CN1916166B - Method for preparing epithelium of autologous cornea - Google Patents
Method for preparing epithelium of autologous cornea Download PDFInfo
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- CN1916166B CN1916166B CN2006101286980A CN200610128698A CN1916166B CN 1916166 B CN1916166 B CN 1916166B CN 2006101286980 A CN2006101286980 A CN 2006101286980A CN 200610128698 A CN200610128698 A CN 200610128698A CN 1916166 B CN1916166 B CN 1916166B
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Abstract
This invention discloses a method for preparing auto-corneal epithelia. The method comprises: (1) performing primary culture of cells of patients' oral mucosa tissue or corneal limbal tissue for 3-15 days; (2) performing proliferation sub-culture for 7-30 days; (3) using fibrinogen as the supporting material to construct tissue engineering corneal epithelia together with the proliferation sub-cultured cells. The method can avoid the problems of immunological rejection, pollution, ununiform cell growth caused by the biological support, and slow degradation faced by presents techniques. The preparation of auto-corneal in this invention is not restricted by corneal epithelia damage, and suitable corneal is available for patients at any situation.
Description
Technical field
The present invention relates to a kind of preparation method of epithelium of autologous cornea.
Background technology
Burn, chemical burn, eye epidermis tumour, supersensitivity or radiation injury, heredity illness in eye, the suppuration of eye surface and other syndromess can cause the cornea epithelial cell shedding, and make a conjunctiva be covered in the cornea surface, produce a large amount of new vessel hyperplasia of cornea, recurrent pterygium even cornea disease such as blind.For the treatment of this class ophthalmic diseases, the excision that generally need undergo surgery, but still can recur and hyperplasia after the excision.Thereby, fundamentally treat this type of disease, must make up the weave construction of cornea and the stem cell of additional corneal limbus.
In order to solve this type of problem, people have invented the biotechnology cornea, it be by the cornea stem cell that will obtain from embryo's corneal limbus tissue or opposite side eye's cornea edge in vitro culture during to third and fourth generation, be structured on the biological support and form.It can be used for treating the corneal transplantation of the patient because corneal injury that a variety of causes causes or cornea come off.
But, embryo's corneal limbus tissue biological engineering cornea epithelium exists many deficiencies: first, because embryo's corneal limbus is tissue-derived in allosome, corneal epithelium prepared therefrom can with the immunocyte generation immunological rejection in patient's corneal limbus, thereby increased patient's the misery and the possibility of operative failure.The second, because the biological support that the preparation cornea adopts is collagen protein, contact lens or amnion, make cornea stem cell and epithelial cell be difficult to even growth on its surface, operation back degradation speed is slow, thereby has limited clinical application.The 3rd, the mouse source property trophocyte that the growth of corneal limbus cell is adopted has the infection that the potential non-human is originated; And the foetal calf serum substratum that adopts, also being easy to infect the transmissible disease of ox etc., these potential sources of infection are threats for the patient.
Application number is that 03150375.6 Chinese patent application discloses a kind of epithelium of autologous cornea and preparation method thereof, this corneal epithelium be with derive from the patient from the corneal epithelial stem cells of body after amplification in vitro and differentiation, be inoculated into to cultivate on the scleroproein biological support to make up and form.This tissue engineering comea has overcome above-mentioned shortcoming, but when bilateral cornea position all went wrong, the corneal limbal stem cell autograft source had just lacked.
Thereby, need a kind of so new organizational project epithelium of autologous cornea, it can overcome the shortcoming of above-mentioned non-auto corneal, and it comes from non-cornea tissue simultaneously, so that when the bilateral cornea tissue all can't obtain, still can construct the epithelium of autologous cornea that can be used for transplanting.In addition, a kind of like this preparation method of organizational project epithelium of autologous cornea can be arranged preferably, this method can adopt non-auto corneal cell, also can prepare corneal epithelium with the auto corneal marginal cell, so that this method can have versatility, is convenient to grasp and operation.
Summary of the invention
Purpose of the present invention provides a kind of preparation method of organizational project epithelium of autologous cornea, this method can adopt the oral mucosa cell from body to prepare corneal epithelium, thereby row except when the bilateral eyes when all going wrong to the restriction in cornea marginal cell source, this method can also be used the corneal limbus cell preparation corneal epithelium from body simultaneously.
The preparation method of organizational project epithelium of autologous cornea provided by the present invention comprises the steps:
(a) former being commissioned to train supported the patient from the oral mucosas tissue of body or cell 3-15 days of corneal limbus tissue;
(b) the gained cell was gone down to posterity amplification cultivation 7-30 days; And
(c) be timbering material with the Fibrinogen, be built into the tissue engineering comea epithelium with the cultured cells that goes down to posterity.
According to an organizational project epithelium of autologous cornea preparation method's of the present invention preferred version, with resulting tissue engineering comea epithelium at 37 ℃, 5%CO
2Down, continue to cultivate 5-45 minute, preferred 10-30 minute, more preferably 20 minutes, be used for transplanting after adding 1ml DMEM again.
A preferred version according to organizational project epithelium of autologous cornea preparation method of the present invention, carry out former being commissioned to train of cell supports and the cultivation of going down to posterity with the cornea nutrient solution, described cornea nutrient solution is selected from DMEM (the lowest base nutrient solution of Dulbecco improvement) or Epilife (available from U.S. CascadeBiologics company), is preferably Epilife; Add the pituitary gland extracting solution of the following material in cornea nutrient solution total amount: 0.1-0.5% (mass percent), the epidermal growth factor of 0.1-5ng/ml, the hydrocortisone of 0.05-0.5 μ g/ml, the Transferrins,iron complexes of 1-20 μ g/ml and the Regular Insulin of 1-20 μ g/ml alternatively again, wherein the further preferred dosage of each material is pituitary gland extracting solution 0.3%, epidermal growth factor 3ng/ml, hydrocortisone 0.2 μ g/ml, Transferrins,iron complexes 10 μ g/ml and Regular Insulin 10 μ g/ml.
According to an organizational project epithelium of autologous cornea preparation method's of the present invention preferred version, described culture vessel is culture dish, six orifice plates etc., the more preferably Tissue Culture Dish of 35mm.
According to an organizational project epithelium of autologous cornea preparation method's of the present invention preferred version, step (a) is described to be to take from the oral mucosas tissue piece of patient from the 1-3 square millimeter at oral mucosa positions such as body hypogloeeis, cheek inboard or lingual surface from body oral mucosas tissue; Corneal limbus is organized as the tissue block of the 1-2 mm square of taking from the normal cornea edge of patient.
A preferred version according to organizational project epithelium of autologous cornea preparation method of the present invention, in step (a) before, with gentamicin sterilize described oral mucosas tissue or described corneal limbus tissue, its concentration is 50-200mg/ml, be preferably 100mg/ml, use 0.05% pancreatin/EDTA liquid digestion after 20 minutes again, with DMEM flushing tissue 2 times, to remove the digestive ferment on surface.
A preferred version according to organizational project epithelium of autologous cornea preparation method of the present invention, in step (a), tissue block is shredded and is uniformly distributed in the surface, inside of culture vessel, add the cornea nutrient solution carry out former be commissioned to train foster, changed a nutrient solution in every 2-3 days, changed a nutrient solution in preferred per 3 days, incubation time is 4 days-12 days, preferred 5 days-10 days, more preferably 7 days.
According to an organizational project epithelium of autologous cornea preparation method's of the present invention preferred version, in step (b), use the cornea nutrient solution at 37 ℃, 5%CO
2Down, changed a nutrient solution in every 2-3 days, reach at cell and go down to posterity after 50%-90% compiles, preferably reach 70% and go down to posterity after compiling, cultivated altogether 9 days-25 days at cell, preferred 12 days-20 days, preferred again 13 days-16 days, more preferably 14 days.
A preferred version according to organizational project epithelium of autologous cornea preparation method of the present invention, in step (c), cumulative volume with system to be made up in the culture vessel is as the criterion, the Fibrinogen that with dosage is 1-8mg/ml earlier is tiled in the culture dish bottom surface, and then be that human blood coagulation, the dosage of 1-5 unit (unit)/ml is calcium ion and about 20000-80000 cell that has gone down to posterity of 1-5 μ mol/ml with dosage, at room temperature mix with the Fibrinogen of completing, making the thickness of mixing liquid in culture vessel is 0.5mm-1.5mm.
A preferred version according to organizational project epithelium of autologous cornea preparation method of the present invention, in step (c), cumulative volume with system to be made up in the culture vessel is as the criterion, the human fibrinogen who earlier with dosage is 5mg/ml is tiled in the culture dish bottom surface, and then is that human blood coagulation, the dosage of 2 units/ml is the CaCl of 2.5 μ mol/ml with dosage
2With about 40000 cells that gone down to posterity, mix with the human fibrinogen who completes down at 20-25 ℃, making the thickness of mixing liquid in culture vessel is 1mm.
According to the preparation method's of an organizational project epithelium of autologous cornea of the present invention preferred version, Fibrinogen or lyophilized human fibrinogen that described Fibrinogen is behaved; Described thrombin extracts from human or animal's blood, be preferably human blood coagulation, optional any or multiple from thrombin II-XII, the thrombin that extracts from animal is meant the thrombin of the animal self of inhuman reorganization, described animal can be pig, ox etc., preferred pig.
A preferred version according to the preparation method of organizational project epithelium of autologous cornea of the present invention, described people's Fibrinogen prepares as follows: get the 200ml human plasma, under 4 ℃, the condition of 3500 commentaries on classics/min centrifugal 30 minutes, abandon supernatant liquor, add 10ml water for injection, make suspension liquid, preserve standby.
Organizational project epithelium of autologous cornea according to the method for the invention preparation can be used as the biomaterial that corneal transplantation is used, and portable is to the patient trauma anterior corneal surface, thus reparation and recovery cornea function.
The preparation method of organizational project epithelium of autologous cornea of the present invention originates as corneal epithelial cell with oral mucosa, the infection in the immunological rejection that has overcome prior art, non-human source and its biological support be unsuitable for cell evenly grow, degrade wait defective slowly in, make the preparation of auto corneal not be subjected to the restriction of bilateral corneal epithelial defect, the cornea that makes the patient under any circumstance can both obtain being fit to.In addition, this method also can be used the corneal limbus cell and prepare corneal epithelium, and its effect is better than existing method.Like this, just can select to use corneal limbus cell/oral mucosa cell, make operation more flexible.
Description of drawings
The invention will be further described below in conjunction with the drawings and specific embodiments.
Figure 1A is the finished product appearing diagram in cell source with the oral mucosa for the present invention, and Figure 1B is the finished product appearing diagram in cell source with the corneal limbus for the present invention;
Fig. 2 A for cultivate successfully be the morphology photo of corneal epithelial cell under 100 times of opticmicroscopes in cell source with the oral mucosa, Fig. 2 B be cultivate successfully be the morphology photo of corneal epithelial cell under 100 times of opticmicroscopes that cell is originated with the corneal limbus;
Fig. 3 A, 4A, 5A, 6A and 7A are when adopting oral mucosa to originate as cell, the cornea situation map before patient's art;
Fig. 3 B, 4B, 5B, 6B and 7B are when adopting oral mucosa to originate as cell, the cornea situation map of patient's postoperative;
Fig. 8 A, 9A, 10A, 11A are when adopting the cornea edge to originate as cell, the cornea situation map before patient's art;
Fig. 8 B, 9B, 10B, 11B are when adopting the cornea edge to originate as cell, the cornea situation map of patient's postoperative.
Embodiment
From normal people's cornea or oral mucosa, isolate stem cell, carry out vitro culture, propagation and differentiation, in two to three weeks, make hundreds of ten thousand times of these cell proliferations, the epithelial cell kind that then these is had stem cell is on a kind of microbial film, thereby grow up to the biotechnology cornea, with its diseased region of being transplanted to the corneal injury patient, repair, make that patient symptom alleviates, vision restoration.
Before determining to get cornea or oral mucosas tissue, by medical worker with qualifications of a licensed doctor to patient or family numbers of patients explanation reasons, strive to such an extent that patient or family members agree, the position of drawing materials of corneal epithelium is determined by the medical worker with qualifications of a licensed doctor, and is drawn materials according to the operation principle of hospital's regulation.The patient checks UP and chemically examines, and comprises the inspection of routine inspection, transmissible disease cause of disease (as HIV, hepatitis virus etc.).The oral mucosa of being got is put into sterile chamber by the professional immediately, and refrigeration (below 10 ℃) immediately, and Standard operation procedure SOP is in accordance with regulations handled then.
Embodiment 1: oral mucosa is the preparation of the organizational project epithelium of autologous cornea in cell source
One, the extraction of oral mucosas tissue's healthy cell and former be commissioned to train foster
1. Operation theatre is drawn materials (hundred grades of purifying airs): the tissue block of being got 1mm * 3mm size by medical practitioner from patient's hypogloeeis oral mucosa.
2. the preservation of the tissue of getting: in sending to the laboratory transportation, tissue block can be put into and contain the 15ml preservation pipe preservation (4 ℃) of preserving liquid, described preservation liquid contains DMEM and 10% serum.
3. sterilization in Bechtop, cleaning, pruning tissue: get fresh tissue or tissue, use the PBS (phosphoric acid buffer) that contains the 100mg/ml gentamicin to wash 3 times, prune unnecessary fat and subcutaneous connective tissue through preserving.
4. tissue digestion (Thermo CO
2Incubator): above-mentioned tissue block is positioned in the 35mm culture dish, adds concentration and be pancreatin/EDTA liquid of 0.05%, digest 20 minutes, and tissue block mentioned with tweezers put into DMEM flushing 2 times, remove surperficial digestive ferment.
5. cellular segregation: postdigestive tissue block is shredded and be uniformly distributed in the inner surface of culture dish of 35mm, add 0.5ml cornea nutrient solution and cultivate, changed nutrient solution once, and cultivated harvested cell after 5 days in per three days.
6. cell is identified
1) morphology: adopt and be inverted observation by light microscope.Cell is multiangular more, and the angle is the obtuse angle, and cell is round property.The cell appearance transparent, cell is tightly linked into single or multiple lift.Be the normal oral mucosa epithelial cell of typical case.
2) cell phenotype: adopt the antibody (American I CN company) of Keratin keratoprotein 3 to carry out cellular immunization antigen association reaction, be accredited as the normal oral mucosa epithelial cell of typical case.
3) carinogenicity: adopt the nude mice test, see " the consideration main points that U.S. FDA is identified and accused with cell strain about biological products production ", as a result non-carcinogenesis.
4) aseptic detection: undertaken by " Chinese biological goods rules " (2000 editions) general rule " biological products sterility test rules " A/B item, the result meets aseptic requirement.
5) cell survival rate: adopt trypan blue (trypan blue) staining inspection, the cell of nuclear staining is a dead cell, and non-staining cell is a viable cell, counts total cell count and dead cell number, calculates cell survival rate.
Cell survival rate=[(total cell count-dead cell number)/total cell count] * 100%
After measured, cell survival rate surpasses 80%.
Two, cell cultures goes down to posterity
Add Epilife (U.S. Cascade Biologics company, catalogue sign indicating number M-EPI-500-CA) in cell, the 35mm culture dish is gone in inoculation, at Thermo CO
2Incubator, 37 ℃, 5%CO
2Under cultivate, changed liquid 1 time in per 3 days, reach 70% at epithelial cell and go down to posterity after compiling cell, cultivated 14 days.
Three, make up the organizational project epithelium of autologous cornea
1. prepare the human fibrinogen
With 200ml blood plasma 4 ℃ centrifugal, 3500 commentaries on classics/min, centrifugal 30 minutes, abandon supernatant liquor, add 10ml water for injection, make suspension liquid, preserve standby.
2. structure corneal epithelium
Adopting people's Fibrinogen is material, is 5mg/ml according to concentration, adds 2unit/ml people's thrombin (purchasing the company in Sigma) and 2.5 μ mol/ml CaCl
2, add about about 40000 cells that are passaged to the third generation simultaneously, mix under the room temperature, pour at once in the culture dish of 35mm, form the thick liquid level of 0.8mm.Culture dish is put into 37 ℃, 5%CO
2After 20 minutes, add 1ml DMEM nutrient solution and get final product in the incubator.
Four, using method and effect
Before the use, with the nutrient solution flushing that does not contain serum three times.Before the transplanting of cornea, need the external coat doctor patient to be carried out the cyclic diseased region excision of 360 degree, organizational project auto corneal of the present invention is made four pins be sutured in the ring-type cutting part, oppress with contact lens, adopt two pins to sew up in eyelid, dermal sutures out after one week has newborn corneal epithelium to form, and the function and the eyesight of cornea improve.
The storage of product and transportation should be carried out in 2 ℃-10 ℃, shady and cool, dry, the environment that cleans, avoid sunlight direct projection, non-corrosiveness gas, no weight.
The present invention is mainly used in burn, chemical burn, and eye epidermis tumour, supersensitivity, radiation injury, heredity illness in eye, a cornea epithelial cell shedding that suppurate in the surface and other syndromess cause, conjunctiva are covered in the cornea surface and losing one's sight of causing.
Embodiment 2: corneal limbus is the preparation of the organizational project epithelium of autologous cornea in cell source
One, the extraction of corneal limbus tissue health cell and former be commissioned to train foster
1. Operation theatre is drawn materials (hundred grades of purifying airs): get 1mm * 1mm size tissue block by medical practitioner from patient's the normal cornea edge of opposite side.
2. the preservation of the tissue of getting: in sending to the laboratory transportation, tissue block can be put into and contain the 15ml preservation pipe preservation (4 ℃) of preserving liquid, described preservation liquid contains DMEM and 10% serum.
3. sterilization in Bechtop, cleaning, pruning tissue: get fresh tissue or tissue, use the PBS that contains the 100mg/ml gentamicin to wash 3 times, prune unnecessary fat and subcutaneous connective tissue through preserving.
4. tissue digestion (Thermo CO
2Incubator): above-mentioned tissue block is positioned in the 35mm culture dish, adds concentration and be pancreatin/EDTA liquid of 0.05%, digest 20 minutes, and tissue block mentioned with tweezers put into DMEM flushing 2 times, remove surperficial digestive ferment.
5. cellular segregation: postdigestive tissue block is shredded and be uniformly distributed in the inner surface of culture dish of 35mm, add 0.5ml cornea nutrient solution and cultivate, changed nutrient solution once, and cultivated harvested cell after 14 days in per three days.
6. cell is identified
1) morphology: adopt and be inverted observation by light microscope.Cell is multiangular more, and the angle is the obtuse angle, and cell is round property.The cell appearance transparent, cell is tightly linked into single or multiple lift.Be the normal corneal epithelial cell of typical case.
2) cell phenotype: adopt the antibody (American I CN company) of Keratin keratoprotein 3 to carry out cellular immunization antigen association reaction, be accredited as the normal corneal epithelial cell of typical case.
3) carinogenicity: adopt the nude mice test, see " the consideration main points that U.S. FDA is identified and accused with cell strain about biological products production ", as a result non-carcinogenesis.
4) aseptic detection: undertaken by " Chinese biological goods rules " (2000 editions) general rule " biological products sterility test rules " A/B item, the result meets aseptic requirement.
5) cell survival rate: adopt the inspection of trypan blue staining, the cell of nuclear staining is a dead cell, and non-staining cell is a viable cell, counts total cell count and dead cell number, calculates cell survival rate.
Cell survival rate=[(total cell count-dead cell number)/total cell count] * 100%
After measured, cell survival rate surpasses 80%.
Two, cell cultures goes down to posterity
Cell cultures: add Epilife in cell, the 35mm culture dish is gone in inoculation, at ThermoCO
2Incubator, 37 ℃, 5%CO
2Under cultivate, changed liquid 1 time in per 3 days, reach 70% at epithelial cell and go down to posterity after compiling cell, cultivated 14 days.
Three, make up the organizational project epithelium of autologous cornea
1. prepare the human fibrinogen
With 200ml blood plasma 4 ℃ centrifugal, 3500 commentaries on classics/min, centrifugal 30 minutes, abandon supernatant liquor, add 10ml water for injection, make suspension liquid, preserve standby.
2. structure corneal epithelium
Adopting people's Fibrinogen is material, is 5mg/ml according to concentration, adds 2unit/ml people's thrombin (purchasing the company in Sigma) and 2.5 μ mol/ml CaCl
2, add about about 40000 cells that are passaged to the third generation simultaneously, mix under the indoor temperature, pour at once in the culture dish of 35mm, form the thick liquid level of 1mm.Culture dish is put into 37 ℃, 5%CO
2After 20 minutes, add 1ml DMEM nutrient solution and get final product in the incubator.
Four, using method and effect
Before the use, with the nutrient solution flushing that does not contain serum three times.Before the transplanting of cornea, need the external coat doctor patient to be carried out the cyclic diseased region excision of 360 degree, organizational project auto corneal of the present invention is made four pins be sutured in the ring-type cutting part, oppress with contact lens, adopt two pins to sew up in eyelid, dermal sutures out after one week has newborn corneal epithelium to form, and the function and the eyesight of cornea improve.
The storage of product and transportation should be carried out in 2 ℃-10 ℃, shady and cool, dry, the environment that cleans, avoid sunlight direct projection, non-corrosiveness gas, no weight.
The present invention is mainly used in burn, chemical burn, and eye epidermis tumour, supersensitivity, radiation injury, heredity illness in eye, a cornea epithelial cell shedding that suppurate in the surface and other syndromess cause, conjunctiva are covered in the cornea surface and losing one's sight of causing.
The clinical report of organizational project epithelium of autologous cornea
One, Case definition
Limbal stem cell lacks Case definition: growth of corneal limbus new vessel or corneal epithelium conjunctivaization are more than or equal to a quadrant.
The long-term damaged Case definition of corneal epithelium: the corneal epithelial defect time is more than 7 days.
Etiological diagnosis:
1. chemical burn and thermal burns such as acid, alkali;
2. drug allergy;
3. a large amount of shallow-layer new vesseles of the corneal limbus due to a variety of causes;
4. recurrent pterygium.
Two, include the case standard in
1. meeting limbal stem cell lacks or the long-term damaged Case definition of corneal epithelium.
2. patient age 〉=12 years old can cooperate every inspection.
3. simple eye ill, branch hole there is not obvious illness in eye history.
4. sign the Informed Consent Form person.
5. basic lacrimal secretion test 〉=5mm (filter paper unified specification).
6. slit lamp examination
Cornea and conjunctiva do not have obvious infection and immune inflammation performance.
Cornea rebirth blood vessel is positioned at subcutaneous and matrix shallow-layer (≤1/4 corneal thickness).
Symblepharon≤2 quadrant.
Fissura palpebrae is fully closed.No entropion trichiasis.
Three, draw materials
Check blood, routine urinalysis, liver, renal function, Australia antigen, hiv antibody before the art.
Draw materials and in regular Operation theatre, to carry out.
The position of drawing materials: the hypogloeeis oral mucosa is got the tissue block of 1mm * 1mm size.The normal cornea edge of opposite side is got 1mm * 1mm size tissue block.
Four, laboratory vitro culture
Place immediately behind the specimen sampling and preserve liquid, be placed in 2 ℃-10 ℃ the insulation can transportation and preserve, in 6 hours, cultivate, cultivate from body oral mucosa/cornea limbal stem cell with the bioengineered tissue technology, and meet its organizational project epithelium of autologous cornea Registering product standard.
Five, reject the case standard
Oral mucosa/cornea limbal stem cell is cultivated failure, and the product of cultivating gained does not meet product standard person.
Six, transplantation
1. some microbiotic collyrium 3 days, 4 times/day before the art.
2. operation should be carried out at regular Operation theatre microscopically.
Corneal epithelium strike off scope should be greater than pathology 2mm
2
4. graft and plant bed should be fitted closely, and do not have hematocele or hydrops are residual.
5. fixedly plant sheet with 8-0 absorbable thread interrupted suture.
6. if any symblepharon, conjunctiva thoroughly should be separated with cornea tissue, fully hemostasis and conjunctiva retreated.
7. art finishes, and the art eye is coated with Tobradex eye ointment and wrapping.
Seven, post surgery treatment
1. wrapped up continuously 2 days, and opened a medicine on the 3rd day.
2. oral antibiotic is 3 days, opens wrapping back point Tobradex 1 week of liquid, 4 times/day.Can be with the time point worker's tear of choosing, but the forbidding epidermal growth factor.
Eight, efficacy assessment standard
(1) produce effects:
1. cornea rebirth blood vessel reduces more than secondary (containing secondary) or disappears.
2. the corneal epithelial defect reparation is more than secondary (containing secondary) or disappear.
3. the conscious irritation of eye alleviates more than secondary (containing secondary) or disappears.
(2) effective:
Cornea rebirth blood vessel reduce one-level or more than.
The corneal epithelial defect reparation one-level or more than.
The conscious irritation of eye alleviate one-level or more than.
(3) invalid: every index does not reach above-mentioned standard person.
Produce effects, effectively criterion: satisfy (1) bar at least and add (2) or (3) bar.
Nine, data management and statistics
1. data gathering
All MethodsThe cases enrolled all must be finished the case observation table, and the investigator will observe, check result is timely, accurate, complete, standard, be recorded in case history and the case observation table truly.
After all data input database and the check and correction, hand over the statistical study personnel to carry out statistical study database, and statistical report is provided, hand over the main investigator of clinical trial to write out the clinical summary report.
2. statistical study
According to data character, to basic data data, efficacy analysis (each index analysis) and safety analysis, carry out descriptive statistics respectively.
The continuous data statistical method: changing conditions adopts paired sample t check before and after in the observation group.
Enumeration data statistical method: adopt definite stochastic method.
Grade type data statistical approach: changing conditions symbolization rank test before and after in the observation group.
3. statistical software: all statistics all utilize SAS JMP5.0 software to carry out statistical analysis, adopt two-tailed test, with P≤0.05 as statistical significance is arranged.
Ten, conclusion
This product has carried out clinical study in Beijing liang man front three hospital, patient and 18 routine corneal limbus that selected altogether 16 routine oral mucosas are the cell source are the patient in cell source, observing its cornea damaged for a long time for corneal epithelium and limbal stem cell shortage patient of postevaluation through six months stimulates the improvement situation of disease, corneal epithelial defect, cornea rebirth blood vessel and eyesight, and the curative effect of each MAIN OUTCOME MEASURES is as follows:
(1) oral mucosa is the cell source
1. cornea irritation: obviously improve 8 examples, improve 6 examples, do not have 2 examples of improvement, improvement rate 87.5%;
2. corneal epithelial defect: obviously improve 8 examples, improve 4 examples, do not have 4 examples of improvement, improvement rate 75%;
3. cornea rebirth blood vessel: obviously improve 6 examples, improve 5 examples, do not have 5 examples of improvement, improvement rate 68.75%;
4. eyesight: obviously improve 2 examples, improve 2 examples, do not have 12 examples of improvement, improvement rate 25%.
Total appraisal:
Produce effects: obviously improve+(2) or (1) obvious 6 examples altogether of improving (3);
Effectively: 4 examples are altogether obviously improved or improved in (3) improvement+(2) or (1);
Promptly overall effective 10 examples, total effective rate 62.5%.
In this Figure of description 3A-7A and 3B-7B exemplified before patient's art that oral mucosa is the cell source respectively and the cornea situation map of postoperative to explain, wherein:
Fig. 3 A and Fig. 3 B derive from patient Lee * *, the woman, 32 years old, the diagnosis: thermal burn, the course of disease 40 days; Fig. 4 A and Fig. 4 B derive from patient Yan * *, the woman, 38 years old, the diagnosis: viral keratitis causes the corneal epithelium erosion, the course of disease 34 months; Fig. 5 A and Fig. 5 B derive from the patient and open * *, man, 28 years old, recurrent pterygium, course of disease June; That Fig. 6 A and Fig. 6 B derive from the patient is normal * *, man, 26 years old, Peracetic Acid burn, the course of disease 30 days; Fig. 7 A and Fig. 7 B derive from the patient and open * *, the man, 40 years old, pteryium, course of disease June.
As can be seen from the figure behind operation transplantation organizational project auto corneal of the present invention, patient's the state of an illness is improved.
(2) corneal limbus is the cell source
1. cornea irritation: obviously improve 8 examples, improve 7 examples, do not have 3 examples of improvement, improvement rate 83.3%;
2. corneal epithelial defect; Obviously improve 8 examples, improve 6 examples, do not have 4 examples of improvement, improvement rate 77.8%;
3. cornea rebirth blood vessel: obviously improve 6 examples, improve 7 examples, do not have 5 examples of improvement, improvement rate 72.2%;
4. eyesight: obviously improve 2 examples, improve 3 examples, do not have 13 examples of improvement, improvement rate 27.8%.
Total appraisal:
Produce effects: obviously improve+(2) or (1) obvious 6 examples altogether of improving (3);
Effectively: 7 examples are altogether obviously improved or improved in (3) improvement+(2) or (1);
Promptly overall effective 13 examples, total effective rate 72.2%.
In this Figure of description 8A-11A and 8B-11B exemplified before patient's art that corneal limbus is the cell source respectively and the cornea situation map of postoperative to explain, wherein:
Fig. 8 A and Fig. 8 B derive from patient's history * *, man, 40 years old, diagnosis: blood vessel hyperplasia, course of disease March; Fig. 9 A and Fig. 9 B derive from the patient horse * *, the woman, 28 years old, the diagnosis: recurrent pterygium, blood vessel hyperplasia, the course of disease 5 months; Figure 10 A and Figure 10 B derive from the patient plum * *, man, 35 years old, molten iron burn, the course of disease 20 days; Figure 11 A and Figure 11 B derive from patient Yin * *, man, 46 years old, alkali burn, the course of disease 30 months.
As can be seen from the figure behind operation transplantation organizational project auto corneal of the present invention, patient's the state of an illness is improved.
Claims (6)
1. the preparation method of an epithelium of autologous cornea comprises the steps:
(a) former being commissioned to train supported the cell of patient from the oral mucosas tissue of body: from body oral mucosas tissue is to take from the tissue block of patient from the 1-3 of body hypogloeeis, cheek inboard or lingual surface square millimeter, tissue block is shredded and is uniformly distributed in the surface, inside of culture vessel, add the cornea nutrient solution carry out former be commissioned to train foster, changed a nutrient solution in every 2-3 days, incubation time is 5-10 days;
(b) to the gained cell amplification cultivation that goes down to posterity: with the cornea nutrient solution at 37 ℃, 5%CO
2Down, changed a nutrient solution in every 2-3 days, reach at cell and go down to posterity after 50%-90% compiles, cultivated 12-20 days; And
(c) with the Fibrinogen be timbering material, be built into the tissue engineering comea epithelium with the cultured cells that goes down to posterity: the cumulative volume with system to be made up in the culture vessel is as the criterion, the Fibrinogen that with dosage is 1-8mg/ml earlier is tiled in the culture dish bottom surface, and then be that human blood coagulation, the dosage of 1-5 unit/ml is calcium ion and 20000-80000 cell that has gone down to posterity of 1-5 μ mol/ml with dosage, at room temperature mix with the Fibrinogen of completing, making the thickness of resulting mixing liquid in culture vessel is 0.5mm-1.5mm;
Wherein, in step (a) before, with the gentamicin described oral mucosas tissue of sterilizing, its concentration is 50-200mg/ml, uses pancreatin/EDTA liquid digestion of 0.05% after 20 minutes again, with DMEM flushing tissue 2 times;
Described cornea nutrient solution is DMEM or Epilife.
2. method according to claim 1 is characterized in that, with resulting tissue engineering comea epithelium at 37 ℃, 5%CO
2Continue to cultivate 5-45 minute down.
3. method according to claim 2, it is characterized in that described cornea nutrient solution is that to be added with in the mass percent of cornea nutrient solution total amount be the DMEM or the Epilife of the Regular Insulin of the Transferrins,iron complexes of hydrocortisone, 1-20 μ g/ml of epidermal growth factor, the 0.05-0.5 μ g/ml of pituitary gland extracting solution, the 0.1-5ng/ml of 0.1-0.5% and 1-20 μ g/ml.
4. method according to claim 3, it is characterized in that described cornea nutrient solution is the DMEM or the Epilife of the Regular Insulin of the Transferrins,iron complexes of the hydrocortisone that is added with epidermal growth factor that mass percent in cornea nutrient solution total amount is 0.3% pituitary gland extracting solution, 3ng/ml, 0.2 μ g/ml, 10 μ g/ml and 10 μ g/ml.
5. method according to claim 1 is characterized in that, earlier is that the human fibrinogen of 5mg/ml is tiled in the culture dish bottom surface with dosage, and then is that human blood coagulation, the dosage of 2 units/ml is the CaCl of 2.5 μ mol/ml with dosage
2With 40000 cells that gone down to posterity, mix with the human fibrinogen who completes down at 20-25 ℃, making the thickness of mixing liquid in culture vessel is 1mm.
6. the preparation method of an epithelium of autologous cornea comprises the steps:
(a) the normal cornea edge of opposite side that exsomatizes from the patient is got 1mm * 1mm size tissue block, with the PBS flushing that contains the 100mg/ml gentamicin 3 times, use 0.05% pancreatin/EDTA liquid digestion after 20 minutes again, organize 2 times with the DMEM flushing, tissue block is shredded and is uniformly distributed in the surface, inside of culture vessel, add the cornea nutrient solution and carry out formerly being commissioned to train fosterly, changed a nutrient solution, and cultivated harvested cell after 14 days in per 3 days;
(b) in cell, add cornea nutrient solution Epilife, at 37 ℃, 5%CO
2Under cultivate, changed liquid 1 time in per 3 days, reach 70% at cell and go down to posterity after compiling cell, cultivated 14 days; And
(c) adopting people's Fibrinogen is material, is 5mg/ml according to concentration, adds 2unit/ml people's thrombin and the CaCl of 2.5 μ mol/ml
2, add 40000 cells that are passaged to the third generation simultaneously, mix under the indoor temperature, pour at once in the culture dish of 35mm, form the thick liquid level of 1mm; Culture dish is put into 37 ℃, 5%CO
2After 20 minutes, add 1ml DMEM nutrient solution and get final product in the incubator.
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| EP3400287B1 (en) * | 2016-01-04 | 2020-12-30 | The Board of Trustees of the Leland Stanford Junior University | Gene therapy for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes |
| CN107326003B (en) * | 2017-06-18 | 2020-09-25 | 广东博溪生物科技有限公司 | 3D model constructed in vitro by using serum-free culture solution and construction method thereof |
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