CN105535946A - Application of transglutaminase in strengthening corneal mechanical properties and biological preparation - Google Patents
Application of transglutaminase in strengthening corneal mechanical properties and biological preparation Download PDFInfo
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- CN105535946A CN105535946A CN201510920073.7A CN201510920073A CN105535946A CN 105535946 A CN105535946 A CN 105535946A CN 201510920073 A CN201510920073 A CN 201510920073A CN 105535946 A CN105535946 A CN 105535946A
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Abstract
Aiming at the deficiency of a conventional corneal crosslinking technology, the invention provides a new biological preparation which can significantly strengthen corneal mechanical properties; the preparation is a biological enzyme (transglutaminase) having a cross-linking function and can be used for strengthening the corneal biomechanical properties. The biological preparation can catalyze a specific amino acid in collagen fibers in corneal stromas to produce an isopeptide bond, so the connection of the collagen fibers is strengthened, and mutual connection between layers of fiber small plates in the corneal stromas is enhanced, and the purpose of strengthening the mechanical properties of the corneal stromas is reached. The stromal cell activity of an eye corneal full layer after mediated crosslinking with the biological preparation is still better, obvious cell apoptosis does not appear, and the security is higher. Compared with a current clinical used corneal crosslinking treatment scheme, the biological preparation has better crosslinking effect and fewer side effects and complications, and has quite strong clinical application prospects.
Description
Technical field
The present invention relates to the application of T-5398 in reinforcement cornea mechanical property and the biological preparation of preparation thereof, belong to biological preparation/keratopathy field.
Background technology
Keratoconus (Keratoconus) be a kind of without inflammation, cornea Progressive symmetric erythrokeratodermia prolapse, cornea irregular astigmatism thinning with corneal stroma, correcting defects of vision drops to the oculopathy of feature.Also be the Typical Representative of keratectasia class disease.
Keratoconus is multiple is born in youngster, and sickness rate is about 1/2000, and the keratoconus of about 21% finally need adopt corneal graft to treat, and accounts for all corneal transplantation patients 16%.The primary treatment of traditional keratoconus has correction of refractive errors and the large class of corneal transplantation two, the former comprises and wears frame eyeglasses and corneal contact lens, cornea internal ring implantation, excimer laser cornea surface cut art (PRK) etc., these methods can reach the object of correction of refractive errors to part-conical cornea patient, but can not stop the progress of the state of an illness.Latter is subject to the restriction of organ origin, and needs of patients uses anti-rejection medication, and application is restricted.
The cause of disease of keratoconus is still not fully aware of so far, but the reduction of the mechanical strength of cornea is considered to one of its Important cause of disease.Histology's upper discovery circular cone place fibrolamellar reduces, and collagen fiber diameter does not change.Therefore think it may is that fibre board ply adhesion is inadequate, the mutual slippage of flaggy, causes cornea thinning.Then, due to Central corneal district or other central area progressive thinning, cause cornea irregular astigmatism degree to increase, myopia degree is also constantly deepened, vision Progressive symmetric erythrokeratodermia decline.From this angle, the mechanical strength increasing cornea becomes new treatment means and the thinking of this disease for the treatment of.
Collagen cross-linking treatment (cross-linkingtreatment) be make collagen fiber by biochemical method be interconnected reinforcement, increase the treatment technology of tissue intensity, physical crosslinking (photochemical crosslinking), chemical crosslinking, biochemical crosslinked (enzyme is cross-linked) three kinds can be divided into according to the difference of cross-linking method.In current research both domestic and external, physical crosslinking (photochemical crosslinking) treatment has shown good curative effect to the Therapy study of keratoconus.Wherein ultraviolet light-riboflavin corneal cross-linking method is ratified by U.S. FDA, applies clinically.
The method adopts riboflavin as photosensitizer, irradiate with the ultraviolet light,long wave (UVA) harmless to internal structures of eyeball, the irradiation of light is utilized to make photosensitizer that oxygen molecule is energized into triplet state, it produces the oxygen-derived free radicals (ROS of very strong oxidation activity when getting back to singletstate, reactiveoxygenspecies) (Fig. 6), make the terminal generation condensation reaction of collagen fiber, the side direction added between collagen fiber connects, thus make the mechanical strength of cornea obtain enhancing, keratoconus is stopped progress even having some patients myopia degree to obtain to alleviate.The effect of chemical crosslinking corneal rests on laboratory stage at present, and what have scholar to use the acyclic β nitroethyl alcohol (BNA) of short chain, genipin for porcine cornea is crosslinked, finds the elastic modelling quantity increase to a certain degree of in vitro animal corneal.
Above-mentioned two kinds of cross-linking methods all have some limitations.
Although ultraviolet light-riboflavin cross-linking method technology is applied to clinical nearly ten years, and is described as " healing of keratoconus is wished ".But remain in many deficiencies: 1) due to the riboflavin absorption effect of cornea, before the method can only make cornea, the Collagen of 250 ~ 300um is cross-linked, therefore, its Clinical efficacy is limited, multinomial research all show ultraviolet light riboflavin crosslinked after cornea steepest diopter to decline only about 2D.2) need corneal epithelium be removed during the method treatment, the generation of patient's postoperative pain, blurred vision, cornea haze and infection can be increased.3) also there is safety issue in the method, and Short Term Clinical research report corneal thickness is cross-linked corneal endothelial damage following cell density lower than the capable ultraviolet riboflavin of keratoconus of 400um and obviously declines.4) to the damage of stromal cell: basic research and the clinical observation of cornea Laser Scanning Confocal Microscope all show there is a large amount of Apoptosis in cornea in the corneal stroma deeply reaching 300um, need to reach six months recovery.5) ultraviolet light riboflavin collagen cross-linking is inapplicable for the patient of photosensitivity body constitution.6) equipment price is expensive.7) for preoperative steepest corneal diopter more than 58D, age more than 35 years old, the vision keratoconus that is greater than 0.8, ultraviolet light riboflavin is cross-linked the risk that various complication occurs Post operation obviously to be increased.The problems referred to above limit the promotion and application of technique.Chemical crosslink technique is simple, and cross-linking agent source is wide, but the side effect of its chemical cross-linking agent treatment is difficult to avoidance, it is illustrated the bio-toxicity needs of its hetero-organization of eye.By contrast, biochemical crosslinked (enzyme is cross-linked) becomes a more satisfactory selection.
Biochemical being cross-linked utilizes activated enzyme, and the specific amino acid substrate of catalysis, makes to produce covalent bond between the aminoacid of peptide chain, reach crosslinked object.T-5398 (TGase) is exactly a kind of outstanding cross-linking enzyme.
The TGase of current discovery comprises 8 hypotypes altogether, and wherein most type extensively exists in human body, and participates in important physiological function.The biological function of TGase is that transglutamin-ase 9 reaction occurs between the sour residue of catalytic proteins glutamic acid and the ammonium residue of lysine, generates isopeptide bond: ε-(gamma-glutamyl)-lysine key (Fig. 2).This isopeptide bond forms rear exceptional stability, effectively can increase the resistance of tissue to physics, chemistry, thus increase the mechanical strength of tissue.
This reaction has the feature of specificity, high efficiency; Enzymic catalytic reaction, as a kind of common physiological phenomenon, uses the tissue injury produced less in local organization; Use enzymic catalytic reaction, the equipment needed for it is simple, simple to operate, has promotional value.
The key component of corneal stroma, accounts for cornea entirely thick more than 90%, is the stable principal element of cornea mechanical structure.It comprises 200-250 layer fibers platelets arranged in parallel, and main component is type i collagen fiber.Type i collagen fiber is the high molecular weight protein of the helical structure comprising two α 1 (I) peptide chain and α 2 (I) peptide chain, in 1464 aminoacid of wherein α 1 (I) peptide chain, comprise lysine 57 (5.24%), 75, glutamic acid (6.94%), in 1366 aminoacid of α 2 (I) peptide chain, comprise lysine 50 (4.95%), 66, glutamic acid (6.58%).Therefore on the whole, corneal stroma has lysine and the glutamic acid of more than 5%, can become the substrate of TGase catalytic reaction.
Summary of the invention
The object of the invention is the deficiency for above-mentioned existing treatment keratoconus technology, a kind of biological preparation obviously can strengthening cornea mechanical property is provided, especially the application in cornea mechanical property strengthened by a kind of biological preparation containing T-5398, T-5398 can the cross-linking reaction of lysine in catalysis corneal stroma and glutamic acid, make cornea can reach effectively crosslinked, mechanical property is obviously strengthened.
One aspect of the present invention provides a kind of biological preparation for strengthening cornea mechanical property, comprises T-5398 and pharmaceutical carrier.
Wherein, described T-5398 is used for the cross-linking reaction of lysine in catalysis corneal stroma and glutamic acid.
Particularly, described T-5398 can derive from and buy or adopt prior art to prepare.
Especially, by described T-5398 and pharmaceutical carrier being merged, form the biological preparation of collyrium form.
Particularly, by described T-5398 and pharmaceutical carrier being merged, form the biological preparation of spongaion form.
The present invention provides a kind of T-5398 strengthening the application in cornea mechanical property on the other hand.
The key component of corneal stroma comprises 200-250 layer fibers platelets arranged in parallel, it is the principal element determining that cornea mechanical structure is stable, the main component of fibers platelets is type i collagen fiber, and the peptide chain of type i collagen fiber comprises lysine and the glutamic acid of more than 5%.And T-5398 is by the cross-linking reaction of the lysine in catalysis corneal stroma and glutamic acid, makes to be cross-linked with each other by aminoacid reaction between each layer fibers platelets, thus enhance the mechanical property of corneal stroma.
Advantage of the present invention and Advantageous Effects as follows:
1, T-5398 of the present invention is by the cross-linking reaction of the lysine in catalysis corneal stroma and glutamic acid, makes to be cross-linked with each other by aminoacid reaction between each layer fibers platelets in corneal stroma, thus enhances the mechanical property of corneal stroma.
2, the Young's modulus of the transglutaminase mediated crosslinked cornea of the present invention is adopted not adopt the Young's modulus of the transglutaminase mediated crosslinked cornea of the present invention on average to improve 44%, the obvious steepening of stress-strain diagram, illustrate that the mechanical property of cornea is obviously strengthened after T-5398 biochemistry is crosslinked.
3, adopt the stromal cell activity of the transglutaminase mediated crosslinked rear cornea holostrome of the present invention still better, do not occur obvious apoptosis, illustrate that T-5398 of the present invention is to the basic not damaged of cornea substrate.
Accompanying drawing explanation
Fig. 1 is the stress-strain curve diagram studying eye and contrast eye in embodiment 1;
The TUNEL coloration result figure of matched group corneal stroma in Fig. 2 position embodiment 2;
Fig. 3 is the TUNEL coloration result figure that embodiment 2 mesophytization is cross-linked group corneal stroma;
The DAPI coloration result figure of matched group corneal stroma in Fig. 4 position embodiment 2;
Fig. 5 position embodiment 2 mesophytization is cross-linked the DAPI coloration result figure of group corneal stroma;
Fig. 6 is riboflavin-ultraviolet light cross-linking principle schematic;
Fig. 7 is the reaction of TGase catalyzing and condensing and isopeptide bond generation figure.
Detailed description of the invention
In the embodiment of the present invention, T-5398 provides T-5398 for German Ruibio company, and 2.5KU/25g packs; New zealand white rabbit provides the test new zealand white rabbit purchased from Fang Yuan edge company for BJ Univ Hospital's experimental animal room, body weight 2.5-3.0kg, this zoopery meets " People's Republic of China's management of laboratory animal regulations " and the pertinent regulations of NIH " experimental animal feeding management and guide ".
Embodiment 1 T-5398 is tested rabbit corneal cross-linking effect
Get 12 new zealand white rabbits, with its right eye for research eye, the biochemical cross-linking reaction 30min that research eye adopts 0.8U transglutaminase mediated, after raising 2 weeks, observes its cornea character; With its left eye for contrast eye.Measure the Young's modulus of research eye and contrast eye respectively, the results are shown in Table 1.Measure the ess-strain of research eye and contrast eye respectively, draw stress-strain diagram, see Fig. 1.
Table 1TGase is to rabbit corneal cross-linking effect
As can be seen from Table 1, the Young's modulus of seminar's cornea comparatively matched group on average improves 44%, as seen from Figure 1, stress-strain diagram comparatively matched group (OD) steepening of seminar (OS) cornea, illustrate relative to matched group, the stress of seminar's cornea, by obviously promoting, therefore can draw, after T-5398 biochemistry is crosslinked, the mechanical property of cornea is obviously strengthened.
The damage measure of the crosslinked corneal substrate of embodiment 2 T-5398 biochemistry
Get 12 new zealand white rabbits, be divided into two groups, often organize 6, wherein one group is biochemical crosslinked group, adopts the biochemical cross-linking reaction 30min that 0.8U is transglutaminase mediated, after raising 2 weeks, observes the apoptosis of its keratocyte; Another group is matched group, adopts ultraviolet light-riboflavin to be cross-linked, after raising 2 weeks, observes the apoptosis of its keratocyte.The TUNEL coloration result of different group white rabbit corneal stroma is shown in Fig. 2 and Fig. 3, and the DAPI coloration result of different group white rabbit corneal stroma is shown in Fig. 4 and Fig. 5.
Fig. 2 is the cornea of matched group, can find out that front 1/2 substrate all occurs apoptosis, and Fig. 3 is the cornea of biochemical crosslinked group, can find out that obvious apoptosis does not appear in cornea.Fig. 4 is the cornea of matched group, can find out that first half keratocyte activity reduces greatly, and Fig. 5 is the cornea of biochemical crosslinked group, can find out that the stromal cell activity of cornea holostrome is still better.
Claims (5)
1. for strengthening a biological preparation for cornea mechanical property, it is characterized in that, comprising T-5398 and pharmaceutical carrier.
2. biological preparation according to claim 1, is characterized in that, described T-5398 is used for the cross-linking reaction of specific amino acids (lysine and glutamic acid) in catalysis corneal stroma.
3. biological preparation according to claim 1, is characterized in that, by described T-5398 and pharmaceutical carrier being merged, forms the biological preparation of collyrium form.
4. biological preparation according to claim 1, is characterized in that, by described T-5398 and pharmaceutical carrier being merged, forms the biological preparation of spongaion form.
5. T-5398 is strengthening the application in cornea mechanical property.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113633760A (en) * | 2020-04-27 | 2021-11-12 | 北京大学第一医院 | Application of transglutaminase in medicine for inhibiting or delaying myopia |
| CN114225016A (en) * | 2021-12-02 | 2022-03-25 | 北京大学第一医院 | Method for inhibiting myopia and keratoconus progression |
Citations (3)
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| CN1390133A (en) * | 1999-09-15 | 2003-01-08 | 布鲁斯·H·德伍尔夫森 | Composition for stabilizing corneal tissue during or after orthokeratology lens wear |
| WO2007120457A2 (en) * | 2006-04-13 | 2007-10-25 | Euclid Systems Corporation | Composition and method for stabilizing corneal tissue after refractive surgery |
| CN102892880A (en) * | 2010-01-14 | 2013-01-23 | 奥加诺吉尼西斯公司 | Bioengineered tissue constructs and methods for producing and using thereof |
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2015
- 2015-12-14 CN CN201510920073.7A patent/CN105535946A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1390133A (en) * | 1999-09-15 | 2003-01-08 | 布鲁斯·H·德伍尔夫森 | Composition for stabilizing corneal tissue during or after orthokeratology lens wear |
| WO2007120457A2 (en) * | 2006-04-13 | 2007-10-25 | Euclid Systems Corporation | Composition and method for stabilizing corneal tissue after refractive surgery |
| CN102892880A (en) * | 2010-01-14 | 2013-01-23 | 奥加诺吉尼西斯公司 | Bioengineered tissue constructs and methods for producing and using thereof |
Non-Patent Citations (5)
| Title |
|---|
| LOUIS TONG等: "Molecular mechanism of teansglutaminase-2 in corneal epithelial migration and adhesion", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| LOUIS TONG等: "Transglutaminase Participates in UVB-Induced Cell Death Pathways in Human Corneal Epithelial Cells", 《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》 * |
| NIKOLAO KOPSACHILIS等: "A Novel Mechanism of UV-A and Riboflavin-Mediated Corneal Crosslinking Through Induction of Tissue Transglutaminases", 《CORNEA》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113633760A (en) * | 2020-04-27 | 2021-11-12 | 北京大学第一医院 | Application of transglutaminase in medicine for inhibiting or delaying myopia |
| CN114225016A (en) * | 2021-12-02 | 2022-03-25 | 北京大学第一医院 | Method for inhibiting myopia and keratoconus progression |
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Application publication date: 20160504 |