Objective To investigate the expression of eotaxin-1, eotaxin-2 and eotaxin-3 in ARPE-19 human RPE cells after exposure to light. Methods Cultured human RPE cells (5th~10th generations) were divided into lightinduced group and control group. Cells light-induced group were exposed to the blue light at the intensity of (600plusmn;100) Lux for 12 h to establish the light damaged model. Eotaxin-1, eotaxin-2 and eotaxin-3 mRNA and protein were determined by real time polymerase chain reaction and Western blot at 0, 3, 6, 12, 24 hours after light-induced. Results In light-induced groups, mRNA levels of eotaxin-1 and eotaxin-2 were increased at 0 h (t1=6.05.t2=12.561) and 3 h （t1=2.95.t2=3.67） significantly(P＜0.05), but the mRNA level of eotaxin-3 had not changed （t3=1.57 and 1.00 respectively，P＞0.05） at that time. At 6 h （t1=4.73，t2=18.64，t3=28.48）, 12 h （t1=3.11，t2=20.62，t3=18.50）, 24 h （t1=8.25，t2=38.27，t3=18.60）, mRNA levels of eotaxin-1, 2, 3 were increased significantly （P＜0.05）. Except for the eotaxin-3 protein had not changed at 3 h （t3=1.28，P＞0.05）, protein expression of eotaxin-1, 2, 3 were increased significantly （P＜0.05） at 0 h （t1=4.85，t2=5.45，t3=6..21）, 3 h （t1=5.64，t2=4.55）, 6 h （t1=31.60，t2=6.63，t3=7.15）, 12 h （t1=14.09，t2=18.22，t3=15.76）, 24 h （t1=6.96，t2=10.47，t3=12.85）. Conclusion Eotaxin-1, eotaxin-2 and eotaxin-3 expression were increased after Light-damage, corresponding to the time after light exposure. Eotaxin-3 was the most prominent isoform.
Objective To investigate the protective effects of riluzole, a sustained activator of K2P subfamily member TRAAK potassium channel, in human retinal pigment epithelium (hRPE) cells with oxidative induce by tert-butyl hydroperoxide (t-BHP) in vitro, and to evaluate the possible involvement of K2P in the cytoprotective function of retina degeneration diseases. Methods The third to fifth passage of the primary cultured hRPE cells were used in the following experiments．hRPE cells were divided into seven groups: normal control group.t-BHP (300 mu;mol/L) group.t-BHP with riluzole (2, 5, 10, 20 mu;mol/L) group and riluzole (10 mu;mol/L) group. The apoptosis was measured by the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, annexinV/PI double staining flow cytometry. Changes of cells and nuclei morphology were observed under a phase contrast microscope and a fluorescence microscope after 4prime;, 6-diamidino-2-phenylindole (DAPI) staining. Immunofluorescence 1abelling was carried out to analysis the expression of TRAAK. Results After 24 hours incubation with 300 mu;mol/L t-BHP, the cells viability decreased to (58.7plusmn;12.2)% as compared to the normal control groups. The cell viability of t-BHP with riluzole group at different concentrations was higher than the t-BHP group, while 10 mu;mol/L riluzole showed maximally protective effect on hRPE death induced by t-BHP（t=4.84.P＜0.05）. Riluzole remarkably decreased pyknotic nucleus and cell swelling when compared with t-BHP group. Morphology of cells was fusiform with the uniform elliptic nuclei in normal and riluzole group. The Results of annexinV/PI double staining flow cytometry showed that ratio of normal cells were （97.6plusmn;1.3）%, （70.3plusmn;7.0）%, （86.9plusmn;5.2）%, （93.9plusmn;1.5）% in normal group.t-BHP group.t-BHP with riluzole group and riluzole group respectively. The ratio significant decreased in t-BHP group when it was compared with the other groups (t=7.53, 4.59, 6.49, respectively.P＜0.05). By contrast with normal group and riluzole group, the ratio of normal cells in t-BHP with riluzole group had no statistical significance（t=2.94, 1.91, respectively.P＞0.05）. Riluzole (10 mu;mol/L) also significantly decreased the ratio of early stage apoptotic cells from (25.50plusmn;8.02)% to (1.20plusmn;0.72)% in t-BHP injured groups （t=7.13，P＜0.05）. The ratio of early stage apoptotic cells significant decreased in t-BHP group when it was compared with the normal group and riluzole group （t=7.07, 5.94, respectively.P＜0.05）. By comparison with normal group and riluzole group, there are no statistical significance in t-BHP with riluzole group（t=0.06, 1.18, respectively.P＞0.05）. The mean gray values of TRAAK expression were 0.040plusmn;0.003, 0.041plusmn;0.001, 0.049plusmn;0.001, 0.055plusmn;0.001 in normal group.t-BHP group.t-BHP with riluzole group and riluzole group respectively. TRAAK density was significantly higher in t-BHP with riluzole group and riluzole group（t=7.40, 12.70, respectively.P＜0.05）. Conclusions Riluzole can protect hRPE cells against oxidative injury-induced cell death at early apoptosis stage. The mechanism may relate to that riluzole can promote the expression of K2P TRAAK potassium channel.
Objective To investigate the effect of blue light on mRNA expression of L-type calcium channel subtypes of human retinal pigment epithelial (RPE) cells in vitro. Methods The fourth-generation of human RPE cells were randomly divided into four groups including control group (no light group), light group, light + nifedipine group, and light + (-) BayK8644 group. The cells were exposed to blue light (2000plusmn;500) lux for 6 hours, and then cultured for another 24 hours. Reverse transcription polymerase chain reaction real time (RT-PCR) and fluorescence quantitative PCR technologies were used to analyze mRNA expression of L-type calcium channel subunit of cardiac subtype ( 1C or CaV1.2), neuroendocrine subtype ( 1D or CaV1.3) and retinal subtypes ( 1F or CaV1.4) in each group. Results The length of PCR product of 1C, 1D, 1F subunit and actin was 68, 157, 125 and 186 base pairs respectively. (1) 1C mRNA expression in light, light + nifedipine and light + (-) BayK8644 group was higher than that in control group, the difference was statistically significant (P＜0.05). 1C mRNA expression in light +nifedipine group and light + (-) BayK8644 group was higher than in light group (P＜0.05). 1C mRNA expression in light + (-) BayK8644 group was higher than that in light + nifedipine group (P＜0.05). (2) Comparing with control group, 1D mRNA expression was higher in light, light +nifedipine and light + (-) BayK8644 group, the difference was statistically significant (P＜0.05). Light + (-) BayK8644 group was higher than light group and light + nifedipine group (P＜0.05), light group and the light + nifedipine group was not statistically significant (P＞0.05). (3) 1F mRNA expression in light, light + nifedipine and light + (-) BayK8644 group was higher than those in control group, there was statistically significant (P＜0.05), light +nifedipine group and light + (-) BayK8644 group was higher than light group (P＜0.05), light + nifedipine group and the light + (-) BayK8644 group was not statistically significant (P＞0.05). Conclusions The human RPE cells mRNA expression of L-type calcium channel 1C, 1D and 1F subunit was increased after exposing to blue light. Application of the 1times;10-5 mmol/L (-) BayK8644 can increase mRNA expression of 1C, 1D and 1F subunit.
Objective To investigate the impact of photodynamic therapy (PDT) with verteporfin on the expression of pigment epithelial derivative factor (PEDF) mRNA and vascular endothelial growth factor (VEGF) mRNA in adult retinal pigment epithelial (RPE) cells in vitro. Methods The changes of cellular viability before and after PDT were assessed by methyl thiazolyl tetrazolum (MTT) colorimetric assay. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to detect the expression of PEDF and VEGF mRNA in RPE cells before and after PDT. Results PDT caused the death of RPE cells. The cellular mortality was positively correlated with the power of photocoagulation and the concentration of verteporfin. Conclusion PDT could downregulate the expression of PEDF and VEGF mRNA in adult RPE cells in vitro, which may relate to the cure or relapse of subfoveal choroidal neovascular membrane after PDT. (Chin J Ocul Fundus Dis, 2006, 22: 256-260)
In thiis study,we show thai carbachol stimulates the accumulation of inositol phosphates(InsPs)in human rellnal pigment epithelium (RPE)cells and atropine blocks the carbachol-induced effect ,suggesting the existence of musearinie acelyleholine receptors in human RPE cells. In contrast,noradrenaline,serotonin, cpidermal growth factor (EGF),isoproterenol,and NECA (5'-[N-ethyl]-carboxamido-adenosine)do not influence the basal levels of InsPs.Moreover,isoprmerenol and NECA do not affect the carhaehol elevated levels of InsPs.EGF,howcvcr,does potentiate the carhaehol stimulated elevation of InsPs in a dose-dependent manner ,suggesting an interaction between EGF and musearinie receptors in cultured human RPE cells. （Chin J Ocul Fundus Dis,1994,10:220-222）
Severely coagulated retinae by argon laser of 20 Chinese hamsters were investigated with transmission electron-microscopy. The results revealed destruction of retinal pigment epithelium-Bruch's membrane-choroid capillary complex at the coagulated foci, and leakage of fluid and blood cells through the choroidal vessels into the subretinal space. Several days after laser burn the subretinal fluid was found to subside and the RPE cells surrounding the burned lesions started to proliferate. The smaller lesions were covered by the proliferating RPE 10 days after coagulation, but poor regeneration of RPE in large necrotic areas. Neovascularization was usually associated with obvious defect of Bruch's membrane and restoration of RPE barrier was most likely impossible. （Chin J Ocul Fundus Dis,1992,8:14-16）
ObjectiveTo investigate the effect of blue light on Ca2+-protein kinase C (PKC) signaling pathway in human retinal pigment epithelial (RPE) cells in vitro. MethodsPrimary human RPE cells were cultured in vitro and characterized. The experiments were carried out using the 4th generation of human RPE cells. The PKC protein level was measured by Western blot to determine the most appropriate concentration of phorbol ester (PMA) and calcium phosphate binding protein (calphostin C) on PKC expression. Non-radioactive isotope method was used to determine the effect of blue light on PKC expression of cultured cells. Blue-light damage model of human RPE cells was established by 6 hour irradiation of medical blue-light lamp [20 W, 450-500 nm wavelength, (2000±500) Lux], and 24 hours prolongation of post-exposure culture. The human RPE cells were randomly divided into 5 groups. Group A did not receive light irradiation, group B only received blue light irradiation, group C was blue light irradiation and 0.1 mmol/L nifedipine treatment, group D was blue light irradiation and 100.0 nmol/L calphostin C treatment, group E was blue light irradiation and 100.0 nmol/L PMA treatment. Intracellular Ca2+ concentration was measured by acetoxymethyl ester (Fluo 3-AM) labelling and confocal microscope imaging. ResultsThe PKC protein expression in 100.0 nmol/L or 200.0 nmol/L PMA-treated groups was higher than 0.1, 1.0, 10.0, and 50.0 nmol/L PMA-treated groups, the difference was statistically significant (F=217.537, P<0.05), but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L PMA-treated groups (P=0.072). The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L calphostin C-treated groups was lower than 5.0, 25.0, 50.0, and 75.0 nmol/L calphostin C-treated groups, the difference was statistically significant (F=164.543, P<0.05), but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L calphostin C-treated groups (P=0.385). PKC level in blue light group was higher than non-light group, the difference was statistically significant (t=-9.869, P<0.05). The Ca2+ fluorescence intensity values in group B, C, D and E was higher than group A, the difference was statistically significant (F=26 764.92,P<0.05). The Ca2+ fluorescence intensity values in group E was higher than group B, C and D (P<0.05), and that in group B was higher than group C and D (P<0.05). ConclusionsThe PKC activity and intracellular Ca2+ concentration in human RPE cells increase after blue-light irradiation. Both calcium channel inhibitor nifedipine and PKC inhibitor calphostin C can reduce intracellular Ca2+ concentration in human RPE cells. PMA can induce intracellular Ca2+ concentration in human RPE cells after blue light irradiation.
ObjectiveTo investigate the effects of leptin on the oxidative damage in human retinal pigment epithelial (RPE) cells. MethodsHuman RPE cells (ARPE-19) were cultured in vitro, and randomly divided into control group and insulin resistance group. RPE cells were treated with 0, 10, 100 ng/mL leptin for 24, 48, 72 hours respectively. Then the levels of reactive oxygen species (ROS) expression in RPE cells were detected by 2', 7'-dichlorofluorescin-diacetate (DCFH-DA), and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression in RPE cells were observed by immunocytochemistry (ICC), and the levels of human 8-oxoguanine DNA glycosylase l (hOGG1) expression in lysate were measured by Western blot. ResultsAfter 24, 48, 72 hours, the level of ROS (Control group:F=37.136, 37.178, 49.634; P < 0.05. Insulin resistance group:F=9.822, 28.881, 71.150;P < 0.05), 8-OHdG (Control group:F=88.643, 390.920, 1039.276;P < 0.05.Insulin resistance group:F=273.311, 299.155, 82.237;P < 0.05) and hOGGl (Control group:F=470.062, 1073.113, 295.456;P < 0.05. Insulin resistance group:F=240.032, 592.389, 527.760;P < 0.05) expression increased significantly with the increase of leptin concentration in control group and insulin resistance group. Under the same leptin concentration, the level of 8-OHdG has a trend that it was higher in the insulin resistance group than the control group. After 24 hours, the difference of hOGGl expression between control group and insulin resistance group was not significant (F=23.392, P > 0.05). After 72 hours, the level of hOGGl expression was significantly higher in the insulin resistance group than the control group (F=129.394, P < 0.05). The level of hOGGl expression was significantly higher at 48 hours than that at 24 hours and 72 hours (P < 0.05). ConclusionLeptin could induce the oxidative damage of RPE cells in normal and insulin resistance status. With the increase of leptin concentration and time extended, the degree of oxidative damage and its repair were both increased. The degree of oxidative repair increased with the increase of leptin concentration, but decreased with time extended.
ObjectiveTo observe the effect of TGF-β receptor inhibitor Compound C on the directed differentiation of human embryonic stem cells (hESC) into retinal pigment epithelial (RPE) cells. MethodsH1 hESC were divided into control group and experimental group. When the hESC reached over confluence, the medium was changed to knockout serum replacement medium without bFGF to induce RPE differentiation. The experimental group was supplemented with 1 μmol/L TGF-β receptor inhibitor Compound C at the first six days of induction. Real-time PCR was carried out to examine the expression of paired-box gene 6 (PAX6), microphthalmia-associated transcription factor (MITF), cellular retinaldehyde blinding protein (CRALBP), and RPE65 in both groups at the 1, 3, 5 weeks of the induction process. hESC-derived RPE (hESC-RPE) cells were isolated mechanically and purified. Real-time PCR, Western blot and immunofluorescence were used to characterize the purified hESC-RPE cells. ResultsPigmented colonies were observed in experimental group at the 4 weeks of the induction process, while no pigmented colony could be detected in the control group. All the purified pigmented cells from experimental group showed polygons morphology. Experimental group showed significantly higher expression of RPE marker genes PAX6, MITF, CRALBP and RPE65 than the control group(P<0.05). Compared with the hESC and ARPE-19 cells line, purified hESC-RPE cells showed much higher expression of PAX6, MITF, CRALBP and RPE65(P<0.05).High expression level of PAX6 and RPE65 proteins were observed in hESC-RPE cells. Immunofluorescence verified the expression of PAX6 and ZO-1 in hESC-RPE cells. ConclusionTGF-β receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE.
ObjectiveTo observe the macular choroidal and retinal pigment epithelium (RPE) thickness in tilted disc syndrome (TDS). MethodsThis is a descriptive study. Thirty eyes of 22 TDS patients (TDS group) and 30 eyes of 15 normal subjects (control group) were analyzed. Among TDS group, there were 8 males (11 eyes) and 14 females (19 eyes), the average age was (9.00±2.78) years old. The best corrected visual acuity (BCVA) was 0.3-1.0, and the average spherical equivalent degree was (-3.44±2.22) DS. Among the control group, there were 8 males (16 eyes) and 7 females (14 eyes), the average age was (9.33±1.11) years old. The best corrected visual acuity (BCVA)≥1.0, and the average spherical equivalent degree was (-3.18±1.13)DS. The difference of the spherical equivalent degree between two groups was not statistically significant (t=-1.648, P=0.110). Enhanced depth imaging techniques of frequency-domain optical coherence tomography was used to measure the thickness of choroid and RPE at totally 17 sites. There sites included subfoveal, 4 sites each (500, 1000, 1500 and 2000 μm from the fovea) at the horizontal (nasal/temple) and vertical (superior/inferior) directions. ResultsThe subfoveal choroidal thickness was (235.53±51.77) μm and (273.45±60.3) μm in TDS patients and control respectively, the difference was significant(t=-2.612,P=0.011). The difference of the choroidal thickness of the other 8 horizontal sites (F=24.180) and 8 vertical sites (F=23.390) in TDS group was statistically significant (P=0.000). The TDS choroidal thickness of all horizontal sites except nasal 1000 μm site was thinner than corresponding sites of the control group (P<0.05). The TDS choroidal thickness of the subfoveal site and 4 inferior vertical sites was thinner than corresponding sites of the control group (P<0.05). The subfoveal RPE thickness was (32.56±5.00) μm and (36.58±3.60) μm in TDS patients and control respectively, the difference was significant(t=-3.567,P=0.001). The subfoveal RPE thickness was the thickest among other 16 sites in both groups, and the TDS RPE thickness of all sites was thinner than control group, the difference was statistically significant (P<0.05). ConclusionThe choroidal and RPE thickness of TDS patient was thinner than normal subjects.