| Age-related Macular Degeneration(AMD)is a degenerative disease that causes vision loss or even blindness in elderly people over 50 years of age in developed areas of China and developed countries.The main structures involved in the disease are photoreceptor cells,retinal pigment epithelium,bruch membrane and choroidal capillaries.Early age-related macular degeneration is characterized by the presence of drusen or retinal pigmentary abnormalities.Late or advanced age-related macular degeneration is characterized by large drusen and geographic atrophy extending to the center of the macula or choroidal neovascularization and its sequelae.At present,several risk factors for the development and progression ofage-related macular degeneration have been established,including oxidative damage,advanced age,race,genetics,heredity,diet and so on.AMD damage the macula which is the most important and most sensitives tructure of human vision,causing irreversible loss of vision.The impaired vision result in many elderly people losing their independence at retirement age,which not only affect the quality of life of patients,but also bring a series of social and economic problems to the country.In recent years,antioxidants and mineral supplements have been used to prevent the development of AMD or slow down the progress of dry AMD,but the effect is not obvious;and for wet AMD,anti-vascular endothelial growth factor(VEGF)drugs treatment can effectively prevent some patients with severe visual loss,but repeated injections will increase the patient' s economic burden and treatment risk,and the treatment of some patients with poor results.Therefore,to find more safe and effective treatment and target become one of the key subjects of ophthalmic research.In recent years,a large number of studies have shown that tissue factor may be involved in the pathogenesis of AMD.Many studies suggest that inflammatory response and oxidative stress can cause up-regulation of TF expression,which induced the occurrence of AMD.Moreover,experimental datas show that TF not only involved in the formation of tumor blood vessels,but also involved in the formation of CNV.Tissue factor Target Peptide(TF-TP)is a novel TF targeting small molecule polypeptide synthesized by Professor Rao,a member of the research group.This study will explore the possible application value of TF-TP to AMD.For this purpose,we have carried out two aspects of research work.The first part investigates the effect of TF-TP on the damage of Human Retinal Pigment Epithelium(hRPE)induced by blue light and the related mechanism.The second part observes the effect of TF-TP on choroidal neovascularization induced by laser in C57BL/6 mice.Objective1.To investigates the effect of TF-TP on the damage of Human Retinal Pigment Epithelium cells induced by blue light and the related mechanism.2.To observe the effect of TF-TP on choroidal neovascularization induced by laser in C57BL/6 mice.Methods1.The normal hRPE cells were cultured with different concentrations(10,100,150,200,300 ?mol/L)TF-TP for 24 h,and the cell viability were detected by CCK-8.2.Human RPE cells were isolated from donor eye and cultured.Cultured cells were divided into blank control group,model group and TF-TP treated group.Light-induced RPE cell damage model was established by exposuring the cells in the blue light of(4.0 ± 0.5)mW/cm2 for 12 hours in the model group,and different concentrations(10,100,150,200,300 ?mol/L)of TF-TP was added into the medium to pretreat the cells for 24 hours and than exposed the cells to the blue light for 12 hours in the TF-TP groups.The optimal concentration of TF-TP in hRPE cells was detected by CCK-8 method.And the optimal concentration was used in the following groups:blank control group,blue light irradiation group and 150?mol/L TF-TP group.3.The morphology and ultrastructure in the hRPE cells were observed under the inverted microscope and transmission electron microscope.The apoptosis of the hRPE cells was assayed by Hoechst staining.The expressions of apoptosis-related protein bax,bcl-2 in the cells were determined by Western blot.4.The expression of TF protein in hRPE cells at different blue light irradiation time(Oh,1h,2h,3h,6h,12h,24h)was detected by Western blot.5.The CNV model of mice was established by 532nm laser.The experimental group was divided as follow:normal control group was treated without any treatment.Laser group was induced by laser.Intervention group was given vitreous cavity injection of TF-TP in the first day after administration of 532nm laser,and once every two days for three times.The CNV leakage was estimated by fundus fluorescein angiography(FFA).6.The maximum thickness of CNV was observed by histopathological examination in 14 days after laser irradiation.In addition,the expression of CD31 in the choroidal choroidal tissue was detected by immunohistochemical staining.7.The expression of TF in RPE-choroidal complex of mice was detected by Western Blot.8.Statistical Methods:SPSS 21.0 statistical software was used for statistical analysis.The datas of the test indicators in this study were normal distribution by W test,expressed as x±s.The mean variance of each detection index is uniformLevene by Levene test.The difference between the two groups was compared with the independent sample t test.The overall difference between the multiple groups was compared with the single factor analysis(ANOVA).The LSD-t test was used for each group.P?0.05 was statistically significant.Results1.CCK-8 assay showed that there was no significant difference in the cell viability among blank control group and different concentrations TF-TP groups(F=2.15,P=0.11),suggesting that TF-TP has no toxic and side effect on hRPE cells.2.The cell viability of the blank control group,blue light irradiation group and each concentration TF-TP group were(100.0±0.00)%,(43.79±6.55)%,(57.24±7.89)%,(59.97±4.73)%,63.45±3.57)%,(58.88±5.59)%and(55.95±2.80)%respectively(F=9.16,P=0.00).Compared with the blue light irradiation group,the cell viability of each TF-TP group was significantly higher than that of the control group,and the difference was statistically significant(P = 0.01,0.00,0.00,0.00,0.00).The group of 150 ?mol/L TF-TP had the highest cell viability and could be used as a model for TF-TP protection.3.A lot of shrinkage,deformation,suspension cells were exhibited under the optical microscope,and decrease of microvilli structure,rupture of mitochondrial cristae and vacuolar degeneration of the cells were found in the model group,and the damage of the cells were evidently lightened in the TF-TP group.The apoptosis rate of the cells were(0.98±0.19)%,(9.98±0.82)%and(5.73±0.88)%in the blank group,model group and TF-TP group,respectively,with significant difference among the groups(F=206.18,P=0.00),and the apoptosis rate of the cells in the TF-TP group was significantly lower than that in the model group(P<0.05).Compared with the blank control group,the relative expression of bax and TF was obviously increased and that of bcl-2 was decreased in the model group;while the expression of bax and TF was lower,and that of bcl-2 was higher in the TF-TP group compared with model group(all at P<0.05).4.The relative expression of TF protein in hRPE cells was(0.25±0.01),(0.26±0.02),(0.28±0.02),(0.31 ±0.02),(0.60±0.03),(0.79±0.04)and(0.56±0.03),respectively.The relative expression of TF protein in each group was statistically different(F=276.99,P<0.05).With the prolongation of blue light irradiation time,the relative expression of TF protein increased gradually and peaked at 12h,followed by a drop.5.FFA examination of mice in the normal control group showed that the retinal vascular was filled and distributed radially with the optic disc as the center.In addition,the choroidal vascular showed diffuse background fluorescence and fluorescein leakage was not observed.In the laser group,14 days after photocoagulation,FFA showed that there were obvious fluorescence leakage at the spot of the retina,and the diffuse fluorescence was diffused at the later stage.FFA examination of mice in the TF-TP group showed that mild fluorescence leakage was observed in the retina of the retina,and the high fluorescence was observed at the late stage of angiography,but no diffusion was observed.There was no significant difference in the leakage rate between the laser group and the TF-TP group(x2=0.59,P=0.24).6.The results of HE staining showed that the CNV thickness in the TF-TP intervention group was significantly lower than that in the simple laser group(t=24.39,p<0.05),indicating that TF-TP can significantly inhibit the thickness of CNV after intervention.The results of immunohistochemical staining showed that the average optical density(0.09±0.03)of CD34 immunohistochemistry positive group in the TF-TP intervention group was significantly lower than that of simple laser group(0.16 ± 0.01),with a significant difference between two groups(t=3.93,P<0.05).7.The results of Western Blot showed that the relative expressions of TF protein in normal control group,simple laser group and TF-TP group were(0.14±0.01),(0.40±0.02)and(0.22±0.03),respectively.The relative expression of TF protein in each group was statistically different(F=128.26,P<0.05)by analysis of ANOVA.Furthermore,the relative expression of TF protein in TF-TP intervention group was significantly lower than that in laser group and the difference was statistically significant(P<0.05).Conclusions1.TF-TP can improve the structure of damaged hRPE cells induced by blue light,decrease the apoptosis rate,decrease the expression of TF protein and bax protein,increase the expression of bc1-2 protein,in a word,TF-TP can reduce the damage of hRPE cells induced by blue light.2.TF-TP can reduce the thickness of laser-induced mouse CNV,decrease the positive expression of CD34 and TF protein and inhibit the formation of CNV in mice. |
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