CN104941001A - Biological film corneal patch and preparation method and application thereof - Google Patents
Biological film corneal patch and preparation method and application thereof Download PDFInfo
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- CN104941001A CN104941001A CN201510402587.3A CN201510402587A CN104941001A CN 104941001 A CN104941001 A CN 104941001A CN 201510402587 A CN201510402587 A CN 201510402587A CN 104941001 A CN104941001 A CN 104941001A
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Abstract
The invention discloses a biological film corneal patch and a preparation method and application thereof. Bone mesenchymal stem cells which are derived from allograft are planted on a silk protein biological film to make into the corneal patch used for curing corneal injuries such as corneal ulcer. From a human bone marrow separation step to the step of inoculating mononuclear cells to a-MEM culture media, when the bone mesenchymal stem cells are amplified to the third generation, the bone mesenchymal stem cells are inoculated to the silk protein biological film, and transplantation is conducted after culture is conducted for five days. The animal experiment proves that the corneal patch has good biocompatibility, toxic reactions of a traditional artificial cornea stroma for an organism and induced rejection reaction are avoided, complete degradation is achieved after the transplantation is conducted as time goes by, the cornea can restore to transparency, and requirements of the organism for vision can be met.
Description
Technical field
What the present invention relates to is regenerative medicine and field of tissue engineering technology, relate to a kind of biomembrane cornea paster and its preparation method and application, specifically, relate to and a kind ofly build cornea paster formed and preparation method thereof by human marrow mesenchymal stem cell and fibroin biomembrane, be used for the treatment of corneal injury etc. disease.
Background technology
Cornea is the ledge of eyeball forefront, directly connects with external environment, thus easily suffers extraneous damage.Eye cornea is divided into 5 layers, corneal epithelium, bowman's lamina, hypothallus, descemet's membrane and endodermis.Outermost corneal epithelium has barrier function, is the key keeping good vision.Cornea is positioned at eyeball foremost, and owing to being often exposed in air, easy bacterial infection, its probability that mechanicalness and chemical injury occur is higher, thus causes Corneal inflammation even corneal ulcer.Corneal ulcer is the common eye surface diseases of ophthalmology blinding, and it is long that ulcer involves deep person's healing time, and recurrent exerbation causes the heavy damage of keratocyte to form corneal ulcer and even bores a hole, and treats thorny.And this disease has acatalepsia, the course of disease easily repeatedly, delay feature.For the unmanageable corneal ulcer of medicine, normal row corneal transplantation.
At present, the method of the disease such as traditional treatment corneal ulcer generally has following several clinically: the corneal transplantation that () is traditional, postoperative short-term corneal transparency, its major defect is that only central Φ 7mm transparent optical region is transplanted, invalid to dry cell gap resisting, because rejection is obvious, need life-time service immunosuppressant partner treatment; (2) autologous limbal transplantation, although without immunological rejection, material source is limited, and major defect is increase the ill danger of strong eye, and can not transplant by 360 degree of ring dresses, effect is limited; (3) allogeneic cornea edge is transplanted, and donor material is very limited, and therapy apparatus can be considerably less, and immunological rejection is obvious, needs long-term prescription, costly.Above implantation method, somewhat expensive, can not obtain corneal material in time, and postoperative may there is rejection and limit its treatment corneal ulcer in application.
In recent years, engineered artificial cornea's technology reaches its maturity, for clinical treatment keratopathy provides a new thinking.It take degradation material as support, carry out cell culture in vitro, allow cell carry out three dimensional growth at material surface and inside, to be divided into multi-layer cellular, finally obtain " the artificial donor's cornea " that be similar to donor's cornea, form primarily of seed cell and timbering material.
Desirable structure tissue engineering corneal seed cells should possess following condition: easily separation and Culture, has stronger multiplication capacity and can keep its distinctive biological activity and characteristic for a long time, non-immunogenicity, without allos pollution etc.Mesenchymal stem cells MSCs (Bone Mesenchymal Stem Cells, BMSCs) obtain conveniently, in-vitro multiplication ability is strong, has multi-lineage potential, to the histo-differentiation of each germinal layers such as bone, fat, cartilage, nerve, therefore can be widely used in regenerative medicine.Meanwhile, BMSCs has paracrine function, participates in immunomodulating; Do not express MHC-II or costimulatory molecules, non-immunogenicity; Inhibitory action is had to CD4+/CD8+ T cell, dendritic cell, natural killer cell etc.In the treatment of corneal alkali burn, research report BMSCs has the effect reduced inflammation, thus promotes the injury repairing of cornea.This therapeutical effect of BMSCs, proposes a kind of new approaches for regulating inflammatory reaction and reducing transplant rejection.
Build the timbering material of active artificial cornea for organizational project, its biocompatibility will be got well, and seed cell can within it normal growth and form the normal configuration of approximate intracorporeal organ, and possesses the characteristics such as cornea distinctive transparent, dioptric, oxygen flow.Timbering material comparatively conventional at present has the material such as corneal stroma, amniotic membrane, collagen, natural coral, fibrin of de-epithelium.There is certain shortcoming in existing several biologic bracket material: though natural macromolecular material good biocompatibility, mechanical property is not good enough, degrades too fast, is unfavorable for that cytotrophy material is to material internal diffusion.Due to the impact of ethics, also there is the problem that donor source lacks in amniotic membrane self, and there is the risk propagating the infectious virus such as hepatitis, HIV (human immunodeficiency virus) (HIV) in amniotic membrane, add that the transparency of amniotic membrane is low, implant and be difficult to recover completely the transparency of cornea, have impact on the recovery of patient visual's function, these problems all significantly limit the further genralrlization of amniotic membrane in clinical and application.
Fibroin is a kind of degradable biological macromole, and because its good mechanical properties, adjustable extent are large, bone regeneration capability is good, in field of tissue engineering technology, often by as embedded material for studying its ossification.Fibroin has water solublity, can avoid the use of toxic solvent in preparation process, and biological safety is good, can degradation in vivo.In addition, fibroin itself is native protein polypeptide, good biocompatibility, not easily challenge.Fibroin in vivo with external equal degradable, degradation time stablize, final catabolite is aminoacid or oligopeptide, does not have toxic action to body.
Summary of the invention
For the deficiencies in the prior art, in conjunction with regenerative medicine and tissue engineering technique, the present invention utilizes mesenchymal stem cells MSCs as seed cell; Build fibroin biomembrane as carrier, preparation forms cornea paster.For the treatment of a series of corneal injury diseases such as corneal ulcer, corneal defect, the corneal opacity, closely echoing of seed cell of the present invention and support, not only can provide sufficient nutrition but also effectively can stop new vessels, to Ocular surface healing and reconstruction, there is remarkable result, have good application prospect.
The object of this invention is to provide a kind of completely newly by Marrow Mesenchymal Stem Cells biomembrane cornea paster, this paster is applied to the treatment of the corneal injury diseases such as corneal ulcer.
Another object of the present invention is to provide the preparation method of above-mentioned biomembrane cornea paster.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A kind of biomembrane cornea paster by the seed cell be inoculated on timbering material and is formed three-dimensional biomembrane support structure form, wherein said seed cell is mesenchymal stem cells MSCs, and described biomembrane support is fibroin biomembrane.
Further, the inoculum density of described mesenchymal stem cells MSCs is (1 ~ 10) × 10
6/ cm
2, preferably 7 × 10
6/ cm
2.
Further, the biomembranous mass content of described fibroin is 1% ~ 10%, preferably 2%; Film baking temperature condition is 10 DEG C ~ 60 DEG C, preferably 25 DEG C; The damp condition of film drying is 25% ~ 60%, preferably 50%.This timbering material can maintain position, the form of seed cell, also can participate in the regulation and control of cell differentiation state.
Further, described mesenchymal stem cells MSCs derives from allogeneic.
A preparation method for biomembrane cornea paster, is made up of following step: the preparation of (1) fibroin biomembrane support; (2) separation of mesenchymal stem cells MSCs, cultivation and qualification; (3) inoculation of mesenchymal stem cells MSCs; (4) preparation of biomembrane cornea paster; (5) transplanting of biomembrane cornea paster.
The concrete operation method of described step (1) is: take the Carbon Dioxide sodium solution that appropriate mulberry silk is placed in 0.5%, bath raio 1:50, in 90 DEG C ~ 100 DEG C process 30min, repeats 3 times (using deionized water for the last time).Come unstuck afterwash, obtains refine silkworm silk after 60 DEG C of oven dryings.Be dissolved in the lithium-bromide solution of 9.5mol/L by the raw silk come unstuck, then dialyse solution 3d in deionized water.After the silk protein solution centrifugal segregation minute quantity flocky precipitate of having dialysed, demarcate the concentration of silk protein solution by gravimetric method, then adding appropriate deionized water by solution dilution to the mass fraction of fibroin is 1% ~ 10%.Moved into by 1ml regenerated fibroin solution in the plastic casing of 3cm × 3cm, 10 DEG C ~ 60 DEG C temperature, under 25% ~ 60% relative humidity, placement is spent the night, and makes its natural drying film forming (about needing 24h).The regenerated silk fibroin membrane thickness of gained is about 5 μm, distilled water immersion 48 hours, when 8h, change distilled water.Take out material film, after drying, respectively soak 1d by PBS and culture medium respectively, for subsequent use.
Timbering material of the present invention, wherein the mass fraction of fibroin is 1% ~ 10%, and return in animal body and plant experimental verification, this timbering material has good biocompatibility and suitable vivo degradation speed, and catabolite is nontoxic.
Further, the concrete operation method of described step (2) is: BMSCs is separated and Secondary Culture: get people's bone marrow 5 ~ 10ml, anticoagulant heparin, dilute with the PBS of equivalent, be added on Ficoll lymphocyte separation medium in 2: 1 ratios, centrifugal 30 min of 400g, get middle tunica albuginea layer, washing 3 times with PBS, is 1 × 10 with the a-MEM culture medium adjustment cell concentration containing 10%FBS
6individual/ml is inoculated in 9cm culture dish.Use differential attachment method 37 DEG C, 5%C0
2cultivate 48 hours later half amounts under condition and change liquid, 4d full dose is changed liquid and is discarded not adherent cell.The growth of observation of cell under inverted phase contrast microscope, when cell grows to 85% fusion, digests with 0.25% trypsin-EDTA, goes down to posterity in the ratio of 1: 2, within about 48 hours afterwards, changes liquid once, within 3rd ~ 4, goes down to posterity once.Get the cell after 4 ~ 6 generations of going down to posterity for the propagation of FCM analysis and cell and Morphological Experimental, each group cell is with 2 × 10
5/ hole, is inoculated in and is covered with in 24 orifice plates of each group of material, 37 DEG C, 5%C0
2cultivate under condition, within every 48 ~ 72 hours, change liquid.
The qualification of BMSCs: after the BMSCs PBS in In vitro culture 4 ~ 6 generation is washed paint many times; Add fluorescently-labeled antibody; 4 DEG C, hatch 30min; PBS washes away unconjugated antibody, and 1% paraformaldehyde is fixed; Application FACScan flow cytomery cell surface antigen is expressed.
Further, the concrete operation method of described step (3) is: BMSCs goes down to posterity to increase after the third generation, is inoculated in obtained cell material complex on timbering material; Place certain hour in incubator after, digestion collecting cell, calculating instrument counts, and draws the cell concentration of loss; After cell material complex cultivates 24 hours, cell material complex is rocked in culture fluid, and changes to the cultivation of another culture dish, collect culture dish culture fluid and digest and collect adherent cell, cell in original fluid is counted in the lump, draws the cell number do not adhered to:
Cell adhesion rate=(cell number of the cell number-loss of inoculation-do not adhere to cell number)/(cell number of the cell number-loss of inoculation).
Further, the concrete operation method of described step (4) is: by BMSCs by (1 ~ 10) × 10
6/ cm
2cell is seeded on fibroin biomembrane, puts 5%CO
2, 37 DEG C of incubators add a-MEM culture medium containing 10% hyclone after cultivating 4h, every 2d changes liquid, Dual culture 5d later, forms artificial cornea's paster, for subsequent use.
Further, the concrete operation method of described step (5) is: be transplanted on damaged corneal by the complex paster that the mesenchymal stem cells MSCs of external structure and fibroin biomembrane Dual culture are formed.After entire patient's anesthesia, after eye table being anaesthetized with tetracaine eye liquid, strike off whole corneal epithelium with circular knife, and use normal saline cleaning down.(area is 1.5 × 1.5 cm to the graft getting slightly larger than corneal diameter
2), epithelial surface is covered in eye table upward, sews up patch edges and bulbar conjunctiva continuously with 10-0 nylon wire, and observation transplanting paster is flat to be covered, give erythromycin eye ointment without local after the situations such as hematocele under it, art finishes gives 0.3% tobramycin/0.1% Dexamethasone Eye Drops local use 3 weeks.Result shows, above-mentioned artificial cornea's paster implant after along with passage of time can be degradable, cornea also progressively recovers transparent, can repairing corneal defect better, meets the requirement of body to vision.
The advantage of the artificial cell membrane cornea of the present invention is:
1. traditional artificial corneal stroma uses keratocyte as seed cell, if be good for side angle membrane damage comparatively greatly because this cell derives from auto corneal stromal cell to individuality, and cell culture is consuming time too of a specified duration; If derive from allogeneic cornea stromal cell, there is the probability that rejection occurs.In the inventive method, biological cornea paster adopts BMSCs as seed cell.BMSCs obtains convenient, solves donor source problem, and in-vitro multiplication ability is strong, has multi-lineage potential, there is not immunological rejection, meanwhile, BMSCs has paracrine function, participate in immunomodulating, the effect reduced inflammation in addition, thus promote the injury repairing of cornea.
2. the BMSCs adopted in the inventive method derives from allogeneic, and can complete the preparation of biomembrane cornea paster in advance, corneal is damaged the wounded and treated in time.Avoid because of adopt autologous cell therapy time, autogenous cell obtain and incubation for a long time consuming time, delay treating time, and the situation causing blindness.
3. the biomembrane adopting fibroin to build in the inventive method has water solublity, can avoid the use of toxic solvent in preparation process, and biological safety is good; Itself be native protein polypeptide, there is excellent biocompatibility and histocompatibility, do not cause rejection and inflammatory reaction; Sustenticular cell grows, and promotes to merge, can degraded and absorbed gradually in body, and degraded and the speed absorbed and histiocytic growth and breeding speed match; Final catabolite is aminoacid or oligopeptide, does not have toxic action to body; There are good tenacity and the various process based prediction model similar to human cornea, provide enough spaces for cell grows in the carrier with substance metabolism.It both can be applied to separately the reconstruction of cornea tissue structure, again can with other organization material use in conjunction, in the application of artificial cornea's material, have good prospect.
4. the biomembrane cornea paster of the inventive method structure, only needs paster to be sutured on damaged corneal during use, and operation is simple, is easy to grasp, postoperative conventional antiinflammatory process, and conveniently, therapeutic effect is very effective in nursing.
5. the biomembrane paster that prepared by the inventive method can reduce the demand to donor's cornea, reduces again the risk of the infection that brings of donor graft and immunologic rejection, more can provide the high activity cell of abundant amount, have splendid application prospect.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, and form a application's part, schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1: biomembrane cornea paster preparation flow figure of the present invention;
Fig. 2: P2 takes a picture (× 400) for BMSCs inverted phase contrast microscope;
Fig. 3: A is the blank glass cell climbing sheet group of load BMSCs; B is the fibroin biofilm experiments group of load BMSCs;
Fig. 4: the statistical analysis of corneal healing after transplant operation: transparency, new vessels, survival rate.A survival analysis, B transparency analysis, C new vessels is analyzed;
Fig. 5: the corneal healing after the transplanting of biomembrane cornea paster: PKH26 Cellular tracking.
Detailed description of the invention
Below with reference to the accompanying drawings and in conjunction with the embodiments, describe the present invention in detail.
Shown in Fig. 1 biomembrane cornea paster of the present invention preparation flow figure, the specific embodiment of the invention is as follows:
The external preparation of embodiment 1 artificial cornea and detection
1. raw material: culture medium a-MEM, component is maintain the essential amino acids of Growth of Cells, protein and polysaccharide etc.; Hyclone (FBS), the nutritional labeling that Growth of Cells is required, its product performs in strict accordance with hyclone quality standard; Tripsin-EDTA, peptic cell is used.
2. the preparation of fibroin biomembrane material:
Take the Carbon Dioxide sodium solution that appropriate mulberry silk is placed in 0.5%, bath raio 1:50, in 90 DEG C ~ 100 DEG C process 30 min, repeat 3 times (using deionized water for the last time).Come unstuck afterwash, obtains refine silkworm silk after 60 DEG C of oven dryings.Be dissolved in by the raw silk come unstuck in the lithium-bromide solution of 9.5 mol/L, then dialyse solution 3d in deionized water.After the silk protein solution centrifugal segregation minute quantity flocky precipitate of having dialysed, demarcate the concentration of silk protein solution by gravimetric method, then adding appropriate deionized water by solution dilution to the mass fraction of fibroin is 2%.By 1ml regenerated fibroin solution move into 3cm × 3cm plastic casing at room temperature (25 DEG C, 50% relative humidity) left overnight, make its natural drying film forming (about needing 24h).The regenerated silk fibroin membrane thickness of gained is about 5 μm, distilled water immersion 48 hours, when 8h, change distilled water; Take out material film, after drying, respectively soak 1d by PBS and culture medium respectively, for subsequent use.
3. human marrow mesenchymal stem cell original cuiture and go down to posterity: get people's bone marrow 5 ~ 10 ml, anticoagulant heparin, dilute with the PBS of equivalent, be added on Ficoll lymphocyte separation medium in 2: 1 ratios, centrifugal 30 min of 400 g, getting middle tunica albuginea layer, wash 3 times with PBS, is 1 × 10 with the a-MEM culture medium adjustment cell concentration containing 10%FBS
6individual/ml is inoculated in 9cm culture dish.Use differential attachment method 37 DEG C, 5%C0
2cultivate 48 hours later half amounts under condition and change liquid, 4d full dose is changed liquid and is discarded not adherent cell.The growth of observation of cell under inverted phase contrast microscope, when cell grows to 85% fusion, digests with 0.25% trypsin-EDTA, goes down to posterity in the ratio of 1: 2, within about 48 hours afterwards, changes liquid once, within 3rd ~ 4, goes down to posterity once.Get the cell after 4 ~ 6 generations of going down to posterity for the propagation of FCM analysis and cell and Morphological Experimental, each group cell is with 2 × 10
5/ hole, is inoculated in and is covered with in 24 orifice plates of each group of material, 37 DEG C, 5%C0
2cultivate under condition, within every 48 ~ 72 hours, change liquid.
The qualification of BMSCs: after the BMSCs PBS in In vitro culture 4 ~ 6 generation is washed paint many times; Add fluorescently-labeled antibody; 4 DEG C, hatch 30min; PBS washes away unconjugated antibody, and 1% paraformaldehyde is fixed; Application FACScan flow cytomery cell surface antigen is expressed.
4. the inoculation of BMSCs
BMSCs goes down to posterity to increase after the third generation, is inoculated in obtained cell material complex on timbering material; Place certain hour in incubator after, digestion collecting cell, calculating instrument counts, and draws the cell concentration of loss; After cell material complex cultivates 24 hours, cell material complex is rocked in culture fluid, and changes to the cultivation of another culture dish, collect culture dish culture fluid and digest and collect adherent cell, cell in original fluid is counted in the lump, draws the cell number do not adhered to:
Cell adhesion rate=(cell number of the cell number-loss of inoculation-do not adhere to cell number)/(cell number of the cell number-loss of inoculation).
5. BMSCs is inoculated in fibroin biomembrane support, by cell obtained for step 2 by 7 × 10
6/ cm
2be inoculated on fibroin biomembrane material support, put 5%CO
2, add a-MEM culture medium containing 10% hyclone after 37 DEG C of adherent 4h of incubator, every 2d changes liquid, Dual culture 5d later, forms biomembrane cornea paster.Every day inverted phase contrast microscope observation of cell growing state.
The observation of biomembrane cornea paster and qualification: get to cover and carry BMSCs and cultivate the biomembrane cornea paster of 5 days, do paraffin section, HE dyes observation.
6. result display:
(1) culture identification of BMSCs:
Adherent after primary BMSCs 24h, full extension, in fusiformis, polygon, form is irregular, hematopoietic cell reject after PBS rinses that fraction is rounded.Cultivate after 7d cell merges substantially and go down to posterity, go down to posterity through 2 times, P2 is fibroblast sample for cellular morphology, cell space is spindle shape, and core is oval, and form is consistent, observe under inverted microscope in swirling, radiate (Fig. 2), Growth of Cells is rapid, and 4 ~ 5d goes down to posterity.
Get the cell in Isolation and culture 4 ~ 6 generation, its surface antigen of flow cytomery.Found that, cell surface CD14, CD34, CD45 express feminine gender, and CD29, CD44, CD90 express the positive.Normal mesenchymal stem cell (MSC) does not express CD14, CD34, CD45, and strongly expressed CD29, express CD44, CD90.Above result shows that the cell of this experiment institute separation and Culture is normal marrow mescenchymal stem cell.
(2) the inoculation experiments result of BMSCs shows, (1 ~ 10) × 10
6/ cm
2cell-seeding-density in scope has higher material adherence rate and cell adhesion amount, and cell-seeding-density is 7 × 10
6/ cm
2shi Xiaoguo is best.
(3) observation of artificial cornea's paster and qualification
Get the BMSCs in Isolation and culture 4 ~ 6 generation, by 7 × 10
6/ cm
2density be inoculated on fibroin biomembrane cultivate 3 days, optical microscope Microscopic observation cellular morphology.At fibroin biofilm surface, cellular morphology is elongated, and in shuttle-type, cell is along silkworm silk direction ordered arrangement, and cell quantity is more than blank glass cell climbing sheet contrast (Fig. 3).
(animal) experiment in the body of embodiment 2 artificial cornea
1. make Alkali-burned Rabbit Corneas model
It is that the new zealand white rabbit of 12 weeks carries out zoopery that the healthy age is chosen in this research, and the average weight of rabbit is 3kg, is provided by Southern Yangtze University's animal experimental center.Slit lamp examination all rabbit eyes prosthomere and accessory organs of eye before alkali burn, get rid of Eye disease, intraperitoneal anesthesia is carried out by 40 ~ 50 mg/kg with 1% pentobarbital sodium, after the capable eye table anesthesia of tetracaine eye liquid, cut-off footpath is the circular filter paper sheet of 7mm, every sheet instillation 17ul 0.5mol/L NaOH solution, be covered in whole cornea to cornea edge 20s, then normal saline flushing 1min is used, with ofloxacin eye drops eye dripping 3 times/day after wound, in erythromycin eye ointment 1 time/evening, regularly carry out slit lamp observation anterior ocular segment situation of change.After corneal alkali burn, 2w carries out cornea scoring, and standards of grading see the following form (table 1), chooses corneal fluorescein dyeing scoring >=3 points, and the rabbit of cornea rebirth blood vessel and cornea opacity >=2 point enters experiment.
The standards of grading of rabbit after table 1 corneal alkali burn
2. artificial cornea's paster is transplanted
1) labelling BMSCs
With the BMSCs that PKH26 labelling is cultivated.(PKH26 is mainly used in cells in vitro labelling, body outer cell proliferation, Cellular tracking experiment.) sterile working: (a) trypsinization BMSCs forms single cell suspension, and serum-free medium washes 1 time; B () 600rpm/ minute centrifugal 5 minutes, form cell mass, abandon supernatant, above cell mass, remaining liq is less than 25ul; C () adds 1ml diluent, re-suspended cell ensures completely discrete; D () prepares PKH26 dye liquor, be placed in clean centrifuge tube, 1% of dyestuff addition < cumulative volume; E 1ml is contained the diluent of cell by (), add in 1ml dye liquor, and even rapid mixing sample hatches 2 ~ 5 minutes for 25 DEG C, and regularly vibrate centrifuge tube gently, 25 DEG C of fully mixings; F () adds equivalent serum and stops staining reaction, hatch 1 minute; G reactant liquor that the dilution of () equivalent culture medium stops; H () 800rpm/ minute centrifugal 6 minutes, abandon supernatant; (i) cell mass proceeds to new centrifuge tube, 1000rpm, centrifugal 2 minutes; J () adds 10ml cell culture medium, re-suspended cell, cellar culture.
2) the multiple cornea paster of biomembrane is cultivated
Fibroin biomembrane support under 37 DEG C of conditions, in a-MEM culture medium submerged culture 3 days.After PKH26 labelling BMSCs, with 7 × 10
6/ cm
2density be seeded in fibroin biomembrane timbering material surface.Be placed in 5%CO
2, add a-MEM culture medium containing 10% hyclone after 37 DEG C of adherent 4h of incubator, every 2d changes liquid, Dual culture 5d later.
3) artificial cornea's paster is transplanted
36 the corneal alkali burn rabbits entering experiment are divided into 3 groups at random, namely A group corneal alkali burn group is matched group (12), B group is simple fibroin film transplantation group (12), C group is BMSCs-fibroin biomembrane cornea paster transplantation group (12).
Operation method: corneal alkali burn rabbit ketamine and chlorpromazine (by 1:1 mixing) anaesthetize sb. generally, and after carrying out the anesthesia of eye table, strike off whole corneal epithelium, and use normal saline cleaning down with circular knife with tetracaine eye liquid.(B group is simple fibroin film transplantation group to the graft getting slightly larger than corneal diameter, C group BMSCs-fibroin biomembrane cornea paster transplantation group), epithelial surface is covered in eye table upward, sew up continuously with 10-0 nylon wire and plant sheet edge and bulbar conjunctiva, observe, cover if graft is flat, it is lower to situations such as hematoceles, local gives erythromycin eye ointment again, and art finishes local and gives 0.3% tobramycin/0.1% Dexamethasone Eye Drops, uses 3 weeks.
3. biomembrane cornea paster transplants the clinical score of rear alkali burn corneal healing
Postoperative Continuous Observation 4 months, takes a picture and slit lamp observation substantially to the cornea (often organizing 12 samples) of two groups of transplantation group and alkali burn group.Monthly 1 transparency to the cornea (often organizing 12 samples) of three groups and new vessels degree are observed and repeated measure, and survival analysis, calculates the cumulative survival rate of 3 group corneas.Finally carry out regression analysis, whether the cornea opacity of the rear different time points (1 month, 2 months, 3 months, 4 months) of evaluation transplanting and new vessels degree affect the cornea survival rate of 4 months.
4. transplant after cornea draw materials, immunohistochemical staining, HE dyeing
Put to death corneal alkali burn rabbit respectively at postoperative 2w, 4w, 10w, get art eye, after fixing 24h with eyeball fixative, dehydration makes paraffin section, partial row immunohistochemical staining, and partial row HE dyes, and observes cornea situation of change after transplanting, and takes.
CK12, CK13 immunohistochemical staining (doing the dyeing of single labelling respectively): (1) paraffin section dimethylbenzene takes off cured to water; (2) PBS rinses 5min × 3 time; (3) 3%H
2o
2incubated at room 30min, to eliminate the activity of endogenous peroxydase; (4) distilled water flushing, PBS soaks 5min; (5) citrate buffer carries out antigen retrieval, rinses after room temperature cooling with PBS; (6) 10% lowlenthal serums are closed, incubated at room 30min, and incline serum deprivation, do not wash; (7) CK12(rabbit anti-mouse polyclonal antibody 1:50 is added), 4 DEG C are spent the night; (8) PBS rinses 5min × 3 time; (9) goat antirabbit biotin labeling two anti-(1:50) is added, incubated at room 30min; (10) PBS rinses 5min × 3 time; (11) drip horseradish peroxidase, hatch 30min for 37 DEG C; (12) PBS rinses 5min × 3 time; (13) DAB chromogenic reagent, after Microscopic observation is painted, fully rinses with tap water; (14) haematoxylin is redyed; (15) neutral gum mounting, basis of microscopic observation.CK13 therewith staining procedure is identical, and primary antibodie is mouse monoclonal anti-human's antibody, and two resist for goat anti-mouse biotin labeling two anti-(1:50), and two kinds of immunohistochemical staining negative controls are PBS and replace primary antibodie.
HE dyes: (1) cut into slices the 20min that respectively dewaxes in dimethylbenzene I, II; The each 10min of (2) 100% ethanol I, II, 95%, 85%, 70% ethanol, at different levels is 5min, proceeds to dye liquor finally by distilled water; (3) hematoxylin dye liquor dyeing 5 ~ 10min; (4) wash unnecessary dye liquor on slide with water, 1% hydrochloride alcohol (70% alcohol) color separation for a moment; (5) running water 20min; (6) distilled water is short washes; (7) 0.5% eosin stain dyeing 1 ~ 5min; (8) successively through 70%, 85%, 95%, 100% dehydration of alcohol, at different levels is 2 ~ 3min; (9) dimethylbenzene transparent (secondary), altogether about 10min.(10) mounting: wipe section unnecessary dimethylbenzene around, drip appropriate canada balsam rapidly, then add coverslip sealing.
5. result display
1) healing state of rear alkali burn cornea is transplanted in observation of substantially taking a picture:
Transplant latter 1 month, the cornea large area of biomembrane cornea paster transplantation group recovers translucent, and local edema, new vessels stretches into corneal graft.Transplant latter 2 months, the cornea of biomembrane cornea paster transplantation group presents the white casse of small size, limitation, and new vessels large area disappears.Transplant latter 3 ~ 4 months, the cornea large area of biomembrane cornea paster transplantation group recovers transparent; The Central corneal district of the independent transplantation group of fibroin film still has white macula sample muddy; Matched group alkali burn cornea then forms white cicatrix and pannus widely, thoroughly can not see intraocular structure.As can be seen here, biomembrane cornea paster transplantation group therapeutic effect is best.
2) statistical analysis of corneal healing after transplanting
After operation transplantation, in 4 months, repeated measure 3 organizes turbidity and the new vessels degree of cornea, and result shows that the cornea of two transplantation group has and recovers transparent and the trend (p<0.05) of new vessels minimizing.Wherein, compared with B group, the rear cornea transparent recovery of C group (BMSC-fibroin biomembrane corneal transplantation group) transplanting is very fast, comparatively light (p<0.05) (Fig. 4 B-C) of vascularization degree.The survival analysis of 4 months draws the Cumulative survival rate of transplantation group and alkali burn group cornea respectively: C group 90%, B group 70%, A group 40%.Cornea after the transplanting of C group has the longer time-to-live (p<0.05) than the cornea of alkali burn, and cornea shows the transparency of longer time.Meanwhile, the cornea survival rate after the transplanting of C group compares with the cornea survival rate of B group, also has significant difference (p<0.05) (Fig. 4 A).Regression analysis shows, the cornea opacity transplanting latter 1st month and the cornea Cumulative survival rate relevant (p<0.05) transplanted in latter 4 months.This shows, the cornea that in 3 groups, performance in 1st month is more muddy after the transfer, and its risk survival occurring failed at 4 months is higher, and relative risk is 3.359 times of transparency cornea.Equally, the cornea opacity transplanting latter 2nd month and the cornea Cumulative survival rate relevant (p<0.05, RR=3.898) transplanted in latter 4 months.
3) healing of alkali burn cornea after transplanting: PKH26 Cellular tracking and histopathology
Biomembrane cornea paster transplantation group, transplants latter 1 month, has PKH26 labeled cell to there is (Fig. 5); Transplant latter 2 ~ 3 months, the inflammatory infiltration of cornea alleviates, and new vessels reduces; Transplant latter 4 months, the collagen of cornea in rule arranges and cladding cell.
4) cornea tissue immunochemistry dyeing
Carry out the dyeing of immunohistochemistry list labelling to postoperative 2w, 4w, 10w cornea of rats serial section, it is ripe corneal epithelial cell that CK12 expresses positive explanation cell, and CK13 expresses the positive, illustrates that cell is conjunctival epithelial cell.
Matched group: postoperative 2w, 4w, 10w:CK13 antigen presentation is all positive, and CK12 and CK13 expression position does not overlap, and illustrates that matched group corneal epithelium is that conjunctival cells covers, without corneal epithelial cell growth.
Simple fibroin film transplantation group: postoperative 2w: it is positive that majority organizes CK13 to express; Postoperative 4w: almost all organize CK13 to be positive; Postoperative 10w:CK13 stained positive, illustrates that epithelium is that conjunctival epithelium covers.
Biomembrane cornea paster transplantation group: postoperative 2w:CK12 stained positive, epithelium is cell monolayer; Postoperative 4w:CK12 is positive, and cell is grown to multilayer structure; Postoperative 10w:CK12 stained positive, epithelial cell structure is normal, does not express CK13, illustrates that corneal epithelium covers anterior corneal surface, and after transplanting, cell is still in continuation differentiation, propagation.
5) cornea tissue HE dyes
Matched group: postoperative 2w, topical epithelial has cell to cover, arrangement disorder, degeneration, cell infiltration, and new vessels is formed; Postoperative 4w, have epithelium to cover (being accredited as conjunctival epithelium through immunohistochemical staining), matrix structure is disorderly, and new vessels is many; Postoperative 10w, epithelial structure is disorderly, and substrate arrangement disorder, has cicatrization, and new vessels is visible.
Simple fibroin film transplantation group: postoperative 2w, part epithelial defect, part epithelial hyperplasia, cell infiltration, visible new vessels, proliferation of fibroblast, substrate thickens; Postoperative 4w, part epithelium covers, structure disturbance, parakeratosis, and substrate thickens, cell infiltration, new vessels; Postoperative 10w, the epithelial cell number of plies reduces, and misaligned, stromal thickness is normal, visible new vessels.
Biomembrane cornea paster transplantation group: postoperative 2w, epithelium is hypertrophy slightly, the more cell infiltration of substrate, visible new vessels; Postoperative 4w, most of epithelium covers, the cell number of plies close to normal, substrate hyperemia comparatively 2w time obviously reduce; Postoperative 10w, epithelium is complete, and stromal thickness is normal, and arrangement in order, remains a little lymphocyte, a small amount of new vessels.
Conclusion: after operation transplantation, through gross examination of skeletal muscle, HE, immunohistochemical detection and cornea scoring statistical analysis, BMSC-fibroin biomembrane cornea paster transplantation group curative effect is significantly better than simple fibroin film transplantation group and matched group, wherein BMSCs is divided into epithelial cell at corneal damage position, repair damaged corneal, reach the effect for the treatment of.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a biomembrane cornea paster, it is characterized in that, by the seed cell be inoculated on timbering material and is formed three-dimensional biomembrane support structure form, wherein said seed cell is mesenchymal stem cells MSCs (BMSCs), and described biomembrane support is fibroin biomembrane.
2. a kind of biomembrane cornea paster according to claim 1, is characterized in that, described mesenchymal stem cells MSCs derive from allogeneic; Inoculum density is (1 ~ 10) × 10
6/ cm
2, preferably 7 × 10
6/ cm
2.
3. a kind of biomembrane cornea paster according to claim 1, is characterized in that, the biomembranous mass content of described fibroin is 1% ~ 10%, preferably 2%; Film baking temperature condition is 10 DEG C ~ 60 DEG C, preferably 25 DEG C; The damp condition of film drying is 25% ~ 60%, preferably 50%.
4. the preparation method of a kind of biomembrane cornea paster according to claim 1, is characterized in that, be made up of following step: the preparation of (1) fibroin biomembrane support; (2) separation of mesenchymal stem cells MSCs, cultivation and qualification; (3) inoculation of mesenchymal stem cells MSCs; (4) preparation of biomembrane cornea paster; (5) transplanting of biomembrane cornea paster.
5. the preparation method of a kind of biomembrane cornea paster according to claim 4, it is characterized in that, the concrete operation step of the preparation of described step (1) fibroin biomembrane support is: take the Carbon Dioxide sodium solution that appropriate mulberry silk is placed in 0.5%; bath raio 1:50; in 90 DEG C ~ 100 DEG C process 30min, repeat 3 times (using deionized water for the last time); Come unstuck afterwash, obtains refine silkworm silk after 60 DEG C of oven dryings; Be dissolved in the lithium-bromide solution of 9.5mol/L by the raw silk come unstuck, then dialyse solution 3d in deionized water; After the silk protein solution centrifugal segregation minute quantity flocky precipitate of having dialysed, demarcate the concentration of silk protein solution by gravimetric method, then adding appropriate deionized water by solution dilution to the mass fraction of fibroin is 1% ~ 10%; Moved into by 1ml regenerated fibroin solution in the plastic casing of 3cm × 3cm, 10 DEG C ~ 60 DEG C temperature, under 25% ~ 60% relative humidity, placement is spent the night, and makes its natural drying film forming (about needing 24h); The regenerated silk fibroin membrane thickness of gained is about 5 μm, distilled water immersion 48 hours, when 8h, change distilled water; Take out material film, after drying, respectively soak 1d by PBS and culture medium respectively, for subsequent use.
6. the preparation method of a kind of biomembrane cornea paster according to claim 4, is characterized in that, the concrete operation step of the separation of described step (2) mesenchymal stem cells MSCs, cultivation and qualification is:
The first step, gets people's bone marrow 5 ~ 10ml, anticoagulant heparin, dilute with the PBS of equivalent, be added on Ficoll lymphocyte separation medium in 2: 1 ratios, centrifugal 30 min of 400g, getting middle tunica albuginea layer, wash 3 times with PBS, is 1 × 10 with the a-MEM culture medium adjustment cell concentration containing 10%FBS
6individual/ml is inoculated in 9cm culture dish; Use differential attachment method 37 DEG C, 5%C0
2cultivate 48 hours later half amounts under condition and change liquid, 4d full dose is changed liquid and is discarded not adherent cell;
Second step, the growth of observation of cell under inverted phase contrast microscope, when cell grows to 85% fusion, digests with 0.25% trypsin-EDTA, goes down to posterity in the ratio of 1: 2, within about 48 hours afterwards, changes liquid once, within 3rd ~ 4, goes down to posterity once;
3rd step, get the cell after 4 ~ 6 generations of going down to posterity for the propagation of FCM analysis and cell and Morphological Experimental, each group cell is with 2 × 10
5/ hole, is inoculated in and is covered with in 24 orifice plates of each group of material, 37 DEG C, 5%C0
2cultivate under condition, within every 48 ~ 72 hours, change liquid;
4th step, the qualification of BMSCs: after the BMSCs PBS in In vitro culture 4 ~ 6 generation is washed paint many times; Add fluorescently-labeled antibody; 4 DEG C, hatch 30min; PBS washes away unconjugated antibody, and 1% paraformaldehyde is fixed; Application FACScan flow cytomery cell surface antigen is expressed.
7. the preparation method of a kind of biomembrane cornea paster according to claim 4, it is characterized in that, the concrete operation step of the inoculation of described step (3) mesenchymal stem cells MSCs is: BMSCs goes down to posterity to increase after the third generation, is inoculated in obtained cell material complex on timbering material; Place certain hour in incubator after, digestion collecting cell, calculating instrument counts, and draws the cell concentration of loss; After cell material complex cultivates 24 hours, cell material complex is rocked in culture fluid, and changes to the cultivation of another culture dish, collect culture dish culture fluid and digest and collect adherent cell, cell in original fluid is counted in the lump, draws the cell number do not adhered to.
8. the preparation method of a kind of biomembrane cornea paster according to claim 4, is characterized in that, the concrete operation step of the preparation of described step (4) biomembrane cornea paster is: by BMSCs by (1 ~ 10) × 10
6/ cm
2cell is seeded on fibroin biomembrane, puts 5%CO
2, 37 DEG C of incubators add a-MEM culture medium containing 10% hyclone after cultivating 4h, every 2d changes liquid, Dual culture 5d later, forms artificial cornea's paster, for subsequent use.
9. the preparation method of a kind of biomembrane cornea paster according to claim 4, it is characterized in that, the concrete operation step of the transplanting of described step (5) biomembrane cornea paster is: be transplanted on damaged corneal by the complex paster that the mesenchymal stem cells MSCs of external structure and fibroin biomembrane Dual culture are formed; After entire patient's anesthesia, after eye table being anaesthetized with tetracaine eye liquid, strike off whole corneal epithelium with circular knife, and use normal saline cleaning down; (area is 1.5 × 1.5 cm to the graft getting slightly larger than corneal diameter
2), epithelial surface is covered in eye table upward, sews up patch edges and bulbar conjunctiva continuously with 10-0 nylon wire, and observation transplanting paster is flat to be covered, give erythromycin eye ointment without local after the situations such as hematocele under it, art finishes gives 0.3% tobramycin/0.1% Dexamethasone Eye Drops local use 3 weeks.
10. a kind of biomembrane cornea paster described in claim 1-3 and the application of a kind of biomembrane cornea paster in corneal injury disease prepared by claim 4-9.
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| WO2020107496A1 (en) * | 2018-12-01 | 2020-06-04 | 铭道创新（北京）医疗技术有限公司 | Flow cytometry testing method for lymphocyte in immune cell |
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