CN1235582C - Medicines for preventing and treating retina and cornea blood vessel diseases - Google Patents
Medicines for preventing and treating retina and cornea blood vessel diseases Download PDFInfo
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Abstract
The present invention belongs to the technical field of biological medicines, which relates to medicines for preventing and treating retina and cornea neovascularization. The medicines are 4', 5, 7-trihydroxyisoflavone (Genistein, Gen for short) pure products, or bean and relevant vegetable crude extracts containing the Gen, or compound preparations of which the main components are the Gen. The present invention can be prepared into dosage forms, such as, tablets, capsules, oral liquid, vein and muscle injection, liposome, microcapsules, eyedrops, etc. Through whole animal experiments and culture cell experiments, the medicines can obviously inhibit the retina neovascularization of a disease model of a C57BL/6 mouse, and can also inhibit the retina neovascularization of a rabbit and a rat caused by a plurality of factors. The medicines can also inhibit the rabbit's retinal pigment epithelium VEGF and HIF1 protein expression caused by physical anoxia and chemical anoxia. Through the experiments, the medicines can perform the functions of preventing and curing the neovascularization of the retinas and the corneas by inhibiting inducing factor expression of the neovascularization.
Description
One, technical field
The invention belongs to the biological medicine technology field.
Two, background technology
The formation of new vessels is the severe complication of a lot of ophthalmic diseases, proliferative diabetic retinopathy, retinal vessel barrier disease, senile macular disciform degeneration, corneal transplantation, glaucoma all can cause the angiogenesis at positions such as retina, cornea, but the 26S Proteasome Structure and Function of a series of pathological change heavy damage eyes such as oozing out of accompanying with it, hemorrhage, hypertrophy, visual function is suffered damage, and serious causing lost one's sight.
Still do not treat at present the active drug and the method for ophthalmic angiogenesis clinically.The laser light of Cai Yonging is coagulated therapy and can be caused the serious irreversible damage of retina clinically.Once the someone considered to adopt the dexamethasone treatment, but effect and not obvious, and toxic and side effects is very big, has limited its application.So searching can prevent and treat the ophthalmic angiogenesis and the little medicine of toxic and side effects has important social and economic implications.
The formation of new vessels, comprise degraded, endothelial cell migration, the endotheli ocytosis of vascular endothelial cell basement membrane, the several steps such as maturation of new vessels, these steps are subjected to the adjusting of multiple somatomedin, as VEGF (VEGF), oxygen deficient induction factor 1 (HIF1), fibroblast growth factor (FGF), transforming growth factor (TGF-α), tumor necrosis factor (TNF-α), epidermal growth factor (EGF) etc.But in these factors, having VEGF only has strong short division, migration to vascular endothelial cell specifically.Ophthalmic angiogenesis and vegf expression have high dependency on time, space, existing more and more evidences shows that VEGF plays key effect in the angiogenesis within the eye.Human VEGF gene is made up of 8 exons and 7 introns, is about 14kb.Because RNA cut mode difference, VEGF family has 4 members at least, is respectively VEGF121, VEGF165, VEGF189, VEGF206.Wherein VEGF165 is topmost isomer.Vegf receptor comprises that fms sample tyrosine protein kinase (FLT) and kinases insert functional domain receptor (KDR) on the endotheliocyte, thereby VEGF starts system in the cell by inducing the autoreceptor phosphorylation.
4 ', 5,7-trihydroxy-isoflavone (Genistein, write a Chinese character in simplified form Gen) be a kind of isoflavonoid that mainly from plants such as beans, extracts, recently discover that Gen can suppress the growth and the transfer of kinds of tumors, but the biochemical site and the mechanism of action of its effect are not clear, several possibility mechanism that propose at present comprise the inhibition tyrosine kinase activity, the activity that suppresses topoisomerase II, antioxidation etc.Gen can be absorbed rapidly by passive mechanism at intestinal, is converted into genistein-7-oxygen-β glucosidase (genistein-7-O-β glucuronide) conjugate at liver, enters duodenum through bile, is heavily absorbed by intestinal again, forms enterohepatic circulation.The existing health food listing of Gen, Shang Weiyou is as the report of drug use.Gen belongs to low toxicity material, and adjusting and abundant soybean resource that it is useful to the human physiological metabolism are for wide prospect has been showed in its development and use.
Three, summary of the invention
The objective of the invention is to utilize Gen prevention and treatment retina and corneal vascularization, this medicine is the pure product of Gen, or contains the crude extract of beans and the corresponding plants of Gen, or is the compound preparation of main component with Gen.Can be prepared into dosage forms such as tablet, capsule, oral liquid, vein and intramuscular dose, liposome, microcapsule and eye drop.
Through whole animal test and cultured cell test confirmation, this medicine can obviously suppress the retinal vessel new life of disease model C57BL/6 mice, also can suppress the corneal vascularization of rabbit and rat due to the multiple factor.This medicine can also suppress rabbit retinal pigment epithelium VEGF and the HIF1 protein expression due to physics anoxia and the chemical hypoxia, shows that the inducible factor of this medicine by angiogenesis inhibiting expressed bring into play prevention and treat retina and corneal vascularization.
Content comprises:
(1) Gen and soybean isoflavone are to mice retinal vessel new life's effect
The relative anoxia of C57BL/6 mice causes the newborn model of retinal vessel.Observe the section of normal control group mice eye, rarely seen minute quantity has the vascular endothelial cell of breaking through internal limiting membrane, the models treated group breaks through the vascular endothelial cell number of internal limiting membrane obviously more than normal group, Gen administration group and obviously reduce than the models treated group to the vascular endothelial cell number that the soybean isoflavone group breaks through internal limiting membrane, and be dose dependent.The expression of VEGF and HIF1 is obviously strong than normal group in the models treated group retinal endothelial cell.Gen administration group and to soybean isoflavone group and models treated group relatively, the expression of VEGF and HIF1 obviously weakens.Show that Gen and soybean isoflavone can suppress mice retinal vessel new life.
(2) inhibitory action of Gen and soybean isoflavone corneal angiogenesis
Observe rabbit corneal angiogenesis progress due to the suture, find behind the cornea suture the 3rd day, cornea hyperemia, and have new vessels to stretch in the limbus of corneae, new vessels appears in every suture upper end, about length 0.6mm.Observed to the suture the 20th day, the number and the total length of visible administration group new vessels obviously reduce than matched group, and the process of angiogenesis also slows down.Show that Gen and soybean isoflavone can suppress rabbit corneal angiogenesis due to the suture.
Rat corneal vascularization progress due to the observation alkali burn, periphery has new vessels to immerse during 1 week, and administration group new vessels number obviously reduces than matched group, and vascular occlusions are wire after 2 weeks, until disappearance.Matched group vascular occlusion occurs after 2 weeks, about 5 weeks back blood vessel disappearance.Show that Gen and soybean isoflavone can obviously suppress the corneal vascularization due to the alkali burn.
(3) Gen influence that rabbit RPE cell VEGE due to chemical hypoxia and the physics anoxia is expressed
Adopt immunofluorescence tissue chemical technology and laser confocal microscope, represent the expression of VEGF with fluorescence ratio, semi-quantitative analysis cobaltous chloride and physics anoxic treatment different time are to the inducing action of retinal pigment epithelium (RPE) the cell VEGE protein expression of In vitro culture, and Gen is to the influence of the vegf expression of cobaltous chloride and physics hypoxia inducible.Find that matched group has the expression of low-level vegf protein, along with cobaltous chloride and the prolongation of physics anoxic treatment time, the variation of fluorescence ratio shows as time dependence, peaks at the 6h point.Gen (50,100,200 μ mol/L group) compares with the DMSO matched group, all can obviously suppress the expression of the VEGF of cobaltous chloride and physics hypoxia inducible, and this kind inhibitory action is concentration dependent.Above result shows that the RPE cell VEGE that Gen can suppress cobaltous chloride and physics hypoxia inducible expresses, and shows that this medicine brings into play prevention and treatment retina and corneal vascularization by the inducible factor vegf expression of angiogenesis inhibiting.
Four, specific embodiments
(1) Gen and soybean isoflavone are to mice retinal vessel new life's effect
1. material and method
1.1 material and key instrument: Gen, 98%, available from Sigma company, be dissolved in 1% sodium carboxymethyl cellulose (CMCC); Soybean isoflavone (Gen content 70%), extract this chamber, is dissolved in 1%CMCC; The vegf protein monoclonal antibody is available from NEOMARK company: the HIF-1 protein monoclonal antibody is available from NOVUS company; Goat anti-mouse IgG-FITC, goat anti-mouse igg-CY3 are available from Sigma company; Oxygen analyser is available from instrucment and meter plant of Shanghai International Automobile City Tourist Festival student's federation; LSM510 type laser confocal microscope is available from German Zeiss company.
1.2 Animal Model Making: select the female Mus (Shanghai Chinese Academy of Sciences animal center provides) of nonparous adult C57BL/6, raise with cage with 1 public Mus respectively for 2 one group, be divided into the oxygen supply group at random, three oxygen supply are to Gen group (Gen 50,100,200mg/kg), and three oxygen supply are to soybean isoflavone group (70mg/kg, 140mg/Kg, 280mg/Kg) and matched group.Every group of 10 the newborn C57BL/6 mices that on average are born.With oxygen supply group, oxygen supply to Gen group and oxygen supply to each 10 mice of soybean isoflavone group in giving birth to back 7d with putting into airtight drying bottle with the female Mus of cage.Insert 100% moistening oxygen in the drying bottle, the volume fraction of keeping oxygen in the oxygen case is 75 ± 5%, regulates once, and monitors with oxygen analyser in every 3-6 hour.Open the oxygen case once a day with food, change water.10 mices live in the normal oxygen environment matched group.
1.3 the hypertrophy of retina section counting retinal vessel: every group of mice all put to death eyeball excise and label orientation in giving birth to back 21d.The eyeball of extracing is immersed in the 4% neutral formalin solution 24 hours, conventional dehydration, paraffin embedding.The continuous 6 μ m section of the parallel optic nerve of sagittal plane, every eye is randomly drawed 5 tangent planes (having the tangent plane of optic nerve section not to be logged in interior), snow husband's periodic acid-haematoxylin (PAS) dyeing, the microscopically counting is broken through the vascular endothelial cell check figure order of internal limiting membrane.
1.4 immunofluorescence detects the expression of VEGF, HIF1 in the retina: from oxygen supply group, oxygen supply administration group, to the retina section of randomly drawing 10 no dyeings soybean isoflavone group and the matched group, after the conventional dewaxing, 100 ℃ of antigen retrieval are 15 minutes in the citrate buffer, give VEGF respectively, 4 ℃ of overnight incubation of HIF1 monoclonal antibody.PBS washes 3 times, each 5 minutes.Then give two anti-hatching 1 hour of FITC or CY3 labelling, PBS washes 3 times, each 5 minutes.Under laser confocal microscope, observe imaging.
1.5 statistical analysis: adopt the non-paired t test method to compare oxygen supply group and matched group respectively, oxygen supply group and oxygen supply administration group break through the cell nuclei of inner limiting membrane.P＜0.05, expression has significant difference.
2. result
2.1 the new life of counting retinal vessel
Observe the section of normal control group mice eye, rarely seen minute quantity has the vascular endothelial cell of breaking through internal limiting membrane, the models treated group breaks through the vascular endothelial cell number of internal limiting membrane obviously more than normal group, Gen administration group and obviously reduce than the models treated group to the vascular endothelial cell number that the soybean isoflavone group breaks through internal limiting membrane, and be dose dependent.
2.2 the expression of VEGF, HIF1 in the retina
The expression of VEGF, HIF1 is obviously strong than normal group in the anoxic treatment group retinal endothelial cell.Gen administration group and to soybean isoflavone group and anoxic treatment group relatively a little less than the fluorescence intensity, and is dose dependent.
(2) inhibitory action of Gen and soybean isoflavone corneal angiogenesis
1. materials and methods
1.1 material and key instrument: Gen, 98%, be dissolved in 1%CMCC or 0.05%DMSO, soybean isoflavone (Gen content 70%), extract this chamber, is dissolved in 1%CMCC or 0.05%DMSO.
1.2 large ear rabbit weighs 2.0～2.2kg.Two are carried out the cornea suture, under the tetracaine topical anesthesia, stitch 3 pins with the 1-0 silk thread above cornea.Rabbit is divided into 7 groups at random, 5 every group.Matched group is irritated stomach 0.5%CMCC, and the administration group is irritated stomach Gen 15,30,60mg/kg and filling stomach soybean isoflavone 25,50,100mg/kg respectively.
1.3Wistar rat weighs 300～350g, the corneal central area is implemented the alkalescence burn after the general anesthesia, with diameter is the circular filter paper of 5mm, is dipped in 1.0M sodium hydroxide solution 10 μ l, places rat cornea central authorities, take off after 10 seconds, immediately with physiological saline solution flushing 1 minute.Matched group eye drip 0.05%DMSO, every day 3 times, each 0.1ml, the administration group is eye drip Gen 0.02mg/ml, 0.04mg/ml, 0.08mg/ml respectively, soybean isoflavone 0.03,0.06,0.12mg/ml, every day 3 times, each 0.1ml.
2. result
2.1Gen and soybean isoflavone is to the influence of rabbit corneal angiogenesis due to the suture
Observe the rabbit corneal angiogenesis with operating loupe, measure the number and the total length of new vessels.Find behind the cornea suture the 3rd day, cornea hyperemia, and have new vessels to stretch in the limbus of corneae, 3～7 new vesselses appear in every suture upper end, about length 0.6mm.Observed to the suture the 20th day, the number and the total length of visible administration group new vessels obviously reduce than matched group, and the process of angiogenesis also slows down.Show that Gen and soybean isoflavone can suppress rabbit corneal angiogenesis due to the suture.
2.2Gen and soybean isoflavone is to the influence of rat corneal vascularization due to the alkali burn
With slit lamp observation cornea rebirth blood vessel progress, periphery has new vessels to immerse during 1 week, and administration group new vessels number obviously reduces than matched group, and vascular occlusion is wire after 2 weeks, until disappearance.Matched group vascular occlusion occurs after 2 weeks, about 5 weeks back blood vessel disappearance.Show that Gen can obviously suppress the corneal vascularization due to the alkali burn.
(3) Gen influence that rabbit RPE cell VEGE due to chemical hypoxia and the physics anoxia is expressed
1. material and method
1.1 material and instrument; Dulbecco`s modified eagle medium (DMEM) culture medium, trypsin is available from GIBCO company; Viviparous Ox blood serum is available from PVG high bridge veterinary station; Dimethyl sulfoxide (DMSO) is available from AMRESCO company; The vegf protein monoclonal antibody is available from NEOMARK company; Goat anti-mouse IgG-FITC is available from Sigma company; Poly-D-lysine is available from Shanghai biotechnology company limited; Gen is available from Sigma company, with DMSO hydrotropy (DMSO final concentration＜0.05%); TY80B type decolorization swinging table is available from biotechnology development company of Nanjing University; 6 well culture plates are available from Denmark NUNC company; LSM510 type laser confocal microscope is a German Zeiss company product.
1.2 the cultivation of rabbit RPE cell: pneumatic needle takes off lagophthalmos after putting to death 2～3 of grey rabbits at once, is soaked in 0.1M PBS (NaCl 8.00g, KCl 0.2g, Na
2HPO
4H
2O 1.56g, K
2HPO
40.2g be dissolved in the 1000ml distilled water, pH 7.4) in, 4 ℃ of refrigerator 1h.Cut off eyeball under the aseptic condition, discard anterior ocular segment and vitreous body, complete absorption cerebral stratum of retina.Use D-Hanks liquid (NaCl 8.00g, KCl 0.40g, Na in the eyecup
2HPO
4H
2O 0.06g, KH
2PO
40.06g, NaHCO
30.35g be dissolved in the 1000ml distilled water, pH 7.4) wash two times, add 0.25% trypsin, 37 ℃ of digestion 30min, make the RPE cell separation with dropper piping and druming, collecting cell adds the DMEM culture fluid and (contains the 100U/ml penicillin sodium, 100 μ g/ml streptomycins behind the centrifuge washing, 20% new-born calf serum, pH7.4), be inoculated in the 50ml culture bottle, every 3d changes liquid once.When cell grows to nearly fusion state, use 0.25% trypsinization, go down to posterity.3～4 generation cell be used for the experiment.
1.3 cobaltous chloride simulation anoxia: after the clean routine disinfection processing of 25 * 25mm coverslip, be soaked in 2～4h in 0.1% poly-D-lysine, take out and dry 2～4h, it is standby to put into 6 well culture plates.To reach on the coverslip of RPE cell inoculation in 6 well culture plates of the third generation, treat that cell covers with 50% of slide～60% and o'clock begins experiment.The culture fluid that inclines after D-Hanks liquid flushing 2～3 times, adds and contains the DMEM culture fluid that final concentration is 125 μ mol/L cobaltous chlorides, continues respectively to cultivate 0,1,2,4,6h.The culture fluid that inclines is used for immunofluorescence test.
1.4 physics anoxia: will reach on the coverslip of RPE cell inoculation in 6 well culture plates of the third generation, and treat that cell will cover with 50% of slide～60% and o'clock begins experiment.The culture fluid that inclines after D-Hanks liquid flushing 2～3 times, places 95%N
2, 5%CO
237 ℃ of incubators in, continue to cultivate 0,1,2,4 respectively, 6h.The culture fluid that inclines is used for immunofluorescence test.
1.5 second quantitative observation of immunohistochemical test and laser confocal microscope: will handle and the pretreated RPE cell of Gen places on the decolorization swinging table with 4 ℃ of PBS flushings 2 times, 5min at every turn through anoxia and cobaltous chloride respectively.100% methanol room temperature is 10min fixedly.Wash cells 2 times with 4 ℃ of PBS.Add monoclonal antibody (dilution in 1: 200) the 50 μ l of mouse anti vegf protein, hatch 1h for 4 ℃.Wash cells 4 times with 4 ℃ of PBS.Add goat anti-mouse IgG-FITC (dilution in 1: 200) 50 μ l, hatch 1h for 4 ℃.Wash cells 4 times with 4 ℃ of PBS.After every testing sample adds 200 μ l PBS, place respectively under the laser confocal microscope that connects computer, * 40 object lens, 7% optical filter, excitation wavelength is 488nm, selected optimum cell sweep parameter behind the prescan, and in experiment, remain unchanged.After each visual field laser scanning 2 times, can note down the average fluorescent strength in the full cell, and store the experimental data of being gathered.This experiment is with the index of the average fluorescent strength in the full cell as the judgement vegf expression.
2, result
2.1RPE cell culture and morphologic observation
Former rabbit RPE cell of being commissioned to train foster is flat polygon more, is full of black or brown pigment granule in the endochylema.After cell enters division stage, the endochrome granule with division repeatedly gradually dilution reduce, cell is transparent gradually, to third and fourth generation, no tangible pigment granule in the cell forms the monolayer spindle cell of transparent and homogeneous.
2.2 cobaltous chloride simulation anoxia is to the influence of RPE cell VEGE protein expression
After 125 μ mol/L cobaltous chlorides are handled, choose 0,1,2,4, the 6h cell.Can be observed under the laser co-focusing fluorescence microscope: compare with not adding an anti-group, in the anoxia 0h group endochylema more weak fluorescence is arranged, prompting has the expression of low-level vegf protein.Then along with the prolongation of anoxia time, the increase of fluorescence ratio shows as time dependence, and cobaltous chloride handles behind the 4h that the increase of fluorescence ratio eases up in the endochylema, forms plateau, and 6h peaks in anoxia.(Fig. 1)
2.3Gen influence to cobaltous chloride simulation anoxia 4h RPE cell VEGE protein expression
Handling RPE cell 4h with 125 μ mol/L cobaltous chlorides+DMSO is matched group, the fluorescence ratio that 125 μ mol/L cobaltous chloride+final concentrations are respectively behind 50 μ mol/L, 100 μ mol/L, the 200 μ mol/L Gen processing RPE cell 4h obviously reduces, and successively decreases with increasing successively of concentration.Average fluorescent strength ratio Analysis prompting: Gen can significantly suppress the proteic expression of RPE cell VEGE behind the cobaltous chloride simulation anoxia 4h, and is concentration dependent.(Fig. 2)
2.4 the physics anoxia is to the influence of RPE cell VEGE protein expression
Six orifice plates of inoculation RPE are placed 95%N
2, 5%CO
237 ℃ of incubators in, continue to cultivate 0,1,2,4 respectively, 6h.Under the laser co-focusing fluorescence microscope, can be observed: compare with not adding an anti-group, in the anoxia Oh group endochylema more weak fluorescence is arranged.Along with the prolongation of anoxia time, the increase of fluorescence ratio shows as time dependence.(Fig. 3)
2.5Gen influence to physics anoxia 4h RPE cell VEGE protein expression
Handling RPE cell 4h with anoxia+DMSO is matched group, and the fluorescence ratio that final concentration is respectively behind 50 μ mol/L, 100 μ mol/L, the 200 μ mol/L Gen processing RPE cell 4h obviously reduces, and successively decreases with increasing successively of concentration.(Fig. 4)
Five, description of drawings
Fig. 1. cobaltous chloride simulation anoxia is to the influence of RPE cell VEGE protein expression.A: after the cobaltous chloride simulation anoxia 0,1,2,4,6h RPE cell laser co-focusing fluorescence microscopy images.B:RPE cell VEGE protein expression is represented with the fluorescence percentage ratio of matched group.n＝8， x±S.D，
××P＜0.01vs?0h。
The effect of the RPE cell VEGE protein expression that Fig. 2 .Gen causes cobaltous chloride simulation anoxia.Laser co-focusing fluorescence microscopy images behind A:Gen (0 μ mol/L, 50 μ mol/L, 100 μ mol/L, 200 μ mol/L) the processing RPE cell.B:RPE cell VEGE protein expression is represented with the fluorescence percentage ratio of matched group.n＝8，x±S.D，
××P＜0.01vs?0μmol/L?Gen。
Fig. 3. the physics anoxia is to the influence of RPE cell VEGE protein expression.A: after the physics anoxia 0,1,2,4,6h RPE cell laser co-focusing fluorescence microscopy images.B:RPE cell VEGE protein expression is represented with the fluorescence percentage ratio of matched group.n＝8， x±S.D，
××P＜0.01vs?0h。
The effect of the RPE cell VEGE protein expression that Fig. 4 .Gen causes the physics anoxia.Laser co-focusing fluorescence microscopy images behind A:Gen (0 μ mol/L, 50 μ mol/L, 100 μ mol/L, 200 μ mol/L) the processing RPE cell.B:RPE cell VEGE protein expression is represented with the fluorescence percentage ratio of matched group.n＝8， x±S.D，
××P＜0.01vs?0μmol/L?Gen。
Claims (1)
1, a kind of 4 ', 5,7 trihydroxy-isoflavone Genistein write a Chinese character in simplified form the pure product of Gen or contain the beans of Gen and the extract of corresponding plants or be the application of compound preparation in preparation prevention and treatment retina and corneal vascularization medicine of main component with Gen.
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| CN 02148598 CN1235582C (en) | 2002-12-23 | 2002-12-23 | Medicines for preventing and treating retina and cornea blood vessel diseases |
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|---|---|---|---|
| CN 02148598 CN1235582C (en) | 2002-12-23 | 2002-12-23 | Medicines for preventing and treating retina and cornea blood vessel diseases |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100427083C (en) * | 2005-11-16 | 2008-10-22 | 江南大学 | Method for preparing flavone aglycone micro capsule |
| CN106822182A (en) * | 2016-12-27 | 2017-06-13 | 广州姿生生物科技有限公司 | A kind of cell extract and application thereof |
| CN107137421A (en) * | 2017-05-25 | 2017-09-08 | 青岛市中心医院 | Dextran sulfate is preparing the application in suppressing corneal vascularization medicine |
| CN109953948A (en) * | 2018-12-29 | 2019-07-02 | 广东宏盈科技有限公司 | A kind of isoflavones eye drops and its preparation method and application |
| CN109925307A (en) * | 2018-12-29 | 2019-06-25 | 广东宏盈科技有限公司 | A kind of isoflavones and sodium hyaluronate pharmaceutical composition and its application |
| CN115778937A (en) * | 2022-10-24 | 2023-03-14 | 中国人民解放军海军军医大学第一附属医院 | Application of chrysin in preparation of medicine for inhibiting retinal neovascularization |
| CN115737661A (en) * | 2022-10-24 | 2023-03-07 | 中国人民解放军海军军医大学第一附属医院 | Application of baicalin in preparation of medicine for inhibiting retinal neovascularization |
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