CN1631138A - Cornea middle term preserving fluid - Google Patents
Cornea middle term preserving fluid Download PDFInfo
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- CN1631138A CN1631138A CN 200410091007 CN200410091007A CN1631138A CN 1631138 A CN1631138 A CN 1631138A CN 200410091007 CN200410091007 CN 200410091007 CN 200410091007 A CN200410091007 A CN 200410091007A CN 1631138 A CN1631138 A CN 1631138A
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Abstract
Disclosed is a cornea middle term preserving fluid which comprises RPMI 1640 culture liquid, chondroitin sulfate, hyaluronic acid, HEPES cushioning liquid, dexamethasone, low-molecular-weight seaweed polysaccharides, Tobramycin or vancomycin, the preserving fluid has stabilized cell activity during storage period, and higher endothelial cell metabolizing function can be maintained than the US's Opthiol.
Description
Technical field
The present invention relates to a kind of improvement of ophthalmology medical product, specifically is that a kind of cornea is preserved liquid mid-term.It belongs to the Chemical composition that technical field.
Background technology
In the cornea activity store method of prior art: traditional wet room preservation method is arranged, and this method is simple, and shortcoming is the preservation of cornea time to lack very much (only 2 days).Have patent documentation CN1262866A to disclose a kind of cornea activity preservative fluid, it comprises, MEM cell culture medium, chondroitin sulfate, D-40, dexamethasone, antibiotic and HEEPES liquid.Though this preservation liquid has the better preserve effect, the component that has may be higher because of the chondroitin sulfate cellulose content, makes to preserve in the liquid electronegatively, and the negative electrical charge corneal endothelial cell of forceful electric power amount is harmful to.Also have a kind of hyaluronic corneal storage medium that contains, its patent No. is: ZL97193593.9.This patent corneal storage medium, main component is a hyaluronic acid, its composition is single, in preserving 15 days, endothelial cell represent lactate dehydrogenase, succinate dehydrogenase, the glucose of cornea activity to take off-the activity level decline of 6-phosphate dehydrogenase, and the preservation of long period can cause the corneal graft oedema, the effective active density of corneal endothelium is descended, cause the operation difficulty, postoperative is prone to plant the sheet endothelial function and loses compensatory.At present clinical commonly used be that the Optisol that U.S. CHIRON company produces preserves the active better preserve liquid of endothelial cell.It is Tc-199 that this preservation liquid is mainly filled a prescription, MEM, and HEPES, gentamicin, chondroitin sulfate and Dextron, the expensive raw material price that wherein has, import corneal storage medium 20ml/50 dollar (every bottle) only is used to preserve a donor's cornea.Therefore China presses for a kind of better preserve effect that has, and the cornea that has China's independent intellectual property rights simultaneously and be fit to national conditions is economically preserved liquid mid-term.
More than the common issue with that also exists of these corneal storage mediums, be exactly the activity of only considering the corneal graft endothelial cell, do not relate to the survival condition of corneal epithelial cell.In general, the corneal graft epithelial cell time-to-live that is stored in the above-mentioned corneal storage medium is 4～6 days, surpasses 7 days epithelial cells and all comes off.Though the corneal graft epithelium layer is important not as endothelial layer, in case the corneal graft epithelial cell comes off, oedema takes place in corneal graft most probably, not only influences the preservation quality of endothelial cell layer, can influence the effect of corneal graft simultaneously.
Summary of the invention
The object of the invention is exactly in order to address the above problem, thus the technical scheme that provides a kind of not only economy, preservation effect but also good cornea to preserve liquid mid-term.
The present invention seeks to realize by following technical measures, develop a kind of cornea and preserved liquid mid-term, it is to contain in the preservation liquid in 1000ml: RPMI1640 culture fluid 4.2-6.2g, chondroitin sulfate 15-35mg, hyaluronic acid 5-25mg, HEPES buffer solution 3.76-5.76g, dexamethasone 5-50mg.Also contain low molecular weight seaweed polysaccharide 10-30mg, tobramycin or vancomycin 10-150mg; This preservation liquid is light red orange, transparent slight thick liquid, and its pH value is 7.0-7.6, osmotic pressure 300-400mOsm/KgH
2O is 2-6 ℃ of preservation.
Also add MEM medium 3.7-5.7g in the described preservation liquid.
Also add hydrocortisone 0.1-5mg/mL in the described preservation liquid.
Vancomycin in the described preservation liquid, also with the tobramycin use in conjunction, vancomycin 10-80mg wherein, tobramycin 25-50mg.
The specification of the tobramycin that described preservation liquid adds is: 25-50 ten thousand units.
Described low molecular weight seaweed polysaccharide, its molecular weight is 25000-100000Da, it comprises: carragheen, the sea lettuce polysaccharide in the green alga, the algal polysaccharide sulfate in the brown alga, its concentration is 2-20mg/mL.
Described preservation liquid also can add L-glutaminate 0.175-0.575mg/mL, adds when when preparation adds or use.
The invention has the advantages that: owing to adopt RPMI1640 culture fluid 4.2-6.2g, chondroitin sulfate 15-35mg, hyaluronic acid 5-25mg, HEPES buffer solution 3.76-5.76g, dexamethasone 5-50mg, low molecular weight seaweed polysaccharide 10-30mg and tobramycin or vancomycin 10-150mg preserve the important prescription of liquid mid-term as cornea of the present invention.Its composition is simple relatively, because chondroitin sulfate, low-molecular-weight algal polysaccharides and hyaluronic synergy, can reduce the consumption of chondroitin sulfate, thereby reduce the amount that cornea is preserved negative electrical charge in the liquid mid-term, make donor's cornea during preservation be unlikely oedema and thicken.During preservation the mechanism that thickens of the cornea generation oedema of Li Tiing is because Na
+-K
+-ATP enzyme function reduction has changed the aquation of cornea, causes the endothelial cell function reduction, and the content of the endogenous glucose in the corneal stroma is reduced, and the oedema of corneal stroma increases.When preserving cornea under 4 ℃ of conditions, inner skin cell function obviously descends, and can not resist the suction negative pressure of the poly-glycosaminoglycan formation that exists in the corneal stroma, if there is not certain colloid osmotic pressure in the cornea surrounding environment, will inevitably cause corneal edema.And the cornea tissue of oedema can cause the reduction of interior composition of more tissue such as poly glucosamine.So at the mechanism that thickens during the above-mentioned cornea {in vitro} conservation; cornea of the present invention is preserved to utilize in the liquid mid-term to has and water forms viscous gel; hyaluronic acid lubricated and the protection cytosis is arranged; have low-molecular-weight algal polysaccharides that increases and keep the colloid osmotic pressure effect and chondroitin sulfate with osmotic dehydration; three's synergistic combination effect, and this synergistic combination is used as the peroxidization that a kind of antioxidant prevents cell membrane lipid.Especially the algal polysaccharides to add can prolong the time-to-live of preserving corneal graft epithelium in the liquid, prevents the generation of the corneal graft oedema that causes because of exuviation.Cornea of the present invention is preserved the synergistic combination that makes full use of chondroitin sulfate, hyaluronic acid, algal polysaccharides three in the liquid and is produced certain colloid osmotic pressure mid-term, to keep cell membrane stability, alleviates the self-dissolving of endothelial cell.Therefore, this synergistic combination effect can obviously improve the colloid osmotic pressure that cornea of the present invention is preserved liquid mid-term, forms the special matrix of endothelial cell, keeps its specific biochemical environment, help keeping desirable corneal thickness, improve the preservation effect of endothelial cell activity.
Experiment showed, that cornea survival preservation liquid of the present invention has raising endothelial cell activity and the preservation effect that prolongs the exfoliation of corneal epithelium time really, during preservation with in performing the operation can keep intimate physiological corneal thickness.Cooperate to add dexamethasone and/or hydrocortisone again, lysosome membrane and other biological film that it can stabilized cell strengthen the defensive ability/resistance ability of cell contratoxin and infringement, keep the form and the function of endothelial cell better.The present invention adds L-glutaminate before preserving the liquid application, can reduce the oxidative damage of corneal epithelium and endothelial cell, replenishes epithelium and the compensatory essential amino acid of endothelial cell, prolongs the time that comes off of epithelium and the activity change of preservation endothelium.Cornea of the present invention is preserved the effective vancomycin of adding or vancomycin and tobramycin use in conjunction in the liquid mid-term, can prevent the pathogen that donor carries or preserve the infected by microbes that the operational pollution in the liquid manufacturing process causes.
The present invention can make the holding time of cornea activity reach more than 2 weeks, endothelial cell activity is stable between storage period, variation is little, can compare favourably with the Optisol that U.S. CHIRON company produces aspect the preservation of corneal graft endothelial cell, and be better than Optisol aspect the epithelial holding time.Enzyme histochemistry checks the prolongation that shows with the holding time, and the Opthiol that the production of the comparable U.S. of liquid CHIRON company is preserved in cornea survival of the present invention keeps higher endothelial cell metabolic function.Through clinical practice, preservation of cornea re-uses after more than 7 days, plants sheet endothelial cell number average postoperative 1-4 week and surpasses 2000/mm
2Cornea of the present invention is preserved the liquid needed raw material and be easy to get mid-term, and is cheap, and good economy performance is fit to preservation method in domestic mid-term, can become a kind of desirable corneal storage medium in mid-term in current China eye bank, fills the domestic gaps.
Embodiment
For illustrating clearly that cornea of the present invention preserves the technical scheme of liquid mid-term, existing by the specific embodiment table, it is described in detail.Embodiments of the invention see Table 1.
Table 1 cornea is preserved the prescription of liquid mid-term:
Cornea of the present invention is preserved the research that liquid is used for the cat exfoliation of corneal epithelium time mid-term:
To be stored in each liquid respectively 5 days, 7 days, 15 days each 10 of cat corneas carry out penetrating keratoplasty, observe the survival condition of postoperative different time corneal graft epithelium.Found that the epithelial time-to-live maintains 4～6 days in the Optisol preservation liquid.And the corneal graft epithelial cell time-to-live of preserving in corneal storage medium of the present invention surpasses 7 days, the longlyest reaches 10 days.
Cornea of the present invention is preserved liquid and be applied to the activity test of rabbit corneal endothelium mid-term:
In 125 rabbit corneals, respectively get 25, assign to and be stored in each 10ml of 4 prescriptions of the present invention and Optisol and preserve in liquid, 4 ℃ of preservations of refrigerator, the holding time is respectively 5 days, 7 days, 10 days, 15 days and 20 days.Respectively get 5, row 0.25% awl orchid adds 1% alizarin red combined staining, observes endothelial cell activity density and activity rate with binocular microscope, the results are shown in Table 1 at every turn.Find out that by table 1 four prescriptions of this discovery all have the active preservation effect of endothelial cell preferably, are close with Optisol.But the holding time of corneal epithelial cell is obviously long than Optisol.The endothelial cell activity rate of also finding out this discovery simultaneously is less over time, stable in properties, and the activity rate of depositing 20 days reaches more than 90.5%.
Cornea of the present invention is preserved liquid and be applied to rabbit corneal endothelial cell enzyme staining mid-term:
Before the blue dyeing of the awl of being expert at; be stored in each liquid 7 days, 15 days each 4 of the rabbit corneals of going bail for; section; other gets 4 sections of normal rabbit cornea and compares group; row enzyme staining, promptly survey three kinds of enzymes of agaric acid dehydrase, lactate dehydrogenase and glucose-6 one phosphoric acid deoxygenases, when surveying every kind of enzyme; get from every cornea and cut into slices 4, hatch with a kind of Incubating Solution.Three kinds of enzyme dyeing all present reaction.The printing opacity density value of every kind of enzyme (OPTDM value) is inverse ratio with the activity of enzyme.As seen from Table 2, with the prolongation of holding time, the present invention can keep higher endothelial cell metabolic function than Optisol.
Cornea of the present invention is preserved the liquid clinical practice mid-term in the experiment that people's cornea is preserved:
Successively to 67 parallactic angle film patients (be simple eye morbidity } carry out corneal transplantation, use 67 people's corneas, the cornea that is stored among the Optisol that the present invention and U.S. CHIRON company produce is respectively 37 and 30, wherein there are 2 because of the transplanting of full-shape film, do not remain the edge cornea and carry out interior leather dyeing observation.
Clinical observation result can be found out, 37 corneas that are stored in this discovery preservation liquid were on average preserved 6.5 days, all obtained transparent healing effect, it plants sheet endothelial cell activity rate all more than 90%, postoperative 1-4 week endothelial cell is checked, planted the sheet endothelial cell and count 2204.56/mm of average out to
2
The corneal transplantation postoperative 1-4 that the present invention preserves plants sheet endothelial cell number entirely at 1500/mm in week
2More than.
Table 2 rabbit corneal endothelial cell is preserved activity rate relatively (%)
| Preserve liquid | Holding time (my god) | ||||
| ????5 | ????7 | ????10 | ????15 | ??20 | |
| Example 1 | ????100 | ????99.2 | ????98.3 | ????92.4 | ??90.5 |
| Example 2 | ????100 | ????99.3 | ????98.6 | ????93.6 | ??91.3 |
| Example 3 | ????99.8 | ????99.3 | ????98.5 | ????94.0 | ??91.0 |
| Example 4 | ????99.3 | ????98.9 | ????98.4 | ????94.1 | ??91.2 |
| Example 5 | ????99.7 | ????99.3 | ????98.4 | ????94.1 | ??91.2 |
| Example 6 | ????99.4 | ????99.3 | ????98.6 | ????94.2 | ??91.0 |
| Example 7 | ????99.5 | ????99.4 | ????98.7 | ????94.2 | ??91.2 |
| Example 8 | ????99.3 | ????98.7 | ????98.3 | ????93.5 | ??90.0 |
| Example 9 | ????99.2 | ????98.5 | ????98.1 | ????93.2 | ??90.1 |
| Example 10 | ????99.6 | ????99.4 | ????98.8 | ????94.1 | ??90.8 |
| Example 11 | ????99.5 | ????99.4 | ????98.7 | ????94.0 | ??90.3 |
| Example 12 | ????99.8 | ????99.6 | ????98.7 | ????93.7 | ??90.5 |
| Example 13 | ????99.9 | ????99.3 | ????98.9 | ????93.8 | ??90.5 |
| Example 14 | ????100 | ????99.3 | ????98.8 | ????94.5 | ??91.2 |
| Example 15 | ????99.8 | ????99.5 | ????98.9 | ????93.9 | ??90.6 |
| Example 16 | ????99.9 | ????99.6 | ????98.9 | ????94.2 | ??91.0 |
| ??Optisol | ????99.3 | ????99.3 | ????98.7 | ????94.2 | ??90.3 |
Annotate: cornea of the present invention is preserved liquid example formulations 4 and do not add algal polysaccharides mid-term, so the chondroitin sulfate and the hyaluronic amount that add are more relatively, may cause the negative electrical charge content in the corneal storage medium to increase, and has influenced the activity of endothelial cell.Because the shortage of algal polysaccharides, the time-to-live of corneal graft epithelium shortens, and takes place because of exuviation causes the corneal graft oedema.Example formulations 4 is compared with the Optisol that U.S. CHIRON company produces, and the endothelial cell of different time is preserved activity rate and all decreased.
Cornea of the present invention is preserved in liquid example formulations 8 and the example formulations 9 and all do not add dexamethasone or hydrocortisone mid-term, causes the lysosome membrane of keratocyte and other biological membrane stability to reduce, and the defensive ability/resistance ability of contratoxin and infringement descends.Example formulations 8 is compared with the Optisol that U.S. CHIRON company produces with example formulations 9, and the endothelial cell of each time is preserved activity rate and all decreased.
Cornea of the present invention is preserved in liquid example formulations 12 and the example formulations 13 and all do not add L-glutaminate mid-term, the Optisol that produces with U.S. CHIRON company compares, though preserving activity rate, the endothelial cell of each time obviously do not change, but the time of corneal graft exuviation shortens, and may cause the oxidative damage of corneal epithelial cell relevant with the shortage of L-glutaminate.
Those of ordinary skill in the art can understand, and in protection scope of the present invention, makes amendment for the foregoing description, and it all is possible adding and replacing, and it does not all exceed protection scope of the present invention.
Claims (7)
1, a kind of cornea is preserved liquid mid-term, it is to contain in the preservation liquid in 1000ml: RPMI1640 culture fluid 4.2---6.2g, chondroitin sulfate 15---35mg, hyaluronic acid 5---25mg, HEPES buffer solution 3.76-5.76g, dexamethasone 5---50mg is characterized in that: also contain low molecular weight seaweed polysaccharide 10---30mg, tobramycin or vancomycin 10-150mg; This preservation liquid is light red orange, transparent slight thick liquid, and its pH value is 7.0---7.6, and osmotic pressure 300-400mOsm/KgH
2O, 2---6 ℃ of preservations.
2, preserve liquid mid-term according to the described cornea of claim 1, it is characterized in that: also add MEM medium 3.7-5.7g in the described preservation liquid.
3, preserve liquid mid-term according to the described cornea of claim 1, it is characterized in that: also add hydrocortisone 0.1-5mg/mL in the described preservation liquid.
4, preserve liquid mid-term according to the described cornea of claim 1, it is characterized in that: the vancomycin in the described preservation liquid, also with the tobramycin use in conjunction, wherein vancomycin 10---80mg, tobramycin 25---50mg.
5, preserve liquid mid-term according to claim 1 or 4 described corneas, it is characterized in that: the specification of the tobramycin that described preservation liquid adds is: 25-50 ten thousand units.
6, preserve liquid mid-term according to the described cornea of claim 1, it is characterized in that: described low molecular weight seaweed polysaccharide, its molecular weight are 25000-100000Da, it comprises: carragheen, sea lettuce polysaccharide in the green alga, the algal polysaccharide sulfate in the brown alga, its concentration is 2-20mg/mL.
7, preserve liquid mid-term according to the described cornea of claim 1, it is characterized in that: described preservation liquid also can add L-glutaminate 0.175-0.575mg/mL, adds when when preparation adds or use.
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| CN 200410091007 CN1270605C (en) | 2004-11-15 | 2004-11-15 | Cornea middle term preserving fluid |
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| CN 200410091007 CN1270605C (en) | 2004-11-15 | 2004-11-15 | Cornea middle term preserving fluid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101502257B (en) * | 2009-03-13 | 2013-01-02 | 厦门大学 | Corneal midterm preservation solution and preparation method thereof |
| CN104094925A (en) * | 2014-07-18 | 2014-10-15 | 广州优得清生物科技有限公司 | Lamellar corneal preserving solution |
| CN105770994A (en) * | 2016-05-09 | 2016-07-20 | 拜欧迪赛尔（北京）生物科技有限公司 | Preparation method and application of bioengineering decellularized eye conjunctiva |
| CN106135197A (en) * | 2016-07-04 | 2016-11-23 | 拜欧迪赛尔（北京）生物科技有限公司 | A kind of cornea mid-term preservation liquid of serum-free composition |
| CN106421908A (en) * | 2016-07-01 | 2017-02-22 | 山东省眼科研究所 | Method for preparing decellularized cornea |
| CN106614519A (en) * | 2016-11-24 | 2017-05-10 | 山东省眼科研究所 | Decellularized corneal limbus protective liquid and preparation method of decellularized corneal limbus |
| CN106729999A (en) * | 2016-11-24 | 2017-05-31 | 山东省眼科研究所 | A kind of method for building tissue engineering comea edge |
| CN107164306A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | Cornea model Cord blood solid medium and preparation method and application method |
| CN111955455A (en) * | 2020-08-06 | 2020-11-20 | 温州医科大学 | Preservation solution for maintaining cornea activity |
| CN115067321A (en) * | 2022-06-30 | 2022-09-20 | 上海市伤骨科研究所 | Nutritive capsule for medium-and long-term three-dimensional preservation of corneal tissue and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502257B (en) * | 2009-03-13 | 2013-01-02 | 厦门大学 | Corneal midterm preservation solution and preparation method thereof |
| CN104094925A (en) * | 2014-07-18 | 2014-10-15 | 广州优得清生物科技有限公司 | Lamellar corneal preserving solution |
| CN104094925B (en) * | 2014-07-18 | 2015-11-18 | 广州优得清生物科技有限公司 | A kind of lamellar cornea conserving liquid |
| WO2016008220A1 (en) * | 2014-07-18 | 2016-01-21 | 广州优得清生物科技有限公司 | Lamellar cornea preserving solution |
| CN105770994A (en) * | 2016-05-09 | 2016-07-20 | 拜欧迪赛尔（北京）生物科技有限公司 | Preparation method and application of bioengineering decellularized eye conjunctiva |
| CN106421908A (en) * | 2016-07-01 | 2017-02-22 | 山东省眼科研究所 | Method for preparing decellularized cornea |
| CN106421908B (en) * | 2016-07-01 | 2018-01-16 | 深圳艾尼尔角膜工程有限公司 | A kind of preparation method of de- cell cornea |
| CN106135197A (en) * | 2016-07-04 | 2016-11-23 | 拜欧迪赛尔（北京）生物科技有限公司 | A kind of cornea mid-term preservation liquid of serum-free composition |
| CN106729999A (en) * | 2016-11-24 | 2017-05-31 | 山东省眼科研究所 | A kind of method for building tissue engineering comea edge |
| CN106614519A (en) * | 2016-11-24 | 2017-05-10 | 山东省眼科研究所 | Decellularized corneal limbus protective liquid and preparation method of decellularized corneal limbus |
| CN106614519B (en) * | 2016-11-24 | 2019-11-19 | 山东省眼科研究所 | A kind of preparation method of de- cell corneal limbus protection liquid and de- cell corneal limbus |
| CN106729999B (en) * | 2016-11-24 | 2019-11-19 | 山东省眼科研究所 | A method of building tissue engineering comea edge |
| CN107164306A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | Cornea model Cord blood solid medium and preparation method and application method |
| CN111955455A (en) * | 2020-08-06 | 2020-11-20 | 温州医科大学 | Preservation solution for maintaining cornea activity |
| CN115067321A (en) * | 2022-06-30 | 2022-09-20 | 上海市伤骨科研究所 | Nutritive capsule for medium-and long-term three-dimensional preservation of corneal tissue and preparation method thereof |
| CN115067321B (en) * | 2022-06-30 | 2023-08-08 | 上海市伤骨科研究所 | Nutritional capsule for medium-long-term three-dimensional preservation of cornea tissue and preparation method thereof |
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