CN105770994B - A kind of bioengineering takes off preparation and the purposes of cell eye conjunctiva - Google Patents
A kind of bioengineering takes off preparation and the purposes of cell eye conjunctiva Download PDFInfo
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- CN105770994B CN105770994B CN201610297081.5A CN201610297081A CN105770994B CN 105770994 B CN105770994 B CN 105770994B CN 201610297081 A CN201610297081 A CN 201610297081A CN 105770994 B CN105770994 B CN 105770994B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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Abstract
The invention provides the preparation method of acellular conjunctiva that is a kind of efficient and can keeping eye conjunctiva function and characteristic.The fresh eye conjunctiva containing only seldom subconjunctival tissue is obtained, eye conjunctiva takes off whole using the hybrid protection liquid for including one or more conjunctiva structural defence agent in cell processes；Using the cell in high static pressure technology pre-separation conjunctival tissue；Residual cells core is digested using composite nucleic acid enzyme method；Loose crushing cell sheet is removed using detergent；Rinsed using hybrid protection liquid.On the basis of the eye conjunctiva carried out using the present invention after de- cell processing can be purified ensureing nucleus, complete conjunctival epithelium fiber layer structure, distinctive fibre structure arrangement and biological nature are kept.
Description
Technical field
The invention belongs to the preparation field of bioengineering medical material, the specially cell free preparation method of eye conjunctiva.
Background technology
Eye conjunctiva defect and relevant disease are the main blinding illness in eye in China.Eye conjunctiva environment and function are normally eyes
The normal indispensable condition of ball visual performance.Because China is manufacturing industry and large agricultural country, produce with agricultural work by
The illness in eye that physically or chemically conjunctiva defect caused by property eye traumas causes eyelid ball to be adhered is of common occurrence.In addition, in clinical ophthalmology,
Often meet such as cut off it is pteryium after, if the improper recurrence for easily causing the triangular mass of mucous membrane growing from the inner corner of the eye in the conjunctiva part of processing institute defect with
The related case of eye conjunctiva.These eye illnesses are all very intractable, currently without good treatment means.
Treating the means of related eye conjunctiva defect disease instantly both at home and abroad is, patients with mild can autologous Conjunctival Transplantation repaiied
It is multiple；Severe patient can only then be treated by amnion transplantation at present, but amnion is only capable of maintaining for two to three weeks will in ocular
Voluntarily dissolve, therefore can not can only at all solve the problems, such as conjunctiva defect as the substitute of interim conjunctiva.Allogeneic knot
Film is transplanted due to immunological rejection, and there has been no successful case at present.So it is treatment knot to find reliable and secure conjunctiva replacement
Film defect relevant disease the only way out.
In recent years, regenerative medicine and bioengineered tissue achieved in terms of de- cellular biological material significant progress and
Development, many de- cell tissue products also achieve preliminary therapeutic effect in animal level and clinical practice, and often at present
The method for removing cells seen, in order to reach the complete effect of de- cell, more extreme means are substantially taken, such as anxious freeze thawing
Method, highdensity chemistry and enzyme preparation are reused after soaking for a long time.The diagonal membrane matrix warp of these methods of previously reported application
After de- cell, existing poor transparency, part mechanical structure destroys the problem of caused resistance declines.Just because of take off at this stage
The limitation of cell means, based on acellular conjunctiva in terms of research also without breakthrough progress.
Previously reported tissue method for removing cells uses basic buffer solution, such as PBS, HBSS, cell culture fluid more, typically
Need first will cell free tissue be immersed in 24-72 hours in these liquid and be swelled tissue, then pass through physics, chemistry or life
Thing method is by after clasmatosis, then soaks and eluted.These method for removing cells frequently can lead to tissue immanent structure by
Destroy, lose the original basic biological function of de- cell tissue.
In view of the above-mentioned problems, the new de- cell eye conjunctiva preparation method that research and development are comprehensive, is avoided because of physics, chemistry and life
Thing etc. handles complete to eye conjunctiva epithelium fiber layer structure, distinctive fibre structure arrangement and biological nature damages, and is
Urgent problem to be solved at present.
The content of the invention
The invention provides one kind can keep complete eye conjunctiva epithelium fiber layer structure, distinctive fibre structure arrangement and raw
The method for removing cells of thing characteristic.
The present invention innovation be in：1st, with low temperature eye conjunctiva fixator and the safe and efficient complete band of acquisition of normal dyeing method
The eye conjunctiva tissue of epithelium；2nd, whole use includes one or more conjunctiva structural defence compositions in the de- cell processes of eye conjunctiva
Hybrid protection liquid；3rd, using the cell in high static pressure technology pre-separation conjunctival tissue；4th, digested using composite nucleic acid enzyme method residual
Remaining nucleus；5th, loose crushing cell sheet is removed using detergent；6th, rinsed and de- using the hybrid protection liquid described in 2
Cell before processing is by clasmatosis.De- cell step sets suitable temperature, time and detergent, digestion enzyme concentration to conjunctiva
Tissue is handled.
Eye conjunctiva tissue is fixed on low temperature eye conjunctiva fixator and with after Trypan Blue, can it is efficient and convenient efficiently
Obtain the eye conjunctiva material that epithelial layer is smooth, tissue is smooth.
The dosage of various protectiveness compositions is that the concentration of chondroitin sulfate is that the concentration of 10-50g/L hyaluronic acids is 5-
50g/L；The concentration of dextran is 5-20g/L；TOB 2.5-5mg/L.
High static pressure pressure condition is 100-300MPa, and high static pressure frequency is 2~5 times, is every time 1~2 minute.
The concentration of DNA enzymatic is 100-1000U/ml, and the concentration of nuclease is 100-1000U/ml.DNA enzymatic or nuclease
The de- nucleus time is 1-4 hours, and treatment temperature is 15-30 degree.
Detergent condition is the concentration 0.1-5% of the concentration 0.1-5% dodecyl sodium sulfates of lauryl sodium sulfate
Triton X-100 concentration 0.5-8%.Detergent processing time is 1-4 hours.
Optionally simultaneously or separately carry out the processing of enzyme and detergent.
Brief description of the drawings
Fig. 1 is the DAPI colored graphs of de- cell rabbit conjunctival section
Fig. 2 is the DAPI colored graphs of normal rabbit conjunctival section
Fig. 3 is de- cell pig eye conjunctiva DAPI colored graphs
Fig. 4 is normal pig eye conjunctiva DAPI colored graphs
Fig. 5 is that eye conjunctiva tissue takes off the whole result using protection liquid of cell
Fig. 6 is that eye conjunctiva tissue takes off the whole result that protection liquid is not used of cell
Fig. 7 is that eye conjunctiva tissue takes off the result that detergent is used in cell processes
Fig. 8 is that eye conjunctiva tissue takes off the result that detergent is not used in cell processes
Fig. 9 is that eye conjunctiva tissue takes off the result that digestive ferment is used in cell processes
Figure 10 is that eye conjunctiva tissue takes off the result that digestive ferment is not used in cell processes
Figure 11 is new zealand white rabbit conjunctiva extirpation experiment model
Figure 12 is new zealand white rabbit conjunctiva defect amnion transplantation group experimental model
Figure 13 is that new zealand white rabbit conjunctiva defect takes off cell eye conjunctiva transplantation group experimental model
Embodiment
The present invention is specifically described below by embodiment, these embodiments are served only for being described in further detail explanation originally
Invention, it is impossible to be interpreted as limiting the scope of the present invention, nonessential adjustment is made within the scope of the present invention to be needed
Present invention side authorizes.
Embodiments of the invention 1
Add the eye conjunctiva tissue that the method for Trypan Blue obtains with low temperature fixator.By using low temperature fixator, energy
Flat apply of eye conjunctiva tissue is fixed on the slab of low temperature, eye conjunctiva and knot are clearly then differentiated by conventional Trypan Blue
Film undertissue, the eye conjunctiva material that last safe and efficient efficiently acquisition epithelial layer is smooth, tissue is smooth.
1st, whole that eye conjunctiva tissue is protected using protection liquid, protection formula of liquid is：RPMI-1640 culture mediums, add
Add chondroitin sulfate 25g/L, D-40 10g/L, TOB 2.5mg/L, tune pH value to 7.2, osmotic pressure is
350mOsm；
2nd, by the fresh eye conjunctiva after subconjunctival tissue has been removed, it is sealed in the polybag for filling protection liquid；
3rd, under the conditions of 300MPa high static pressures, handle 2 times, each time is 2 minutes；
4th, after conjunctiva is taken out, it is placed in the protection liquid of the DNA enzymatics of X-100+1000U/ml containing 1%Triton, temperature is
25 DEG C, shaking table speed is set as 100 revs/min, is handled 3 hours；
5th, after detergent and ferment treatment, take out conjunctiva and be placed in protection liquid and rinse 3 hours.
To remain that the integrality of conjunctival epithelium layer, the elastic of conjunctival tissue and due biology are special in de- cell processes
Property is not damaged
Embodiments of the invention 2
Add the eye conjunctiva tissue that the method for Trypan Blue obtains with low temperature fixator.By using low temperature fixator, energy
Flat apply of eye conjunctiva tissue is fixed on the slab of low temperature, eye conjunctiva and knot are clearly then differentiated by conventional Trypan Blue
Film undertissue, the eye conjunctiva material that last safe and efficient efficiently acquisition epithelial layer is smooth, tissue is smooth.
1st, whole that eye conjunctiva tissue is protected using protection liquid, protection formula of liquid is：
RPMI-1640 culture mediums, add chondroitin sulfate 40g/L, Dextran-20 g/L, TOB
2.5mg/L, tune pH value to 7.2, osmotic pressure 350mOsm；
2nd, by the fresh eye conjunctiva after subconjunctival tissue has been removed, it is sealed in the polybag for filling protection liquid；
3rd, under the conditions of 250MPa high static pressures, handle 5 times, each time is 1 minute；
4th, after conjunctiva is taken out, it is placed in the protection liquid containing 0.8% lauryl sodium sulfate+500U/ml DNA enzymatics, temperature
For 25 DEG C, shaking table speed is set as 100 revs/min, is handled 4 hours；
5th, after detergent and ferment treatment, take out conjunctiva and be placed in protection liquid and rinse 5 hours.
To remain that the integrality of conjunctival epithelium layer, the elastic of conjunctival tissue and due biology are special in de- cell processes
Property is not damaged.
Embodiments of the invention 3
Add the eye conjunctiva tissue that the method for Trypan Blue obtains with low temperature fixator.By using low temperature fixator, energy
Flat apply of eye conjunctiva tissue is fixed on the slab of low temperature, eye conjunctiva and knot are clearly then differentiated by conventional Trypan Blue
Film undertissue, the eye conjunctiva material that last safe and efficient efficiently acquisition epithelial layer is smooth, tissue is smooth.
1st, whole that eye conjunctiva tissue is protected using protection liquid, protection formula of liquid is：RPMI-1640 culture mediums, add
Add chondroitin sulfate 50g/L, D-40 5g/L, TOB 2.5mg/L, tune pH value to 7.2, osmotic pressure is
380mOsm；
2nd, by the eye conjunctiva after subconjunctival tissue has been removed, it is sealed in the polybag for filling protection liquid；
3rd, under the conditions of 200MPa high static pressures, handle 3 times, each time is 2 minutes；
4th, after conjunctiva is taken out, it is placed in the protection liquid containing 2% dodecyl sodium sulfate+2000U/ml DNA enzymatics, temperature
For 30 DEG C, shaking table speed is set as 100 revs/min, is handled 5 hours；
5th, after detergent and ferment treatment, take out conjunctiva and be placed in protection liquid and rinse 3 hours.
To remain that the integrality of conjunctival epithelium layer, the elastic of conjunctival tissue and due biology are special in de- cell processes
Property is not damaged.
Following comparing result is configured according to the de- cell step of embodiment 1.
Fig. 5 and Fig. 6 is that eye conjunctiva tissue takes off result of the cell whole process using protection liquid and whole unused protection liquid respectively
Compare.It is whole using sterilized water for injection replacement protection liquid that protection liquid group is not used.Pass through Fig. 5 and Fig. 6, it is seen that protection is not used
Liquid (sterilized water for injection) is although the de- cell of group is complete, epithelium missing, and uses protection liquid group epithelial structure complete.
Fig. 7 and Fig. 8 is the results contrast using detergent and unused detergent respectively.As shown in Figure 7 and Figure 8, use
The de- cell of detergent group is complete, and detergent group, which is not used, has obvious nucleus remaining.
Fig. 9 and Figure 10 is the results contrast using digestive ferment and unused digestive ferment respectively.It is it can be seen that thin using digestive ferment group
Born of the same parents are de- clean, and digestive ferment group, which is not used, has a large amount of nucleus to colour.
Figure 11, Figure 12 and Figure 13 are new zealand white rabbit conjunctiva extirpation experiment model comparison result respectively.Figure 11 is new west
Blue White Rabbit conjunctiva extirpation experiment model group, that is, cut off the eyelid of rabbit 1/4 and bulbar conjunctiva, and the eyeball surface for occurring defect after 2 weeks is rotten to the corn.
Figure 12 is new zealand white rabbit conjunctiva defect amnion transplantation group experimental model group, that is, after cutting off the eyelid of rabbit 1/4 and bulbar conjunctiva, while
Conjunctiva defect face row amnion transplantation flap coverage, the amnion tissue of amnion group has started to dissolve after 2 weeks, and damaged part does not have
New conjunctival tissue is grown, surface of a wound exposure.Figure 13 is that new zealand white rabbit conjunctiva defect takes off cell eye conjunctiva transplantation group experiment mould
Type, that is, after cutting off the eyelid of rabbit 1/4 and bulbar conjunctiva, while cell eye conjunctiva transplanting flap coverage, 2 Zhou Houyi are taken off in conjunctiva defect face row
The acellular conjunctiva of plant is in place, wound repair.
New zealand white rabbit conjunctiva defect model group, that is, cut off the eyelid of rabbit 1/4 and bulbar conjunctiva, lacked after occurring 2 weeks after 2 weeks
The eyeball surface of damage is rotten to the corn.In amnion transplantation group experimental model, the amnion tissue of amnion group has started to dissolve after 2 weeks, and damaged
Position does not have new conjunctival tissue to grow.Acellular conjunctiva group, dye within postoperative 14 days, be covered in the de- cell of eyeball defective region
Conjunctiva is complete, and fornix is normal.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (5)
- A kind of 1. acellular conjunctiva for keeping complete eye conjunctiva epithelium fiber layer structure, tissue fibers aligned orderly and biological nature Preparation method, it is characterised in that：Whole process uses the hybrid protection for including one or more protection conjunctiva functions and tissue integrity Liquid, preparation method comprise the following steps：(1) use takes conjunctiva fixator to obtain eye conjunctiva at low temperature, and passes through normal dyeing fast resolution institutional framework, it is ensured that Get complete conjunctiva；(2) the fresh eye conjunctiva after subconjunctival tissue will have been removed, has been sealed in the polybag for filling hybrid protection liquid, uses height Cell in static pressure pre-separation conjunctiva；(3) smudge cells core and loose crushing cell are removed using containing the hybrid protection liquid of DNA enzymatic and detergent；(4) rinsing removes unnecessary DNA enzymatic and cell fragment in hybrid protection liquid；(5) structural integrity, tissue fibers aligned orderly, the acellular conjunctiva for possessing biological nature are obtained；Wherein hybrid protection liquid is into being grouped into：RPMI-1640 culture mediums；The concentration of chondroitin sulfate is 10-50g/L；It is transparent The concentration of matter acid is 5-50g/L；The concentration of dextran is 5-20g/L；TOB 2.5-5mg/L, regulation pH value are 6.8- 7.2, osmotic pressure 300-400mOsm；The concentration 100-2000U/ml of the DNA enzymatic, de- nucleus time of DNA enzymatic are 1-4 hours, treatment temperature 15-30 Degree Celsius；The concentration of the detergent lauryl sodium sulfate is 0.1-5%, and the concentration of dodecyl sodium sulfate is 0.1-5%, Triton X-100 concentration is 0.5-8%, and detergent usage time is 1-4 hours.
- 2. preparation method as claimed in claim 1, wherein it is described take conjunctiva fixator it is whole during conjunctiva is taken keep 0~ 4 degrees Celsius of low temperature, conjunctiva can put down to apply to be easy to obtain the complete conjunctiva that epithelial surface is smooth, tissue is smooth in the fixator, is passed through Trypan Blue, which understands, distinguishes conjunctiva and subconjunctival tissue.
- 3. preparation method as claimed in claim 1, high static pressure technology includes different pressure, time and frequency parameter.
- 4. preparation method as claimed in claim 1, wherein rinsing removes unnecessary nuclease and cell in hybrid protection liquid The time rinsed in fragment is 0.5~5 hour.
- 5. preparation method as claimed in claim 3, wherein described high static pressure pressure condition is 100-300MPa, high static pressure frequency Rate is 2-5 times, is every time 1~2 minute.
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| US20080195229A1 (en) * | 2007-02-09 | 2008-08-14 | Quijano Rodolfo C | Decellularized pericardial tissue |
| CN101590292A (en) * | 2009-06-11 | 2009-12-02 | 山东省眼科研究所 | The preparation method of acellular conjunctiva matrix and application |
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