Objective: To investigate the effects of nerve growth factor(NGF)on proliferation and DNAthesis of cultured human fetal retinal pigment epithelium (RPE)cells in vitro.Methods: Primary culture and subculture of human f...Objective: To investigate the effects of nerve growth factor(NGF)on proliferation and DNAthesis of cultured human fetal retinal pigment epithelium (RPE)cells in vitro.Methods: Primary culture and subculture of human fetal retinal pigment epithelium cellswere established in vitro first. Cultured RPE cells were treated with NGF by variousconcentrations 0μg/L, 50μg/L, 100μg/L, 200μg/L and 300μg/L(final concentration)for 48 hs.After 48 hs, cells proliferation was measured with methyl thiazolyl tetrazolium(MTT)assay method and the amount of DNA was determined by the absorbance at 280nm of nucleic acid & protein analysis.Results: The A values of 100 μg/L, 200 μg/L, 300 μg/L NGF was(0. 213 7 ± 0. 23 3),(0. 218 8 ±0. 018 1), (0. 232 2 ±0. 016 4) as compared with(0. 189 7 ±0. 015 2) of Avalue of 0 μg/L NGF respectively, q value was 3.63,4.40, 6. 42 and P value was0. 015, 0. 000, 0. 000(q-test). The DNA concentrations of 100 μg/L, 200 μg/L, 300μg/L and 400 μg/L NGF was (981. 220 4 ± 123.535 7), (1 375. 848 4 ±244. 471 8),(1 658.707 1 ± 176. 938 1), (2 353.086 3 ±609. 906 4) μg/ml as compared with(666. 818 8 ± 141. 330 2) μg/ml of DNA concentration of 0 μg/L NGF respectively, qvalue was 3.63,8.20,11.47,19.46, P value was 0. 024,0. 000,0. 000,0. 000 (q-test).Conclusion: The data suggested that NGF could stimulate the proliferation and DNAsynthesis of cultured of hRPE cells in vitro in a dose-dependent manner.展开更多
AIM： To investigate the effects of high glucose levels and anti-vascular endothelial growth factor（VEGF） agents（bevacizumab,ranibizumab and aflibercept） on retinal pigment epithelium（RPE） cells.METHODS： ARPE-1...AIM： To investigate the effects of high glucose levels and anti-vascular endothelial growth factor（VEGF） agents（bevacizumab,ranibizumab and aflibercept） on retinal pigment epithelium（RPE） cells.METHODS： ARPE-19 cells were cultured at different glucose levels（5.5 mmol/L,25 mmol/L,and 75 mmol/L）.Cell viability was evaluated by MTT assay at 3d after treatment with D-glucose.Cell migration ability was measured by wound healing assay at 3d.A cell death detection kit was used to assess apoptosis at 3 and 14 d.Cell proliferation was assessed by EdU assay at 3d.The culture medium was treated with anti-VEGF agents at clinically relevant concentrations.The experiment was then repeated at a different glucose level.RESULTS： The viability and migration of ARPE-19 cells were significantly decreased in the presence of 75 mmol/L as compared to 5.5 mmol/L glucose.The percentage of TUNEL-positive cells was significantly increased and the proliferative potential was decreased with 75 mmol/L compared to 5.5 mmol/L glucose.There were no significant differences in the results between 25 mmol/L and 5.5 mmol/L glucose.In the presence of 75 mmol/L glucose,the groups treated with anti-VEGF showed decreased cell viability and proliferation and increased apoptosis.However,there were no significant differences between the anti-VEGF groups.CONCLUSION： High glucose level decreases the viability,wound healing ability,and proliferation of RPE cells,while increasing apoptosis.Furthermore,anti-VEGF agents interfered with the physiological functions of RPE cells under high-glucose conditions,accompanied by decreases in cell viability and proliferation.展开更多
AIM: To investigate the regulation of vascular endothelial growth factors(VEGF) and pigment epithelium-derived factor(PEDF) expression by autophagy in retinal pigment epithelium(RPE) cells on exposure to hypoxia. METH...AIM: To investigate the regulation of vascular endothelial growth factors(VEGF) and pigment epithelium-derived factor(PEDF) expression by autophagy in retinal pigment epithelium(RPE) cells on exposure to hypoxia. METHODS: ARPE-19, an RPE cell line, was treated as following: the control group was kept in a normoxic incubator; the hypoxia group was incubated in a hypoxic incubator with 1% O_2/5% CO_2/94% N_2 for 24 h; the hypoxia + 3-methyladenine(3-MA) group was pretreated with 10 mmol/L 3-MA for 1 h and then in the hypoxic incubator for 24 h; and the hypoxia + chloroquine(CQ) group was pretreated with 50 μmol/L CQ for 1 h and then in the hypoxic incubator for 24 h. The morphology and ultrastructure of the cells was observed by an inverted microscope or a transmission electronic microscope(TEM). Western blot was performed to assay the expression of autophagy-associated markers, including microtubule associated protein 1 light chain 3 B(LC3 B), Beclin-1, Atg5 and p62. The concentration of VEGF and PEDF in the culture supernatant was determined by ELISA, and the ratio of VEGF/PEDF was calculated. RESULTS: There were no obvious differences in cell morphology among different groups and autolysosomes could be observed in the cytoplasm in all groups. Compared to the control cells, the LC3 B-II/I ratio and levels of Beclin-1 and Atg5 were significantly increased and p62 level was significantly decreased in the hypoxia group. With the increase of VEGF and decrease of PEDF concentration, the VEGF/PEDF ratio was significantly increased in the hypoxia group compared to the control cells. The LC3 B-II/I ratio was significantly reduced by 3-MA treatment and increased by CQ treatment. The expressions of Beclin-1 and Atg5 were significantly reduced by 3-MA or CQ treatment, while expression of p62 was increased in the 3-MA or CQ treated cells. The concentration of VEGF was significantly decreased and PEDF increased, thereby the VEGF/PEDF ratio was decreased in the hypoxia + 3-MA group and hypoxia + CQ group compared with that in the hypoxia group. CONCLUSION: Hypoxia leads to elevated autophagy in RPE cells, and expression of VEGF and PEDF might be regulated by autophagy on exposure to hypoxia to further participate in regulating the formation of retinal neovascularization.展开更多
Objective To explore whether the serum concentrations of vascular endothelial growth factor （VEGF） and pigment epithelium-derived factor （PEDF） could serve as the predictors of ovarian hyperstimulation syndrome （...Objective To explore whether the serum concentrations of vascular endothelial growth factor （VEGF） and pigment epithelium-derived factor （PEDF） could serve as the predictors of ovarian hyperstimulation syndrome （OHSS） in patients undergoing in vitro fertilization-embryo transfer （IVF-ET）. Methods Enzyme-linked immunoadsordent assay （ELISA） was employed to measure the serum concentrations of VEGF and PEDF on the day of hCG administration, oocyte retrieval and embryo transfer, respectively. Based on OHSS classification of the criteria of Golan, 85 patients were divided into three groups. Patients in group A （n=10） showed symptoms of severe OHSS and patients in group B （n=13） suffered from moderate OHSS. The control group （group C, n=62） contained patients without symptoms of OHSS as well as patients with mild OHSS.Results In groups A, B and C, serum concentrations of PEDF on the day of hCG administration （h-PEDF）（166.54 ± 102.81 pg/ml, 159.45 ±136. 77 pg/ml, 172.05±170.95 pg/ml, P=0.48）, oocyte retrieval （o-PEDF）（176.91 ± 103.37 pg/ml, 122.52± 92.54 pg/ml, 179.82±177.47 pg/ml, P=0.27） and embryo transfer （e-PEDF）（169.02± 240.08 pg/ml, 136.80 ±139.21pg/ml, 157.38 ±222.54 pg/ml, P=0.95）, h-VEGF （175.55 ± 103.54 pg/ml, 218.84 ±179.70pg/ml, 153.39±145.06 pg/ml, P=0.36） and o- VEGF （171.93 ± 128.55 pg/ml, 220.36±149.82 pg/ml, 138. 74 ±% 139.30 pg/ml, P=0. 15） showed no significant differences. There was a statistical difference in serum concentration of e-VEGF between group A （197.04±156.63 pg/ml） and group C （110.69±49.55 pg/ml）（P=0.008）. The serum level of estradiol showed a positive correlation with the count of large follicles （r=0. 744）. The ratios of h-VEGF/h-PEDF, o-VEGF/o-PEDF and e-VEGF/e-PEDF were calculated and showed a clear difference among groups A, B and C （4.04±3.39, 2.10±2.14, 1.05± 4.80, P〈0.001; 4.54 5.69, 2.29 ±1.67, 0.94 ±0.59, P〈0.001; 5.43±6.16, 1.81±1.36, 2.42±2.60, P=0.04）. Conclusion While neither serum concentrations of VEGF nor PEDF can be used as an OHSS predictor, the ratios of h-VEGF/h-PEDF, o-VEGF/o-PEDF and e-VEGF/e-PEDF may have great predictive value.展开更多
In order to elucidate the effect of dexamethasone on the expression of transforming growth factor-betal （TGF-β1） in ciliary pigment epithelial （CPE） cells cultured in vitro, rabbit CPE cells were cultured in vitr...In order to elucidate the effect of dexamethasone on the expression of transforming growth factor-betal （TGF-β1） in ciliary pigment epithelial （CPE） cells cultured in vitro, rabbit CPE cells were cultured in vitro, treated with DMEM medium containing 0, 1 × 10^-8 , 5 × 10^-8 , 10 × 10^-8 and 50 × 10^-8 mol/L dexamethasone respectively for 5 days. The TGF-131 expression was detected by immunohistochemistry Supervision methods and analyzed semi-quantitatively by HMIAS-2000 image system. As opposed to in vivo, rabbit CPE cells expressed TGF-131 under cul- tured circumstance in vitro. The gray scales of the positive yellow staining in the groups of 1 × 10^-8 , 5 × 10^-8 , 10 × 10^-8 and 50 × 10^-8 mol/L dexamethasone were 136.57 ± 4.43, 140.20 ± 6. 10, 142. 98± 2. 99, 146.80±1.68 and 150.05 × 1.94 respectively. When the concentrations of dexamethasone were equal to or higher than 5 × 10^-8mol/L and, the expression of TGF-β1 was inhibited. 10^-7 mol/L dexamethasone showed a significant inhibition. It was suggested that CPE cells possess the potential ability of synthesizing and expressing TGF-β1. The inhibition of TGF-β1 expression by dexamethasone may be beneficial to the treatment of proliferative vitroretinopathy, also exert some influence on the secretion of aqueous humor and ciliary inflammation.展开更多
Choroidal neovascularization （CNV）, a major cause of vision loss, is the result of the increased vascular endothelial growth factor （VEGF） expression in human retinal pigment epithelial （RPE） cells. It is import...Choroidal neovascularization （CNV）, a major cause of vision loss, is the result of the increased vascular endothelial growth factor （VEGF） expression in human retinal pigment epithelial （RPE） cells. It is important to inhibit the expression of VEGF protein in RPE cells.展开更多
文摘Objective: To investigate the effects of nerve growth factor(NGF)on proliferation and DNAthesis of cultured human fetal retinal pigment epithelium (RPE)cells in vitro.Methods: Primary culture and subculture of human fetal retinal pigment epithelium cellswere established in vitro first. Cultured RPE cells were treated with NGF by variousconcentrations 0μg/L, 50μg/L, 100μg/L, 200μg/L and 300μg/L(final concentration)for 48 hs.After 48 hs, cells proliferation was measured with methyl thiazolyl tetrazolium(MTT)assay method and the amount of DNA was determined by the absorbance at 280nm of nucleic acid & protein analysis.Results: The A values of 100 μg/L, 200 μg/L, 300 μg/L NGF was(0. 213 7 ± 0. 23 3),(0. 218 8 ±0. 018 1), (0. 232 2 ±0. 016 4) as compared with(0. 189 7 ±0. 015 2) of Avalue of 0 μg/L NGF respectively, q value was 3.63,4.40, 6. 42 and P value was0. 015, 0. 000, 0. 000(q-test). The DNA concentrations of 100 μg/L, 200 μg/L, 300μg/L and 400 μg/L NGF was (981. 220 4 ± 123.535 7), (1 375. 848 4 ±244. 471 8),(1 658.707 1 ± 176. 938 1), (2 353.086 3 ±609. 906 4) μg/ml as compared with(666. 818 8 ± 141. 330 2) μg/ml of DNA concentration of 0 μg/L NGF respectively, qvalue was 3.63,8.20,11.47,19.46, P value was 0. 024,0. 000,0. 000,0. 000 (q-test).Conclusion: The data suggested that NGF could stimulate the proliferation and DNAsynthesis of cultured of hRPE cells in vitro in a dose-dependent manner.
基金Supported by grants from Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the ministry of Education,Science,and Technology(No.2016R1A2B4008376Seoul,Republic of Korea)partially supported by the Soonchunhyang University Research Fund
文摘AIM： To investigate the effects of high glucose levels and anti-vascular endothelial growth factor（VEGF） agents（bevacizumab,ranibizumab and aflibercept） on retinal pigment epithelium（RPE） cells.METHODS： ARPE-19 cells were cultured at different glucose levels（5.5 mmol/L,25 mmol/L,and 75 mmol/L）.Cell viability was evaluated by MTT assay at 3d after treatment with D-glucose.Cell migration ability was measured by wound healing assay at 3d.A cell death detection kit was used to assess apoptosis at 3 and 14 d.Cell proliferation was assessed by EdU assay at 3d.The culture medium was treated with anti-VEGF agents at clinically relevant concentrations.The experiment was then repeated at a different glucose level.RESULTS： The viability and migration of ARPE-19 cells were significantly decreased in the presence of 75 mmol/L as compared to 5.5 mmol/L glucose.The percentage of TUNEL-positive cells was significantly increased and the proliferative potential was decreased with 75 mmol/L compared to 5.5 mmol/L glucose.There were no significant differences in the results between 25 mmol/L and 5.5 mmol/L glucose.In the presence of 75 mmol/L glucose,the groups treated with anti-VEGF showed decreased cell viability and proliferation and increased apoptosis.However,there were no significant differences between the anti-VEGF groups.CONCLUSION： High glucose level decreases the viability,wound healing ability,and proliferation of RPE cells,while increasing apoptosis.Furthermore,anti-VEGF agents interfered with the physiological functions of RPE cells under high-glucose conditions,accompanied by decreases in cell viability and proliferation.
基金Supported by the National Natural Science Foundation of China(No.81500726)the Medical Research Project of Xi’an Science Technology Bureau [No.201805097YX5SF31(4)]+1 种基金the Health Research Foundation of Shaanxi Province(No.2018D074)the Outstanding Youth Talent Support Plan of Shaanxi Ordinary University
文摘AIM: To investigate the regulation of vascular endothelial growth factors(VEGF) and pigment epithelium-derived factor(PEDF) expression by autophagy in retinal pigment epithelium(RPE) cells on exposure to hypoxia. METHODS: ARPE-19, an RPE cell line, was treated as following: the control group was kept in a normoxic incubator; the hypoxia group was incubated in a hypoxic incubator with 1% O_2/5% CO_2/94% N_2 for 24 h; the hypoxia + 3-methyladenine(3-MA) group was pretreated with 10 mmol/L 3-MA for 1 h and then in the hypoxic incubator for 24 h; and the hypoxia + chloroquine(CQ) group was pretreated with 50 μmol/L CQ for 1 h and then in the hypoxic incubator for 24 h. The morphology and ultrastructure of the cells was observed by an inverted microscope or a transmission electronic microscope(TEM). Western blot was performed to assay the expression of autophagy-associated markers, including microtubule associated protein 1 light chain 3 B(LC3 B), Beclin-1, Atg5 and p62. The concentration of VEGF and PEDF in the culture supernatant was determined by ELISA, and the ratio of VEGF/PEDF was calculated. RESULTS: There were no obvious differences in cell morphology among different groups and autolysosomes could be observed in the cytoplasm in all groups. Compared to the control cells, the LC3 B-II/I ratio and levels of Beclin-1 and Atg5 were significantly increased and p62 level was significantly decreased in the hypoxia group. With the increase of VEGF and decrease of PEDF concentration, the VEGF/PEDF ratio was significantly increased in the hypoxia group compared to the control cells. The LC3 B-II/I ratio was significantly reduced by 3-MA treatment and increased by CQ treatment. The expressions of Beclin-1 and Atg5 were significantly reduced by 3-MA or CQ treatment, while expression of p62 was increased in the 3-MA or CQ treated cells. The concentration of VEGF was significantly decreased and PEDF increased, thereby the VEGF/PEDF ratio was decreased in the hypoxia + 3-MA group and hypoxia + CQ group compared with that in the hypoxia group. CONCLUSION: Hypoxia leads to elevated autophagy in RPE cells, and expression of VEGF and PEDF might be regulated by autophagy on exposure to hypoxia to further participate in regulating the formation of retinal neovascularization.
文摘Objective To explore whether the serum concentrations of vascular endothelial growth factor （VEGF） and pigment epithelium-derived factor （PEDF） could serve as the predictors of ovarian hyperstimulation syndrome （OHSS） in patients undergoing in vitro fertilization-embryo transfer （IVF-ET）. Methods Enzyme-linked immunoadsordent assay （ELISA） was employed to measure the serum concentrations of VEGF and PEDF on the day of hCG administration, oocyte retrieval and embryo transfer, respectively. Based on OHSS classification of the criteria of Golan, 85 patients were divided into three groups. Patients in group A （n=10） showed symptoms of severe OHSS and patients in group B （n=13） suffered from moderate OHSS. The control group （group C, n=62） contained patients without symptoms of OHSS as well as patients with mild OHSS.Results In groups A, B and C, serum concentrations of PEDF on the day of hCG administration （h-PEDF）（166.54 ± 102.81 pg/ml, 159.45 ±136. 77 pg/ml, 172.05±170.95 pg/ml, P=0.48）, oocyte retrieval （o-PEDF）（176.91 ± 103.37 pg/ml, 122.52± 92.54 pg/ml, 179.82±177.47 pg/ml, P=0.27） and embryo transfer （e-PEDF）（169.02± 240.08 pg/ml, 136.80 ±139.21pg/ml, 157.38 ±222.54 pg/ml, P=0.95）, h-VEGF （175.55 ± 103.54 pg/ml, 218.84 ±179.70pg/ml, 153.39±145.06 pg/ml, P=0.36） and o- VEGF （171.93 ± 128.55 pg/ml, 220.36±149.82 pg/ml, 138. 74 ±% 139.30 pg/ml, P=0. 15） showed no significant differences. There was a statistical difference in serum concentration of e-VEGF between group A （197.04±156.63 pg/ml） and group C （110.69±49.55 pg/ml）（P=0.008）. The serum level of estradiol showed a positive correlation with the count of large follicles （r=0. 744）. The ratios of h-VEGF/h-PEDF, o-VEGF/o-PEDF and e-VEGF/e-PEDF were calculated and showed a clear difference among groups A, B and C （4.04±3.39, 2.10±2.14, 1.05± 4.80, P〈0.001; 4.54 5.69, 2.29 ±1.67, 0.94 ±0.59, P〈0.001; 5.43±6.16, 1.81±1.36, 2.42±2.60, P=0.04）. Conclusion While neither serum concentrations of VEGF nor PEDF can be used as an OHSS predictor, the ratios of h-VEGF/h-PEDF, o-VEGF/o-PEDF and e-VEGF/e-PEDF may have great predictive value.
文摘In order to elucidate the effect of dexamethasone on the expression of transforming growth factor-betal （TGF-β1） in ciliary pigment epithelial （CPE） cells cultured in vitro, rabbit CPE cells were cultured in vitro, treated with DMEM medium containing 0, 1 × 10^-8 , 5 × 10^-8 , 10 × 10^-8 and 50 × 10^-8 mol/L dexamethasone respectively for 5 days. The TGF-131 expression was detected by immunohistochemistry Supervision methods and analyzed semi-quantitatively by HMIAS-2000 image system. As opposed to in vivo, rabbit CPE cells expressed TGF-131 under cul- tured circumstance in vitro. The gray scales of the positive yellow staining in the groups of 1 × 10^-8 , 5 × 10^-8 , 10 × 10^-8 and 50 × 10^-8 mol/L dexamethasone were 136.57 ± 4.43, 140.20 ± 6. 10, 142. 98± 2. 99, 146.80±1.68 and 150.05 × 1.94 respectively. When the concentrations of dexamethasone were equal to or higher than 5 × 10^-8mol/L and, the expression of TGF-β1 was inhibited. 10^-7 mol/L dexamethasone showed a significant inhibition. It was suggested that CPE cells possess the potential ability of synthesizing and expressing TGF-β1. The inhibition of TGF-β1 expression by dexamethasone may be beneficial to the treatment of proliferative vitroretinopathy, also exert some influence on the secretion of aqueous humor and ciliary inflammation.
基金This study was supported by a grant from the Scientific Foundationfor Backbone Teachers with Innovating Ability of Common College inHeilongjiang Province (No.1055G059)
文摘Choroidal neovascularization （CNV）, a major cause of vision loss, is the result of the increased vascular endothelial growth factor （VEGF） expression in human retinal pigment epithelial （RPE） cells. It is important to inhibit the expression of VEGF protein in RPE cells.
