CN108795844A - Fibroblastic preparation method and products thereof for cicatrix of skin reparation - Google Patents
Fibroblastic preparation method and products thereof for cicatrix of skin reparation Download PDFInfo
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- CN108795844A CN108795844A CN201810634895.2A CN201810634895A CN108795844A CN 108795844 A CN108795844 A CN 108795844A CN 201810634895 A CN201810634895 A CN 201810634895A CN 108795844 A CN108795844 A CN 108795844A
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- fibroblast
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- skin
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- cicatrix
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- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of fibroblastic preparation method for cicatrix of skin reparation, wherein, the method includes fibroblast is extracted from tissue, fibroblast is obtained by containing fibroblast growth factor, vitamin E, phosphoric acid -3-O- rutinosides and Pfansteihl culture, the fibroblast of preparation has good biological activity, fracture fibr tissue is repaired, the collagen structure of skin base layer is restored, while inhibiting fibrosis and cicatrization caused by excess collagen expression；Fibroblast provided by the invention will have cicatrix of skin treatment and cosmetic result very well.
Description
Technical field
It is obtained the present invention relates to a kind of fibroblastic method for cicatrix of skin reparation and by the method
The fibroblast of cicatrix of skin is treated, particularly, the present invention relates to obtaining fibroblast by vitro culture from tissue,
It is broken fibr tissue with repairing, restores the collagen structure of skin base layer；Excess collagen expression is inhibited to make simultaneously
At fibrosis and cicatrization, will be expected to cicatrix of skin reach more preferably treatment and cosmetic result.
Background technology
Mode of appearance and tissue pathologies change of the cicatrix of skin for normal skin tissue caused after various wounds, meat
Bud tissue is through the ripe fibrous connective tissue formed of reconstruction.Body tissue hyperplasia, connective tissue slightly increase after skin injury
Cicatrization caused by the raw or slight hyperplasia reason of connective tissue.At present for the more serious depressed scar of degree, atrophy
Property scar and hyperplastic scar, operation excision is main means, but any modus operandi cannot all completely remove scar, only
It is endangered caused by improvement or correcting scar to greatest extent, and new scar is also preferably formed after performing the operation.For the scar of lesser extent
Trace patient, laser, freezing or radiotherapy are more commonly used means, and effect is relatively good, but pigmentation and concurrent easily occur
The problem of disease.
The development of biotechnology also provides new means for cicatrix of skin treatment, and such as research finds extrasin beta 4 in scar
It expresses and reduces in pimple tissue, its medicine can be used as；Scar containing active factors, which repairs gel, has anti-fibre
Dimension mother cell hyperplasia, anti-inflammatory and softening, the effect of cicatricial tissue and agate card are dispelled scar essence glue and containing some active factors
Agate card dispel scar essence have prevent damaged skin pigment generate variation, restore the normal skin tone and elasticity of skin.These hands
Duan Jun plays good effect to cicatrix of skin treatment.But for the true cause that cicatrix of skin is formed, inhibit collagen mistake
Degree expression and fibrosis of skin are to treat its crucial problem.
The present invention is protein expression vector and the function of itself using fibroblast, is had very to cicatrix of skin treatment
Good effect.Fibroblast (Fibroblast, FB) has been used to clinical treatment field, is achieved in dermoplastic treatment
Fine curative effect, the limitation of the immunological rejection being not present, ethics and oncogenicity etc., before there is good clinical application
Scape.Thus, the present invention obtains fibroblast using Vitro Culture Techniques, has and repairs fracture fibr tissue, restores skin base
The collagen structure of bottom；Fibrosis and cicatrization caused by inhibiting excess collagen to express simultaneously, have no Tumor formation,
It is expected to generate better effect to cicatrix of skin reparation and beauty.
Invention content
The applicant passes through synthesis and degradation imbalance the study found that as the collagen in cicatrix of skin forming process, glue
The result of former over-deposit；So that collagen overexpression, trauma skin fibrosis and cicatrization.
The present invention is broken up using fibroblast proliferation and the characteristic of " going back to the nest ", autologous fibroblasts can repair fracture
Fibr tissue restores the collagen structure of skin base layer；Simultaneously its secrete cytokines will inhibit collagen expression and
The biological action of fibrosis and its function of fibroblast itself improve local skin microenvironment,
Inventor has been surprisingly found that by being added to phosphoric acid -3-O- rutinosides and Pfansteihl culture at fiber finer
Born of the same parents' proliferative capacity has to be promoted very well, while also inhibiting transforming growth factor (transforming growth significantly
Factor- β, TGF-β) expression, it is most close to be excessively formed relationship with scar, to fibroblast in cicatricial tissue into
Enter in impaired fibroblast, collagen expression and scar is inhibited to be excessively formed, the inhibition that pathologic scar is formed is produced
Significant effect is given birth to.
The present invention provides a kind of fibroblastic preparation methods for cicatrix of skin reparation, which is characterized in that institute
The method of stating includes the fibroblast of tissue extraction, by containing fibroblast growth factor, vitamin E, phosphoric acid -3-O- rutinosides
Under the condition of culture of Pfansteihl, improve fibroblast play more preferably inhibit collagen over-expression expression, prevent at
Fibrocyte fibrosis and improvement fracture fibr tissue reparation, can be used to treat cicatrix of skin.
The fibroblast detects through immunofluorescence technique, marker α smooth muscle actins, I procollagen types
Protein alpha 1 and 90% or more the expression of type III procollagen α 1.
The FBs has following performance：Inhibit cicatricial tissue Collagen of Fibroblasts protein overexpression and fibrosis, suppression
Inflammatory reaction processed repairs fracture fibr tissue, restores the collagen structure of skin base layer；And without tumor.
Fibroblast of the present invention for cicatrix of skin reparation can pass through nothing with subculture in vitro separately culture, i.e. FBs
Serum free culture system liquid (1-500ng/ml fibroblast growth factors, 1-200ng/ml vitamin Es, 0.1-500 μ g/ml phosphoric acid -3-O- rues
Fragrant glucosides, 1-500 μ g/ml Pfansteihls and 1 × serum-free additive) subculture in vitro separately culture, when cell culture was by the 5-6 days, carefully
Intracellular growth fusion reaches 90% or more, and pancreatin (containing 0.05%EDTA) digestion that use quality volume ratio is 0.25% is needed to pass
Generation.
The cellular morphology observed under secondary culture to 3 generations and 10 generation FBs inverted microscopes is taken, there is similar cell shape
State.
The fibroblast for cicatrix of skin reparation externally with fibroblasts from hyperplastic scar co-culture experiments
In inhibit Proliferation of Hypertrophic Scar Fibroblasts and collage synthesis and α-SMA and I-type collagen to express, promote it
Apoptosis occurs；In rabbit scar model body in transplantation experiments, with individual Allogenic Cultured Dermal Fibroblasts Transplantation and saline control group phase
Than inhibiting I-type collagen and the secretion of the TGF-β 1 of 1 gene expression of TGF-β and inflammatory factor, having to cicatrix of skin aobvious
It writes and repairs；Internal experiment in vitro shows without tumor.
The present invention also provides a kind of fibroblastic reagent kit product for cicatrix of skin reparation prepared, features
It is, the kit includes：
1) fibroblast serum free medium；
2) II Collagenase Types；
3) pancreatin；
4) fibroblast growth factor, vitamin E, phosphoric acid -3-O- rutinosides, Pfansteihl；
5) serum-free additive；
6) operation instructions；
The present invention also provides fibroblastic applications for cicatrix of skin reparation, by ensure that in vitro culture
Sufficient amount of fibroblast is obtained, fracture fibr tissue is repaired, restores the function of the collagen structure of skin base layer,
It is played simultaneously to cutaneous scar tissue Collagen of Fibroblasts protein overexpression and fibrosis, it is expected to be used for treatment skin
Scar.
Description of the drawings
Fig. 1 is that human fibroblasts Immunofluorescence test α-SMA, COLI α 1 prepared by embodiment 1 and COLIII α 1 is expressed
It is horizontal
Fig. 2 is the fibroblast of the preparation of embodiment 1 to Proliferation of Hypertrophic Scar Fibroblasts level view
Fig. 3 is the fibroblast of the preparation of embodiment 1 to fibroblasts from hyperplastic scar collage synthesis result figure
Fig. 4 is that rabbit fibroblast prepared by 1 method of embodiment transplants the secretion of TGF-β 1 water in rabbit ear scar model serum
Flat figure
Specific implementation mode
The present invention provides a kind of fibroblastic preparation methods for cicatrix of skin reparation, which is characterized in that from
The fibroblast extracted in tissue, by containing fibroblast growth factor, vitamin E, phosphoric acid -3-O- rutinosides, Pfansteihl
Culture obtains fibroblast, can repair fracture fibr tissue, restore the collagen structure of skin base layer, inhibit simultaneously
Fibrosis and cicatrization caused by excess collagen is expressed, can be used to treat cicatrix of skin.
Fibroblast convenient sources of the present invention, i.e., discarded skin, blood and hair follicle；It is preferably derived from skin
Skin.Due to fibroblast convenient sources, cell culture obtains simply, and can be obtained from individuation can accomplish individuation application.
Fibroblast preparation method of the present invention, i.e.,：Discarded skin is passed through containing 1 dual anti-× PBS of 2 times of mycillins
(pH7.4) washing uses the II Collagenase Type 5-20mL that 1: 1 mass volume ratio is 0.1% afterwards three times, disappears in 4 DEG C of refrigerator casees
Change overnight；It is that 0.25% pancreatin digests 4 hours in 37 DEG C of incubators that dermis layer tissue mass volume ratio is taken after digestion, after digestion
1500rpm is centrifuged 10 minutes, and physiological saline resuspension is added after abandoning supernatant in suction, is filtered with 100 μm of cell sieves；Cell suspension
1000rpm is centrifuged 10 minutes, and cell precipitation is added complete serum-free medium after being washed 2 times with physiological saline resuspension and (contains 1-
500ng/ml fibroblast growth factors, 1-200ng/ml vitamin Es, 0.1-500 μ g/ml phosphoric acid -3-O- rutinosides, 1-500
μ g/ml Pfansteihls and 1 × serum-free additive) it is resuspended, 1-10 × 10 are pressed after tongue disk orchid dyeing counting6Cell/ml is added to
It is cultivated in the coated tissue culture plate of recombinant human fibronectin polypeptide or culture dish, in 37 DEG C, 5%CO2It is small that 24-72 is cultivated in incubator
When；
Culture, which is inhaled after 24-72 hours with 5ml pipettes, abandons culture solution, and fresh serum free culture solution is added and carries out changing liquid, carefully
Born of the same parents' culture plate or culture dish continue to place 37 DEG C, 5%CO2Continue to cultivate in incubator；
Digestion is carried out when fibroblastic growth fusion reaches 80% or more and expands bottle, and culture solution is absorbed, takes the pH value to be
7.4 1 × PBS is added after washed once in 0.25% pancreatin to culture bottle of 0.5ml, and 200 μ l tires are added after 2-3 minutes in digestion
Cow's serum terminates digestion, adds physiological saline, is blown and beaten, is drawn in 15ml centrifuge tubes, then use physiological saline with 5mL pipettes
It washed once, be added in the same 15mL centrifuge tubes, 1000 rpms of centrifugations collect fibroblast in 10 minutes；After centrifugation
It is resuspended, is counted with 1mL fresh mediums, the fresh serum-free mediums of 10mL are added with 5mL pipettes, are added to 1 bottle of fibre even
Continue in the coated culture bottle of albumen at 37 DEG C, 5%CO2It is cultivated in incubator, a fresh medium was changed every 2 days；It waits for into
When fibroblast growth fusion reaches 80% or more, by above-mentioned through collected by trypsinisation fibroblast, continuous passage 3 is commissioned to train
It supports, obtains 109Above fibroblast, is resuspended with 4ml physiological saline, tongue disk orchid dyeing counting obtains largely into fiber
Cell saves backup under the conditions of placing 4 DEG C；
Part fibroblast forms in 40% Fibroblast culture solution, 10% dimethyl sulfoxide (DMSO) and 50% fetal calf serum
Frozen stock solution be stored in -196 DEG C of liquid nitrogen containers, in case from now on recover after use.
Fibroblast detects through immunofluorescence technique, marker α smooth muscle actins, I procollagen types protein alpha 1
It is almost 100% with the expression of type III procollagen α 1.
The cellular morphology observed under secondary culture to 3 generations and 10 generation FBs inverted microscopes is taken, is grown at fusiformis.
Serum-free medium of the present invention is commercially available, including BIOWIT serum free mediums,
OriCellTMSerum free medium, StemXVivo serum free mediums andSerum free medium etc., more preferably
Use OriCellTMSerum free medium.
The present invention externally co-cultures with fibroblasts from hyperplastic scar real for the fibroblast of cicatrix of skin reparation
It inhibits Proliferation of Hypertrophic Scar Fibroblasts and collage synthesis and α-SMA and I-type collagen to express in testing, promotes
Its apoptosis occurs.
The present invention for cicatrix of skin reparation fibroblast in rabbit scar model body in transplantation experiments, and it is individual
Allogenic Cultured Dermal Fibroblasts Transplantation is compared with saline control group, inhibit I-type collagen and 1 gene expression of TGF-β and inflammation because
The TGF-β 1 of son is secreted, and is had to cicatrix of skin and is significantly repaired；Internal experiment in vitro shows without tumor.
The present invention also provides a kind of fibroblastic reagent kit product for cicatrix of skin reparation prepared, features
It is, the kit includes：
1) 500ml fibroblasts serum free medium；
2)104IU/mL II Collagenase Types；
3) 0.25% pancreatin of 100ml；
4) 50 times of active compounds of 10mL：50ng-5 μ g fibroblast growth factors, 25ng-5 μ g vitamin Es, 50 μ g-
250mg phosphoric acid -3-O- rutinosides, 500 μ g-250mg Pfansteihls；
5) 50 times of serum-free additives of 10mL；
6) operation instructions；
Wherein, the operation instructions include above-mentioned method.
The present invention also provides fibroblastic applications for cicatrix of skin reparation, by ensure that in vitro culture
Sufficient amount of fibroblast is obtained, has and repairs fracture fibr tissue and inhibit inflammatory reaction, restore skin base layer
The function of collagen structure；Fibrosis and cicatrization caused by inhibiting excess collagen expression are expected to be used for treatment skin
Skin spot.
Hereinafter, specific embodiments of the present invention are illustrated, but the technical scope of the present invention is not limited to these examples.
The preparation of 1 human fibroblasts of embodiment
Discarded skin uses 1 after 1 × PBS (pH7.4) washings three times dual anti-containing 2 times of mycillins：1 mass body
Product digests overnight than II Collagenase Types (Sigma Co., USA's purchase) 5-20mL for 0.1% in 4 DEG C of refrigerator casees；After digestion
It is 0.25% pancreatin (containing mass volume ratio 0.02%EDTA) (U.S. Thermo Fisher to take dermis layer tissue mass volume ratio
Company buys) it is digested 4 hours in 37 DEG C of incubators, 1500rpm is centrifuged 10 minutes after digestion, and physiology salt is added after abandoning supernatant in suction
Water is resuspended, and is filtered with 100 μm of cell sieves；Cell suspension 1000rpm is centrifuged 10 minutes, and cell precipitation is resuspended with physiological saline and is washed
Fibroblast serum-free medium (fibroblast growth factor containing 1ng-100ng/ml, the purchase of R&D companies of the U.S. are added after 2 times
It buys；0.5ng-100ng/ml vitamin Es, Sigma Co., USA's purchase；10-200 μ g/ml phosphoric acid -3-O- rutinosides (Kang Long
Chemical conversion synthesis and identification)；10-200 μ g/ml Pfansteihls, Sigma Co., USA's purchase；1 × serum-free additive, the U.S.
Thermo Fisher companies buy) it is resuspended, 1-10 × 10 are pressed after tongue disk orchid dyeing counting6Cell/ml is added to recombined human fibre
It is even cultivated in the coated tissue culture plate of albumen or culture dish, in 37 DEG C, 5%CO2It is cultivated 48-72 hours in cell incubator.
The fibroblast culture medium for replacing fresh factor-containing is continuously cultivated again, until cell adherent growth comes out.
When cell growth is closeer, digested using 0.25% (mass volume ratio) pancreatin, with 10ml containing cell because
The fibroblast culture medium of son is transferred to the coated 75cm of new recombinant human fibronectin polypeptide after being resuspended2In culture bottle, in 37 DEG C,
5%CO2Culture is merged until growth in cell incubator reaches 90% or so, and 0.25% pancreatin is used to carry out had digestive transfer culture training
It supports, i.e., 1 bottle passes on 2 bottles, and the fibroblast culture medium for supplementing fresh factor-containing continues to cultivate, when cultivating to 5 generation, at
Fibrocyte is digested using 0.25% pancreatin, that is, is obtained fibroblast, can be resuspended to the tire ox containing 10% dimethyl sulfoxide (DMSO)
It is frozen in liquid nitrogen in serum.
Tongue is taken to expect 1.0 × 10 after blue dyeing counting5For FBs cells kind in the burnt special culture dish of copolymerization, ice PBS washes three
Time, 5 minutes every time；When cell is half-dried, covering fixes 15 minutes with 4% cold paraformaldehyde, is protected from light；After sucking paraformaldehyde,
It is washed three times, every time 5 minutes with 1 × PBS of ice；0.5%Triton X-100 covering cell 10 minutes, 1 × PBS of ice wash three times, often
Secondary 5 minutes；Import fetal calf serum (Fems bovine serum, FBS) room temperature of host identical as secondary antibody is closed 30 minutes；Match
Primary antibody processed dilutes 200 times, i.e. rabbit-anti people α smooth muscle actins (α-Smooth muscle actin, α-SMA), I types with FBS
Procollagen α 1 (type I Collagen alpha-1, COL1 α 1) and 1 (type III of type III procollagen α
Collagen alpha-1, COLIII α 1) it is mostly anti-；It is separately added into primary antibody covering cell, tinfoil wraps up 4 DEG C and is protected from light overnight.It takes
Go out cell and warms to room temperature about 1h again；1 ‰ Tween of ice is washed twice, 5 minutes every time, in shaking table.1 × PBS of ice is washed once, 5 minutes,
In shaking table；Prepare fluorescent marker secondary antibody：Ab50598 Goat anti-rabbit IgG (H&L) TRITC is prepared with 1 × PBS,
Concentration 1: 200；Secondary antibody is added, is incubated at room temperature 1 hour (being protected from light).1 ‰ Tween of ice is washed twice, 5 minutes every time, in shaking table.Ice 1
× PBS is washed once, 5 minutes, in shaking table.DAPI contaminates core, is dripped per ware 1, covers all cell.1 ‰ Tween of ice washes two
It is secondary, 5 minutes every time, in shaking table.1 × PBS of ice is washed once, 5 minutes, in shaking table.Anti- fluorescent quenching mountant is added, is protected from light.Carefully
Born of the same parents' machine testing in confocal fluorescent microscopic (German Lycra company).
For Fig. 1 the results show that human fibroblasts are through above-mentioned Immunofluorescence test, A is α-SMA in confocal fluorescent microscopic
Under the fluorescent red-orange observed, be almost 100% with its fluorescence signal is compared under light field, show fibroblast express alpha-
SMA positive rates are almost 100%；B and C is respectively the orange that COLI α 1 and COLIII α 1 are observed under confocal fluorescent microscopic
Red fluorescence and DAPI dye core blue lights merge picture, are almost 100% with its fluorescence signal is compared under light field, show into fiber
It is almost 100% that cell, which expresses COLI α 1 and 1 positive rates of COLIII α also,.
Fibroblastic reagent kit product for cicatrix of skin reparation prepared by embodiment 2
Fibroblastic kit for cicatrix of skin reparation includes：
1) 500ml fibroblasts serum free medium；
2)104IU/mL II Collagenase Types；
3) 0.25% pancreatin of 100ml；
4) 50 times of active compounds of 10mL：50ng-5 μ g fibroblast growth factors, 25ng-5 μ g vitamin Es, 5mg-
100mg phosphoric acid -3-O- rutinosides, 5mg-100mg Pfansteihls；
5) 50 times of serum-free additives of 10mL；
6) operation instructions；
Wherein, the operation instructions include the method described in embodiment 1-3.
3 human fibroblasts co culture system in vitro Inhibiting proliferation scar fibroblast proliferation of embodiment is tested
The fibroblasts from hyperplastic scar (being bought from U.S. ScienCell) of logarithmic growth phase is with 3 × 105Cell/
Hole concentration is inoculated in 6 hole Transwell culture plates (0.3 μm, Corning companies of the U.S.), the training of UltraCULTURETM serum-frees
It supports, 1.5mL is inoculated with per hole, human fibroblasts, 3 × 105 cells/room are added in cell, and 1.5ml sets 37 DEG C, 5%CO2 incubators
It co-cultures, is grouped.Blank control A groups：It is added without fibroblast in cell and co-cultures (Control), multiple holes 6；It is real
Apply the fibroblast experiment B groups of the preparation of example 1：Normal fibroblast (FB) is added in cell to co-culture, multiple holes 6；And
(comparative example fibroblast is existed the fibroblast control C groups of comparative example culture using Abdian N etc.《Cell tissue bank》
What in October, 2015 delivered《The fibroblasts of adult human dermis growth speed in addition fibroblast growth factor and the culture medium not added
The comparison of rate》Method carry out culture preparation, i.e. Abdian N, Ghasemi-Dehkordi P, Hashemzadeh-
Chaleshtori M, et al.Comparison of human dermal fibroblasts (HDFs) growth rate
in culture media supplemented with or without basic fibroblast growth factor
(bFGF).Cell Tissue Bank.2015 Dec；16(4)：487-95.)：Normal fibroblast (FB) is added in cell
It co-cultures, multiple holes 6；In vitro culture 1-7 days is observed under inverted microscope, takes a picture.
The cytochrome oxidase isozymes of blank control are good, directional, radial.And normal fibroblast experiment B groups and C groups
There is the reduction of fibroblasts from hyperplastic scar number, protrusion shortens, and shape becomes irregular, arranges aobvious confusion, refractivity
It is weaker.
By U.S.'s Bio-Rad cell counters, according to operation instructions respectively on day 1, the 2nd day, the 3rd day, the 4th
It and the 5th day detection Proliferation of Hypertrophic Scar Fibroblasts are active.It is recognised that blank control A groups and experiment B from Fig. 2
Group, comparison C groups are compared, and Proliferation of Hypertrophic Scar Fibroblasts speed faster, between the two and has significant difference (* * P
=0.011)；And test B groups and compared with comparison C groups, Proliferation of Hypertrophic Scar Fibroblasts speed is substantially reduced, between the two
And there is significant difference (* P=0.028), show that fibroblast prepared by the present invention inhibits hyperplastic scar at fiber
Cell Proliferation promotes its apoptosis to occur.
4 human fibroblasts co culture system in vitro of embodiment inhibits cell collagen compound experiment
In 3 co-culture experiments of embodiment, each group is taken to the 50 μ L of supernatant of co-cultivation the 1st day per hole, 100 DEG C are dried, then
4mol/L NaOH, 120 DEG C, 10min is added and sequentially adds oronain T, perchloric acid solution, develop the color about 15min at 65-70 DEG C, uses
Beckman DU-600 spectrophotometric colos survey absorbance value, wavelength 550nm；Hydroxyproline colorimetric determination each group is thin
The content of its hydroxyproline in the supernatant of born of the same parents analyzes fibroblasts from hypertrophic scars collage synthesis.
Fig. 3 results are shown：Fibroblast prepared by embodiment 1 has inhibition to make fibroblasts from hypertrophic scars collage synthesis
With FB tests B groups compared with blank control A groups and comparison C groups, and collage synthesis significantly reduces, respectively * * P=0.015 and * P
=0.041；Two groups of experimental data is through examining, and there are statistical significances.
5 rabbit ear cicatrix of skin model skin experiment in vivo of embodiment
24 adult rabbits purchased from Zhongshan University's animal center, half male and half female, 2.0~2.5 kg of weight, rabbit ear
Edge is injected intravenously 3% yellow Jackets (30mg/kg), and performing the operation with the outside of belly of picking up the ears after anesthesia cuts off the complete of a diameter of 10~15mm
Layer skin, strikes off perichondrium.At ear two, each surface of a wound interval 15mm or more, surface of a wound exposure, the 20d after surface of a wound epithelialization, skin
Scar proliferation is formed, and rabbit ear cicatrix of skin model is prepared into.This model continues no less than 3 weeks.
After modeling success, it is randomly divided into tri- groups of A, B and C, every group 6, A groups are to be moved using FBs prepared by 1 method of embodiment
Plant group, B groups are the rabbit FBs transplantation groups (FB) prepared using comparative example method, and skin carries out corium at rabbit ear cicatrix of skin two
Layer is subcutaneously injected 5 × 105Cell, 50 μ l of volume；C groups are saline control group (Control), same to inject 50 μ l physiology
Brine.
Three groups of rabbit modeling ear modeling positions and normal auris dextra are visually observed after the last administration, and take pathological biopsy,
It is fixed with 10% formalin, paraffin embedding, slice, Hematoxylin-eosin (HE) dyeing, using the green skies production in purchase Beijing
Hematoxylin-eosin staining kit is carried out according to operation instructions, and tissues observed changes under light microscope.
Rabbit ear color is pink, poor, soft before modeling, and pathological analysis thereon arrange by visible clearly capillary, follicular orifice
Row are neat, and the 30th day after modeling, the variation of cicatrix of skin animal model local skin pathology is the positive evidence of model success or not,
After model is successfully prepared, skin histology at modulus type, the visible local skin fibroblast proliferation of microscopy, collagenous fibres increase increasing
Slightly, fine and close and disorganized, during which there is a small amount of inflammatory cell.Specific rabbit ear cicatricial tissue variation see the table below 1.
Table 1
Note：Cicatrix of skin local skin pathology variation classification reference standard is "-"：Skin histology is normal, and institutional framework is complete
It is whole；"+"：Fibroblast proliferation, collagenous fibres increase thickening, fine and close and disorganized, during which there is a small amount of inflammatory cell；"+
+"：Fibroblast had significant proliferation, collagenous fibres showed increased thickening is fine and close and disorganized, during which there is inflammatory cell；"++
+"：Fibroblast is largely proliferated, collagenous fibres showed increased thickening, very fine and close and disorganized, during which there is more inflammatory
Cell.
The rabbit FBs transplantation treatment rabbit ear scar models that prepare of the present invention in 1 result of table, after the transfer the 14th Lepus ear scar
Repairing well occurs in trace, i.e., Proliferation of Hypertrophic Scar Fibroblasts is inhibited well, and collagenous fibres substantially reduce change
Carefully, inflammatory cell liquid significantly reduces in tissue.Simultaneously tumor formation phenomenon is not found in each group animal model body.
Rabbit ear scar model uses rabbit FBs experiment in vivo prepared by 1 the method for embodiment, transplants 7 days, 14 days, 30 days
With 60 days after by ear central artery acquire rabbit peripheral blood, by 1000rpm centrifugation obtain serum after ten minutes.Use TGF-β
1 enzyme linked immunosorbent assay (ELISA) (ELISA) kit detects 1 secretion level of TGF-β (being purchased from R&D companies of the U.S.), according to U.S. R&D
1 ELISA kit operation instructions of company's T GF- β are detected.Rabbit FBs transplantation experiments A groups TGF-β 1 is secreted significantly low in Fig. 4
B groups, * P=0.039 are compared in FBs；And the TGF-β 1 that FBs comparisons B component is secreted is horizontal also significantly lower than saline control C
Group, * * P=0.009；Show that FBs prepared by the present invention inhibits the TGF-β 1 of rabbit ear scar model inflammatory factor to secrete, helps
Improve and promote scar repairing in rabbit ear scar inflammatory reaction.
Claims (10)
1. a kind of fibroblastic preparation method for cicatrix of skin reparation, which is characterized in that the method includes tissues
Middle extraction fibroblast is obtained by containing fibroblast growth factor, vitamin E, phosphoric acid -3-O- rutinosides and Pfansteihl culture
Obtain fibroblast.
The fibroblast detects through immunofluorescence technique, marker α smooth muscle actins, I procollagen type protein alphas
90% or more 1 and type III procollagen α 1 expression.
The fibroblast has following performance：Fibrosis and cicatrization caused by inhibiting excess collagen expression, together
When fibroblast repair fracture fibr tissue, restore the collagen structure of skin base layer；And without tumor.
2. according to the method described in claim 1, it is characterized in that, the fibroblast extracted from tissue, passes through 1-
500ng/ml fibroblast growth factors, 1-200ng/ml vitamin Es, 0.1-500 μ g/ml phosphoric acid -3-O- rutinosides and 1-500
Culture obtains fibroblast under the conditions of the serum free medium of μ g/ml Pfansteihls.
3. according to claim 1-2 any one of them methods, which is characterized in that it prepares in fibroblastic condition of culture,
Preferably, 0.1-500 μ g/ml phosphoric acid -3-O- rutinosides and 1-500 μ g/ml Pfansteihls are added.
4. according to the method described in claim 1-3, which is characterized in that prepare in fibroblastic condition of culture, more preferably
Ground adds 10-200 μ g/ml phosphoric acid -3-O- rutinosides and 10-200 μ g/ml Pfansteihls.
5. according to claim 1-4 any one of them methods, which is characterized in that the fibroblast source discard skin,
Blood and hair follicle；It is preferably derived from skin.
6. according to claim 1-5 any one of them methods, which is characterized in that discarded skin passes through dual anti-containing 2 times of mycillins
1 × PBS (pH7.4) washing three times afterwards use 1: 1 mass volume ratio be 0.1% II Collagenase Type 5-20mL, in 4 DEG C of ice
It is digested overnight in case case；It is that 4 are digested in 37 DEG C of incubators is small for 0.25% pancreatin that dermis layer tissue mass volume ratio is taken after digestion
When, 1500rpm is centrifuged 10 minutes after digestion, and physiological saline resuspension is added after abandoning supernatant in suction, is filtered with 100 μm of cell sieves；Cell
Suspension 1000rpm is centrifuged 10 minutes, and cell precipitation is added serum-free medium after being washed 2 times with physiological saline resuspension and (contains 1-
500ng/ml fibroblast growth factors, 1-200ng/ml vitamin Es, 0.1-500 μ g/ml phosphoric acid -3-O- rutinosides, 1-500 μ
G/ml Pfansteihls and 1 × serum-free additive) it is resuspended, 1-10 × 10 are pressed after tongue disk orchid dyeing counting6Cell/ml is added to weight
It is cultivated in the coated tissue culture plate of group people's fibronectin or culture dish, in 37 DEG C, 5%CO2It is small that 24-72 is cultivated in incubator
When；
Culture, which is inhaled after 24-72 hours with 5ml pipettes, abandons culture solution, and fresh serum free culture solution is added and carries out changing liquid, cell training
It supports plate or culture dish continues to place 37 DEG C, 5%CO2Continue to cultivate in incubator；
Digestion is carried out when fibroblastic growth fusion reaches 80% or more and expands bottle, culture solution is absorbed, and it is 7.4 to take pH value
1 × PBS is added after washed once in 0.25% pancreatin to culture bottle of 0.5ml, and 200 μ l fetal calf serums are added after 2-3 minutes in digestion
Digestion is terminated, physiological saline is added, is blown and beaten, is drawn in 15ml centrifuge tubes with 5mL pipettes, then with brine one
It is secondary, it is added in the same 15mL centrifuge tubes, 1000 rpms of centrifugations collect fibroblast in 10 minutes；1mL is used after centrifugation
Fresh medium is resuspended, and counts, and the fresh serum-free mediums of 10mL are added with 5mL pipettes, are added to 1 bottle of fibronectin packet
Continue in the culture bottle of quilt at 37 DEG C, 5%CO2It is cultivated in incubator, a fresh medium was changed every 2 days；Wait for into fiber finer
Intracellular growth fusion is when reaching 80% or more, and by above-mentioned through collected by trypsinisation fibroblast, continuous passage 3 is commissioned to train foster, acquisition
109Above fibroblast, is resuspended with 4ml physiological saline, tongue disk orchid dyeing counting obtains a large amount of fibroblasts, puts
It is saved backup under the conditions of setting 4 DEG C；
The jelly that part fibroblast forms in 40% Fibroblast culture solution, 10% dimethyl sulfoxide (DMSO) and 50% fetal calf serum
Liquid storage is stored in -196 DEG C of liquid nitrogen containers, in case being used after recovering from now on.
7. a kind of fibroblastic preparation method as claimed in any one of claims 1 to 6 for cicatrix of skin reparation,
It is characterized in that, takes the cellular morphology observed under secondary culture to 1 generation and 10 generation fibroblast inverted microscopes, cell is at fusiformis
Growth；In vitro Proliferation of Hypertrophic Scar Fibroblasts and glue are inhibited with fibroblasts from hyperplastic scar co-culture experiments
Original synthesis and α-SMA and I-type collagen expression, promote its apoptosis to occur.
8. according to claim 1-7 any one of them methods, which is characterized in that it is described treatment cicatrix of skin at fiber finer
Born of the same parents compared with individual Allogenic Cultured Dermal Fibroblasts Transplantation and saline control group, inhibit I types in rabbit scar model experiment in vivo
Collagen and the secretion of the TGF-β 1 of 1 gene expression of TGF-β and inflammatory factor, have cicatrix of skin and significantly repair；In vivo
Experiment in vitro shows without tumor.
9. a kind of fibroblastic kit prepared such as claim 1-8 any one of them for cicatrix of skin reparation is produced
Product, which is characterized in that the kit includes：
1) fibroblast serum free medium；
2) II Collagenase Types；
3) pancreatin；
4) fibroblast growth factor, vitamin E, phosphoric acid -3-O- rutinosides, Pfansteihl；
5) serum-free additive；
6) operation instructions；
Wherein, the operation instructions include the method for claim 1-8 any one of them.
10. a kind of such as claim 1-9 any one of them fibroblast and its application.
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| CN114836378A (en) * | 2022-04-11 | 2022-08-02 | 江西龙卿堂科技有限公司 | In-vitro culture method of autologous breast milk stem cells, injection and application of injection in skin injury repair |
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