliferation, thus peomote wound healing and improve wound healing quality.
Detailed description of the invention
Fig. 1 is ESCs identified by immunofluorescence result；
Fig. 2 is the surface of a wound of each group full thickness dermal wounds model SD rat respectively at 0d, 3d, 7d, 14d, 21d days
Healing state；
Fig. 3 is the residual wound rate of each group full thickness dermal wounds model SD rat in different time points；Wherein,*Table
Show compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P < 0.05 (n=6)；From top to bottom
Curve respectively represents Control group, EBCs treatment group, FN+EBCs treatment group full thickness dermal wounds model SD rat not
With the residual wound rate at time point；
Fig. 4 is the healing time of the surface of a wound of each group full thickness dermal wounds model SD rat；Wherein,*It indicates and compares
Group is compared,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P < 0.05 (n=6)；
Fig. 5 is the surface of a wound of the surface of a wound in treatment 3d, 7d and 21d of each group full thickness dermal wounds model SD rat
H&E coloration result；
Fig. 6 is epidermal thickness situation of the surface of a wound in treatment 21d of each group full thickness dermal wounds model SD rat；Its
In,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P < 0.05 (n=6)；
Fig. 7 is epidermal ridge quantity situation of the surface of a wound in treatment 21d of each group full thickness dermal wounds model SD rat；
Wherein,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P < 0.05 (n=6)；
Fig. 8 is CD31 immune group of the surface of a wound in treatment 7d, 14d of each group full thickness dermal wounds model SD rat
Change coloration result；
Fig. 9 is microvessel density situation of the surface of a wound in treatment 7d of each group full thickness dermal wounds model SD rat；
Wherein,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P < 0.05 (n=6)；
Figure 10 is microvessel density feelings of the surface of a wound in treatment 14d of each group full thickness dermal wounds model SD rat
Condition；Wherein,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P < 0.05 (n=6)；
Figure 11 is immunofluorescence dyeing knot of the surface of a wound in treatment 7d of each group full thickness dermal wounds model SD rat
Fruit；
Figure 12 is the surface of a wound of each group full thickness dermal wounds model SD rat in the thin for the treatment of 7d, K15 the expression positive
The density of born of the same parents；Wherein,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P < 0.05
(n=6)；
Figure 13 is that the surface of a wound of each group full thickness dermal wounds model SD rat is positive in treatment 7d, Ki-67 expression
The density of cell；Wherein,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P <
0.05 (n=6)；
Figure 14 is the surface of a wound of each group full thickness dermal wounds model SD rat in treatment 7d, and K15, Ki-67 are expressed
The density of positive cell；Wherein,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#
P < 0.05 (n=6)；
Figure 15 is the surface of a wound of each group full thickness dermal wounds model SD rat in treatment 7d, and CD31 mRNA's is opposite
Express ratio；Wherein,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P < 0.05
(n=6)；
Figure 16 is the surface of a wound of each group full thickness dermal wounds model SD rat in treatment 14d, and CD31 mRNA's is opposite
Express ratio；Wherein,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates compared with EBCs treatment group,#P < 0.05
(n=6)；
Figure 17 is the surface of a wound of each group full thickness dermal wounds model SD rat in treatment 7d, K15 mRNA and Ki-67
The relative expression ratio of mRNA；Wherein,*It indicates compared with the control group,*P < 0.05 (n=6)；#It indicates and EBCs treatment group phase
Than,#P < 0.05 (n=6).
Specific embodiment
The present invention is described in further detail with attached drawing combined with specific embodiments below.
Embodiment 1, for wound repair medicine box preparation
(1) quick separating of epidermal basal cell suspension and extraction:
A. it digests: taking razor graft 1cm × 2cm that thickness is about 0.2mm to be placed in culture dish, TrypLE is added immediately
Select (10 ×) digestive juice digests 15min under the conditions of 37 DEG C；Wherein, TrypLE Select (10 ×) digestive juice is purchased from
Thermo Fisher Scientific company (article No.: A1217701), is preheated to 37 DEG C before use；
B. it separates: Lactated Ringer'S Solution is added and rinses skin graft and terminates digestion, separates epidermis and corium with tweezers, and use nothing
Bacterium scalpel gently scrapes EBCs from basal layer of epidermis, with 1ml syringe and round end needle aspirate EBCs, collects EBCs, then
It is diluted with 2ml Lactated Ringer'S Solution, obtains cell suspension；
C. it extracts and filters: being filtered above-mentioned cell suspension in 50ml centrifuge tube with 100 μm of cell strainers；It rinses again
Culture dish remnants EBCs, the solution eluted are filled into above-mentioned 50ml centrifuge tube with 100 μm of cell strainers, are obtained after purification
EBCs suspension, 1500r/min be centrifuged 5min, abandon supernatant, add 2ml Lactated Ringer'S Solution be resuspended, obtain EBCs suspension；
(2) it takes fibronectin antibacterial liquid (concentration is 20 μ g/ml, is purchased from Zhengzhou Forberun Biotechnology Co., Ltd.), and
EBCs suspension obtained above is sub-packed in the medicine box for being used for wound repair in medicine box to get the present embodiment respectively.
The application method of embodiment 2, the present invention for the medicine box of wound repair:
S1, the fresh granulation wound bed after patient's complete resection scar and take in the wound bed of dermatotome spray fibronectin suppression
To its uniform fold wound bed, (every ml fibronectin antibacterial liquid about covers 10cm to bacterium solution2The surface of a wound), stand 30min；
S2, EBCs suspension is sprayed in the wound bed of fibronectin covering, naked eyes visible cell suspension is uniformly attached at
Wound bed is lost without obvious.
EBCs can be prepared by skin graft separation and Extraction, be also possible to the cell manually cultivated.When thin for what is manually cultivated
When born of the same parents, needs that cell is carried out routine culture in advance, be resuspended after then taking cell, centrifugation to remove supernatant with Lactated Ringer'S Solution, is made
Cell suspension is sub-packed in medicine box with fibronectin antibacterial liquid.When obtaining cell suspension by skin graft, skin graft can be patient certainly
The Razor thin skin of body healthy skin is also possible to artificial skin graft；Separation and Extraction, system can be carried out to cell before prosthesis carries out
Cell suspension is obtained, is sub-packed in medicine box with fibronectin antibacterial liquid；Using skin graft prepare EBCs suspension process can also with it is upper
It states step S1 while carrying out.When skin graft selects self skin graft, preferably carried out simultaneously with step S1, thus, it is possible to repair the surface of a wound
Multiple operation quickly, is disposably completed, and patient is avoided to be subjected to the pain (taking Pi Yici, cell transplantation is primary) of second operation.In addition,
The surface of a wound is treated for the medicine box of wound repair using the present invention, not needing very high cell transplantation density can reach very
Good therapeutic effect takes surface product or cell number is avoided not enough need to carry out culture amplification it is possible thereby to reduce, to avoid adding
The pain of big patient or extended treatment time.The preparation method of the EBCs suspension provided according to the present invention, average every 1cm2Skin graft
Cell number obtained is about 106A, activity reaches 85% or more, average when transplanted simultaneously with fibronectin for wound repairing
Every 1cm2Cell portable is in 40~80cm acquired by skin graft2The surface of a wound.EBCs suspension is attached at the surface of a wound of fibronectin covering
After on bed, the ESCs in EBCs suspension by Preferential adsorption in surface of a wound basal layer, thus facilitate wound healing and regeneration skin group
The normal alignment knitted, wound healing and raising healing quality.
The identification of experimental example one, ESCs purity
1, the quick separating of EBCs and extraction: method is centrifuged, after abandoning supernatant with embodiment 1, the filtering of EBCs cell suspension,
It is resuspended with K-SFM culture medium.
2, the quick adsorption separation of ESCs:
(1) it is coated with: differential attachment method separation and Extraction ESCs from EBCs is used, in advance with the FN of 20 μ g/ml in 37 DEG C of items
Coated cell culture dish 30min under part；
(2) plate is planted: by isolated EBCs with 105/cm2Kind plate, 10~20min of stationary culture under the conditions of 37 DEG C；
(3) not adherent cell is washed away with culture medium, attached cell is ESCs.
3, identified by immunofluorescence ESCs purity
(1) cell climbing sheet: by 104The EBCs that a quick separating extracts is seeded to the 20 coated Room 4 μ g/ml FN and exempts from
On epidemic disease fluorescence glass slide, liquid is changed after adsorbing ESCs, at 37 DEG C, 5%CO2It is incubated overnight in incubator；
(2) it is rinsed 3 times with the PBS of pre-temperature；
(3) fixed: 4% cold paraformaldehyde fixes 20min, and PBS is washed three times；
(4) permeabilization: 0.2%Triton X-100 permeabilization 10min, PBS are washed three times；
(5) it closes: at room temperature, being added permeabilization Block buffer (1%BSA+0.1%TritonX-100), be incubated for 30min；
(6) primary antibody is incubated for: the primary antibody diluted is added, is placed in 4 DEG C of wet box overnight incubations, PBST is rinsed 3 times；
* primary antibody used in this part are as follows: the how anti-Integrin- α 6 of rat anti-human, rabbit-anti people's monoclonal antibody CD71；
(7) secondary antibody is incubated for: being protected from light, fluorescein-labeled the two of the dilution in proportion (l:500) in corresponding primary antibody source are added
It is anti-, it is incubated at room temperature 1h, PBST is rinsed 3 times, each 5min；
* secondary antibody used in this part are as follows: the goat anti-rat IgG of Alexa Fluor 594 (ab150160) label；
The goat anti-rabbit igg of Alex Fluor 488 (ab150081) label；
(8) it contaminates core and mounting: being incubated for 10min, anti-fluorescence quencher mounting with 0.2%DAPI, and shown in laser co-focusing
Micro- microscopic observation is simultaneously taken pictures；
(9) it dyes post analysis: fluorescence results being analyzed using ImageJ software: counting the cell of stained positive and divide
Analyse fluorescence intensity.6 positive signal of Integrin- α is red, is mainly expressed by ESCs, and CD71 positive signal is green；DAPI institute
Contaminating blue pointing object is nucleus.
(10) experimental result: as shown in Figure 1.In Fig. 1, Integrin- α 6 and CD71 are for identifying ESCs；DAPI shows 4,6
Diamidino -2-phenylindone；Merge is first three Zhang Ronghe figure, Integrin- α 6bri CD71dim(the i.e. high table of Integrin- α 6
Up to CD71 low expression) prompt ESCs purity is high, illustrate that fibronectin being capable of quick adsorption ESCs.
Experimental example two: the present invention repairs the experimental study of the SD rat skin full-thickness defects surface of a wound for the medicine box of wound repair
1, the separation and extraction of SD rat EBCs, the separation and identification of ESCs: method is the same as experimental example one.
2, the building of SD rat full skin wound model:
(1) Preoperative Method: purchase 6-7 week old male SD rat 90, continuous raising 1 week adapt to experimental situation to rat
And start to test after meeting experiment condition；
(2) experimental group: SD rat is only randomly divided into 3 groups: FN+EBCs treatment group by every group 6, EBCs treatment group is empty
White control group (Control group)；
(3) anaesthetize: rat gives Isoflurane inhalation anesthesia, general weakness, of flaccid muscles, corneal reflection and pain occurs to it
It is tested when phenomena such as pain reaction disappears；
(4) modeling: applying depilatory cream after softly shaving back wool with electric shaver-for women outside, cleaned after acting on 1min with warm water,
It is successively respectively sterilized twice with Iodophor and alcohol, makes the round full thickness skin that a diameter is 2cm every rat back center
Wound Defect is taken pictures and with vernier caliper measurement surface of a wound size.
SD rat full skin wound is as shown in Figure 2.
3, the processing and observation of SD rat full skin wound model:
(1) handle: FN+EBCs treatment group 30min before spraying EBCs first sprays fibronectin antibacterial liquid, EBCs in the surface of a wound
EBCs is directly sprayed in treatment group, and control group uses PBS or ringer's solution, with 3M transparent membrane flap coverage；
(2) it observes and records: attaching the outer auxiliary material with the surface of a wound in five time point replacements of 0d, 3d, 7d, 14d, 21d and comment
Estimate each group wound healing situation, with vernier caliper measurement surface of a wound size, records and compare residual wound rate and wound healing time.
After EBCs sprays in FN+EBCs treatment group, observation discovery EBCs suspension is uniformly attached on the surface of a wound, is lost without obvious；
And after EBCs treatment group sprinkling EBCs, part cell suspension flows around.
The wound healing situations of SD rat different time points as shown in Fig. 2, different time points residual wound rate such as Fig. 3 institute
Show, wound healing time is as shown in Figure 4.From the result of Fig. 2~Fig. 4: the wound healing of the SD rat of FN+EBCs treatment group
Speed is significantly faster than EBCs treatment group and blank control group, and by the wound healing situation of 21d each group rat it is found that blank pair
More serious according to the surface of a wound skin contraction of group rat, the surface of a wound of EBCs treatment group rat also shows a degree of contraction, and FN+
The Wound Contraction lesser extent of EBCs treatment group rat, no cicatricial tissue are formed, and healing quality significantly improves, and complication is reduced.
4, materials and sample preparation:
(1) it draws materials: putting to death rat in 3d, 7d, 14d, 21d and take the surface of a wound together with the edge tissues of 0.5cm around, will create
Face sample is cut into two parts from centre；
(2) liquid nitrogen cryopreservation: a copy of it surface of a wound sample is cut into two parts from centre again, is wrapped immediately with masking foil respectively
It is placed in liquid nitrogen, is respectively used to real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qRT-PCR) detection；
(3) paraffin section: being laid in Riker mount for another surface of a wound side downward and be placed into 4%PFA, with full-automatic tissue
Dewaterer carries out sequencing dehydration to it, then routine paraffin wax embedding is cut into the serial section of 5 μ m-thicks with paraffin slicing machine
Sample is fixed on anticreep glass slide, after drying after be stored at room temperature.
5, H&E is dyed:
(1) dewaxing and aquation: paraffin section is placed in 60 DEG C of oven 1h, is then placed in dimethylbenzene and impregnates twice, every time
5min；Paraffin section is placed in slide holding frame, is successively immersed in 100%, 95%, 85%, 70% ethyl alcohol dye vat, every time
3min finally embathes 5min with distilled water；
(2) haematoxylin dyeing: slice being placed in haematoxylin dye liquor and disseminates 20min, and distilled water flushing slice is anti-blue；
(3) differentiation is faded: by the 10s that fades in slice 1% ethanol solution hydrochloride of merging, distilled water flushing slice is anti-blue；
(4) Yihong is redyed: will disseminate 2min in the eosin stain of slice merging 0.5%；
(5) it is dehydrated transparent: slice being successively placed in 80%, 90%, 95%, each 3min in 100% ethyl alcohol and dimethylbenzene.
(6) mounting: slice uses resinene mounting after air-drying immediately.
3d, 7d and 21d are treated, the H&E coloration result of the SD rat surface of a wound is as shown in Fig. 5~Fig. 7.By Fig. 5~Fig. 7
Result it is found that the healing speed of the SD rat of FN+EBCs treatment group is dramatically speeded up, epidermal thickness increases, epidermal ridge number
Mesh increases, and wound healing quality significantly improves.
6, immunohistochemical staining:
(1) dewaxing and aquation: method is dyed with H&E；
(2) endogenous peroxydase is removed: by 10min in slice 3% hydrogenperoxide steam generator of merging, to reduce false positive
And background stainings；
(3) antigen retrieval: slice being immersed in the dye vat for filling sodium citrate buffer solution, and dye vat is placed in steam copper,
95 DEG C of effect 30min take out cooled to room temperature；
(4) it draws a circle: drying slice, and iris out the scope of organization with immunohistochemistry pen；
(5) permeabilization is closed: slice is placed in wet box, dropwise addition permeabilization confining liquid (2%BSA, 5% lowlenthal serum, 0.2%
TritonX-100), it is incubated for 30min at room temperature；
(6) primary antibody is incubated for: drawing confining liquid with filter paper, the good primary antibody of proper proportion dilution (1:100), 4 DEG C of incubations are added dropwise
Overnight.Primary antibody involved in this part are as follows: small anti-rat CD31 monoclonal antibody, PBST embathe 3 times, each 5min；
(7) secondary antibody is incubated for: the secondary antibody working solution for diluting (1:500) in proportion being added according to primary antibody source, is incubated at room temperature
1h；
(8) DAB is dyed: the colour developing working solution in DAB kit is added dropwise, at room temperature in being protected from light incubation 5min in wet box, goes
Ionized water embathes slice color development stopping reaction；
(9) redye: haematoxylin solution redyes 5min, and distilled water embathes 5min；
(10) dehydration, transparent and mounting: method is dyed with H&E.
7d, 14d are treated, the CD31 immunohistochemical staining result of the SD rat surface of a wound is as shown in Fig. 8~Figure 10.There is Fig. 8
The result of~Figure 10 is it is found that treat 7d and 14d, and new vessels are compared with EBCs in the surface of a wound of the SD rat of FN+EBCs treatment group
Treatment group and blank control group significantly increase.
7, immunofluorescence dyeing:
(1) dewaxing, aquation, antigen retrieval, draw a circle, permeabilization closing: the same immunohistochemical staining of method；
(2) primary antibody is incubated for: drawing confining liquid with filter paper, the primary antibody that proper proportion dilutes (1:250), 4 DEG C of incubations are added dropwise
Overnight.Primary antibody involved in this part are as follows: small anti-rat monoclonal antibody K15, rabbit-anti rat monoclonal antibody Ki67, PBST embathe 3 times, every time
5min；
(3) secondary antibody is incubated for: the secondary antibody working solution for diluting (l:500) in proportion being added according to primary antibody source, is incubated at room temperature
1h.Secondary antibody involved in this part are as follows: the goat anti-mouse IgG of Alexa Fluor 594 (ab150116) label；Alex
The goat anti-rabbit igg of Fluor 488 (ab150077) label；
(4) it contaminates core: 10min being dyed with 1:1000DAPI, PBST embathes 3 times, each 5min；
(5) it is dehydrated and transparent: the same immunohistochemical staining of method；
(6) slide, anti-fluorescence quencher mounting mounting: are dried with blotting paper.
7d is treated, the immunofluorescence dyeing result of the SD rat surface of a wound is as shown in Figure 11~Figure 14.In Figure 11, K15 is used for
ESCs is detected, Ki67 is for detecting proliferative cell, K15briKi67briIndicate that ESCs proliferative capacity is high；DAPI shows 4,6 diamidinos-
2-phenylindone；Merge is first three Zhang Ronghe figure.By the result of Figure 11~Figure 14 it is found that after treatment 7d, FN+EBCs treatment group
SD rat the surface of a wound in ESCs significantly increase and the proliferative capacity of ESCs significantly increases.
8, qRT-PCR is detected:
(1) total serum IgE is separated from the skin samples of rat by Trizol reagent, and uses PrimeScript RT reagent
Box is transcribed into cDNA to specifications；
(2) qRT-PCR reaction is carried out with SYBR Premix ExTaq, and is detected by Mx3000P real-time PCR system；
(3) specificity of each qRT-PCR reaction is verified by gene solubility curve and amplification curve；GAPDH gene is used
Make the quantitative house-keeping gene of mRNA；Pass through 2-ΔΔCT method calculates the relative expression ratio of mRNA, and primer sequence is shown in Table 1.
1 qRT-PCR primer sequence of table
As a result as shown in Figure 15~Figure 17.By the result of Figure 15~Figure 17 it is found that treating 7d and 14d, FN+EBCs is controlled
CD31 expression significantly increases in the surface of a wound of the SD rat for the treatment of group, and after treating 7d, the expression of Ki67 and K15 are also significantly improved, and say
Bright FN+EBCs treatment peomotes angiogenesis and ESCs proliferation, to peomote wound healing and improve healing matter
Amount.
Above-described is only some embodiments of the present invention.For those of ordinary skill in the art, not
Under the premise of being detached from the invention design, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.
Claims (9)
1. being used for the medicine box of wound repair characterized by comprising self-existent by fibronectin and pharmaceutically acceptable
Fibronectin external preparation and epidermal basal cell suspension made of auxiliary material.
2. the medicine box according to claim 1 for wound repair, which is characterized in that the epidermal basal cell suspension by
Following methods are made:
(1) skin graft is digested with TrypLE Select digestive juice；
(2) it is rinsed skin graft with Lactated Ringer'S Solution and is terminated and digested, separated epidermis and corium, then scrape table from basal layer of epidermis
Scytoblastema floor cells are diluted with Lactated Ringer'S Solution after collecting cell, obtain cell suspension；
(3) cell suspension is purified to get epidermal basal cell suspension.
3. the medicine box according to claim 2 for wound repair, which is characterized in that in the step (1), by skin graft in
Disappeared under the conditions of 37 DEG C with 5~15 times of the TrypLE Select digestive juice that concentration is 1 × TrypLE Select digestive juice concentration
Change 10~20min.
4. the medicine box according to claim 2 or 3 for wound repair, which is characterized in that in the step (3), to thin
The step of born of the same parents' suspension purifies includes: to be filtered with 100 μm of cell strainers.
5. the medicine box according to claim 4 for wound repair, which is characterized in that in the step (1), the skin graft
For Razor thin skin.
6. the medicine box according to claim 1 for wound repair, which is characterized in that the surface of a wound be selected from burn, scald,
The surface of a wound that diabetic ulcer, scar excision, mole excision, subcutaneous Tumor resection are formed.
7. the preparation method of the medicine box according to claim 1 for wound repair, which is characterized in that will be by fibronectin
And fibronectin external preparation and epidermal basal cell suspension made of pharmaceutically acceptable auxiliary material are distributed into medicine box；Wherein institute
The preparation method for stating epidermal basal cell suspension includes the following steps:
(1) skin graft is digested with TrypLE Select digestive juice；
(2) it is rinsed skin graft with Lactated Ringer'S Solution and is terminated and digested, separated epidermis and corium, then scrape table from basal layer of epidermis
Scytoblastema floor cells are diluted with Lactated Ringer'S Solution after collecting cell, obtain cell suspension；
(3) cell suspension is purified to get epidermal basal cell suspension.
8. the preparation method of the medicine box according to claim 7 for wound repair, which is characterized in that the step (1)
In, it is 5~15 times of TrypLE of 1 × TrypLE Select digestive juice concentration that skin graft is used to concentration under the conditions of 37 DEG C
Select digestive juice digests 10~20min.
9. the preparation method of the medicine box according to claim 7 for wound repair, which is characterized in that the step (3)
In, the step of purifying to cell suspension includes: to be filtered with 100 μm of cell strainers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910636265.3A CN110339060A (en) | 2019-07-15 | 2019-07-15 | A kind of medicine box and preparation method thereof for wound repair |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910636265.3A CN110339060A (en) | 2019-07-15 | 2019-07-15 | A kind of medicine box and preparation method thereof for wound repair |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110339060A true CN110339060A (en) | 2019-10-18 |
Family
ID=68176339
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910636265.3A Pending CN110339060A (en) | 2019-07-15 | 2019-07-15 | A kind of medicine box and preparation method thereof for wound repair |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110339060A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102488930A (en) * | 2011-12-15 | 2012-06-13 | 无锡市第三人民医院 | Method for preparing auto and allo-epidermal cell mixed suspension |
| CN104013999A (en) * | 2014-06-11 | 2014-09-03 | 朱家源 | Tissue engineering skin and preparation method thereof |
| CN104768586A (en) * | 2012-09-04 | 2015-07-08 | 人类起源公司 | Methods of tissue generation |
| WO2018106536A1 (en) * | 2016-12-06 | 2018-06-14 | The Regents Of The University Of California | Methods for making and using dedifferentiated and stem-like human cells |
-
2019
- 2019-07-15 CN CN201910636265.3A patent/CN110339060A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102488930A (en) * | 2011-12-15 | 2012-06-13 | 无锡市第三人民医院 | Method for preparing auto and allo-epidermal cell mixed suspension |
| CN104768586A (en) * | 2012-09-04 | 2015-07-08 | 人类起源公司 | Methods of tissue generation |
| CN104013999A (en) * | 2014-06-11 | 2014-09-03 | 朱家源 | Tissue engineering skin and preparation method thereof |
| WO2018106536A1 (en) * | 2016-12-06 | 2018-06-14 | The Regents Of The University Of California | Methods for making and using dedifferentiated and stem-like human cells |
Non-Patent Citations (1)
| Title |
|---|
| HU ZC 等: "Randomized clinical trial of autologous skin cell suspension combined with skin grafting for chronic wounds", 《BR J SURG》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6469655B2 (en) | Method of skin substitute and hair follicle neoplasia | |
| JP5689023B2 (en) | Surgical device for skin treatment or examination | |
| Ehrlich | Understanding experimental biology of skin equivalent: from laboratory to clinical use in patients with burns and chronic wounds | |
| CN101856517B (en) | Tissue engineering material-based culture method and applications of melanophore | |
| CN107812014A (en) | The fibroblastic dosage unit formulations of auto derma | |
| CN105176914A (en) | Simultaneous autologous epidermic cell and fibrocyte preparation method and biological cosmetic product thereof | |
| CN105597148B (en) | A kind of Nerve Scaffold, preparation method and application for repairing of neural injury | |
| JPH02174848A (en) | Cuticle sheet;and production, storage and usage thereof | |
| CN108057116A (en) | Application of the stem cell composition in skin injury medicine | |
| CN113462632B (en) | Balsam pear exosome, extraction method and application thereof in preparation of medicines for treating burns and scalds | |
| CN102631706B (en) | Method for preparing low-immunogenicity pig dermal support | |
| Liu et al. | Reconstruction of a tissue‐engineered skin containing melanocytes | |
| CN104611289A (en) | Method for simultaneously preparing autologous epidermal cells and fibroblasts, and biological beauty product comprising autologous epidermal cells and fibroblasts | |
| CN107683148B (en) | Skin reconstruction method | |
| CN103550828A (en) | Skin renewal method based on hair follicle stem cells and silica gel dressing | |
| CN108066750A (en) | Stem cell and its secretion are used to treat the new application of skin burn | |
| CN103054628A (en) | Method for fast constructing and repairing skin defects by tissue engineering skins | |
| CN110339060A (en) | A kind of medicine box and preparation method thereof for wound repair | |
| CN108066824A (en) | A kind of new method for preparing skin blemish medicine | |
| CN110241073B (en) | Method for rapidly separating and extracting epidermal stem cells | |
| CN109646459A (en) | A kind of water optoinjection instrument injection umbilical cord mesenchymal stem cells preparation and its application | |
| CN105396136B (en) | CCN1（Cyr61）Application in treatment skin injury and atrophoderma relevant disease | |
| CN108542915A (en) | A kind of drug and preparation method thereof promoting wound healing | |
| CN108057131A (en) | A kind of novel agent box containing stem cell | |
| CN105087482B (en) | A kind of cell culture substrate and its application and application method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200930 Address after: Floor 3, No.69, Gongye Road, Shiqiao street, Panyu District, Guangzhou City, Guangdong Province Applicant after: Guangzhou Maishitian Medical Technology Co.,Ltd. Address before: 510080 No. two, No. 58, Zhongshan Road, Guangdong, Guangzhou Applicant before: Zhu Jiayuan |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191018 |
|
| RJ01 | Rejection of invention patent application after publication |