Purpose:To evaluate the function of gap junction-mediated intercellular communication in cultured cells of retinal pigment epithelial(RPE) cells from porcine eyes. Methods:The cultured RPE cells were previously stained by a fluorescent probe 5, 6-carboxy fluorescein diacetate (CFDA) ,and then photobleach the fluorescent molecule in chosed cells. Using laser scanning confocal microscope (LSCM)to observe fluorescence recovery rate of the RPE cells which located in different condition. The function of gap junction communication was evaluated according to the fluorescence recovery rate. Results:The fluorescence recovered after photobleached and the fluorescent density of cells which touching to them descend. The recovery rate per minnte of the cells which the cell number it adjacent to was 1,2 and 3 respectively was 1. 997plusmn;0. 665, 4. 378plusmn;0. 811 and 8. 736plusmn;2. 084. Conclusion:The cultured porcine RPE cells have the function of gap junction communication,and its function proportion is associate to its adjoining cells number. （Chin J Ocul Fundus Dis,1996,12: 41-42）
Objective To clarify the relationship between inhibition of proliferation and cxpression of Ki-67 in cultured human retinal pigment epithelial(RPE) cells. Methods The cultured human RPE cells were treated with daunoblastina at a dose of 180 mu;g/L for 12h.Twenty-four hours later,DNA inhibiting rate was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.The expression of Ki-67 was evaluated by immunocytochemical staining technique and image analysis system.Flow cytometry was used to analyse cell cycle. Results DNA inhibiting rate was directly proportional to the dosage of daunoblastina.The proportion of the cells positive staining to Ki-67 in the control and the daunoblastina-treated group were 89.3% and 45.6%(Plt;0. 01),and the integral optical density values for expression of Ki-67 in the two groups were 68.1plusmn;6.2 and 27.3plusmn;5.5(Plt;0.01),respectively.The percen tage of cells in G2 phase of cell cycle increased from 8.9% to 29.5%. Conclusion G2 block was induced and poliferation was inhibited by daunoblastina in cultured human RPE cells.There is a relatively good correlation between Ki-67 immunostaining and inhibition of RPE cell proliferation. (Chin J Ocul Fundus Dis，2000，16：1-70)
PURPOSE:In search of the mechanism for photic retinal injury. METHODS:A visible light damage model was established in the primary cultured healthy,adult human RPE cells by using intense fluorescence light (2 400 Lx). RESULTS:Electron microscopy revealed swelling of the mitochondria and obscurity of nuclear membranous structure in the light damaged cells. The decrease or dissolution of organelle,vacuolization of cytoplasm and myelinic degeneration were found in some severely damaged cells. The level of intracellular SOD was decreased to 41% of that of the controls (P＜0.05). CONCLUSION:The structure of the RPE was damaged by the light radiation and the level of intracellular SOD was decreased. These suggested the light damage might be associated with the production of free radicals and the lipid perioxide reaction in membranous structure of cell. （Chin J Ocul Fundus Dis,1996,12: 174-175 ）
Objective To investigate the molecular mechanism of apoptosis in cultured human retinal pigment epithelial (RPE) cells. Methods The growth media of confluency human RPE cells were replaced with a daunoblastinacontaining one at a dose of 180mu;g/L,and the cells were incubated for 12 hr at 37℃.After incubation with the drug,the medium was withdrawn,fresh medium was added and incubation was carried out for an additional 24 hr.Apoptosis was monitored by light microscopy,enzyme linked immunosorbent assay(ELISA)and terminal deoxynucleotidyl transferase mediated biotin-dUTP nick-end labelling(TUNEL)staining.The expression of bax and bcl-2 were evaluated by immuncoytochemical staining with anti-human bcl-2 and bax antibodies. Results After the RPE cells treated with daunoblastina,shrinkage of cytoplasm and nucleus was identified.The ratio of nucleus to cytoplasm was increased.TUNEL staining showed that many cells were positive staining.The amount of apoptotic cells was directly proportional to the drug dose.The integral optical desity values for expression of bax inereased by 22.0%(Plt;0.05), and that of bcl-2 did not change significantly(Pgt;0.05). Conclusions During human RPE cell apoptosis induced by daunoblastina,overexpression of bax or low bcl-2/bax ratio were demonstrated.The results suggest that bax and bcl-2 gene expression could play a role in regulation of RPE cell apoptosis. (Chin J Ocul Fundus Dis, 1999, 15: 153-156)
ObjectiveTo construct a lentiviral vector carrying rat sirt1 gene and observe the expression of sirt1 in retinal ganglion cell (RGC) of rat. MethodsRat sirt1 cDNA was inserted into pLV5 vector. After identification by sequencing analysis and PCR, the recombinant sirt1expressinglentivirus vector was packaged by cotransfecting 293T cells with packaged plasmid.Then pLV5-sirt1 was used to infect the cultured Sprague-Dawley rat RGC cell in vitro.The expressions of sirt1 protein and mRNA in infected rat RGC were detected by quantitative real-time PCR and Western blot. ResultsThe sirt1 expression vector pLV5 was successful constructed and sequence was proved to be correct. The expression of sirt1 protein and mRNA in RGC was significantly increased than that in cells infected with control lentiviruses(P < 0.05). ConclusionWe have successful constructed a sirt1 expression lentivirus vector pLV5-sirt1 and it can increase the expression of sirt1 protein and mRNA in the rat retinal ganglion cells.
Objective To investigate the adhesiveness of osteoblasts and vascular endothel ial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro. Methods The BMSCs were isolated from 4-week-old SD rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothel ial cells. The osteoblasts and vascular endothel ial cells after being induced for 7 days in a ratio of 1 to 1 were directlyco-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and prol iferation abil ity were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothel ial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 × 106/mL、0.50 × 106/mL、1.00 × 106/mL、2.00 × 106/mL、4.00 × 106/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope. Results ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the prol iferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P lt; 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) × 106/mL. The cell adherence rate reached the peak when the seeding density was 1.00 × 106/mL. The cell adherence rate decreased when the seeding density was more than 2.00 × 106/mL. There were significant differences in cell adherence rate between modified group and unmodified group at different seeding densities (P lt; 0.05). The prol iferation of the osteoblasts and endothel ial cells presented better growth and histocompatibil ity under scanning electron microscope. Conclusion The growing behavior of two kinds of cells is good in the allogeneic freezedried partially bone coated by 20% Col I , which can be used in reconstrction of vascularized tissue engineered bone.
Objective To detect the apoptosis of vascular endothelial cells and retinal pigment epithelial (RPE) cells in vitro induced by verteporfin-photodynamic therapy. Methods Cultured vascular endothelial cells and human RPE cells were incubated with verteporfin at a concentration of 1.0 mu;g/ml which was equivalent to the initial plasma level of verteporfin in clinical therapy. Each kind of cells were divided into 6 groups according to different time of incubation: 0, 5, 15, 30, 60, and 120 minutes group. After incubated, the cells were illuminated by the laser light with the maximum wavelength of absorption of verteporfin (wavelength: 689 nm, power density: 600 mW/cm2) with the power of 2.4 J/cm 2for 83 seconds. The percentage of cellular apoptosis was measured by flow cytometry 3 hours after PDT, and the measurement was repeated thrice. Results The proportion of cellular apoptosis 3 hours after PDT were 0.01plusmn;0.01, 0.25plusmn;0.02, 0.32plusmn;0.02, 0.41plusmn;0.04, 0.49plusmn;0.03 and 0.61plusmn;0.02, respectively in 0-120 minutes group of vascular endothelial cells; and 0.02plusmn;0.01, 0.22plusmn;0.01, 0.31plusmn;0.02, 0.38plusmn;0.03, 0.47plusmn;0.05 and 0.58plusmn;0.03 respectively in 0-120 minutes group of RPE cells. The proportion of cellular apoptosis of both kinds of the cells increased as the incubation time was prolonged. There was no significant difference of the percentage of cellular apoptosis between the accordant time groups in the two kinds of cells (P＞0.05). Conclusions Cellular apoptosis can be quickly induced by verteporfin-PDT both in human vascular endothelial cells and RPE cells; under the same condition in vitro, PDT has no obvious selection for the apoptosis of the two kinds of cells. (Chin J Ocul Fundus Dis, 2006, 22: 253-255)
OBJECTIVE :To investigale effect of subretinal fluld(SRF)on proliferalion of the cellular elements of PVR. METHOD:The effect of SRF of 28 patients with rhegmatogenous retinal detachment proliferation of the cultured human retinal pigment epithelial cells(RPE),retinal glial cells (RG),and fibroblast (FB)was observed and detected by the methods of cell-counting and 3H-TdR in DNA synthesis. RESULTS:The range of proliferatinn-stimulating activity was 52.5%~233.3%, 36.4% ~ 177.8%，55.4% ~277.8% above the baseline in 1:10 dilution of these 3 kinds ,of cellular elements,and there was no significant difference among them. CONCLUSION ;The stimulating effect of SRF on the cellular proliferation was thougt to be due to the actions from certain growth factors. （Chin J Ocul Fundus Dis,1996,12: 233-235）
Objective To observe the effect of epithelial-mesenchymal transdifferentiation (EMT) of human retinal pigment epithelial (RPE) cells induced by vitreous humor in vitro. Methods The third to fifth passage cultured RPE cells were divided into two groups of treatment by 10% serum containing Dulbecco minimum essential medium (DMEM)/F12 medium (group A), or the same medium supplemented with 25% human vitreous (group B). The morphological changes were observed with a phase contrast microscrope. Cell migration, invasion and contractility were tested using a scratch wound assay, Transwell invasion assay and collagen gel contraction analysis. The expression levels of alpha;-smooth muscle actin (SMA) and Snail1 were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The RPE cells in group A were flat and gathered together. The RPE cells in group B grew as a fan-shaped projection at one edge and cone-shaped tail at the opposite edge, or spindle-shaped, and appeared to separate. In group A, filamentous actin distributed mainly at the margin of the cells with the distribution an oval shape. In group B, filamentous actin reorganized and formed fan-like flat pseudopodia at one edge of the cells. Compared to group A, the migration and invasion of the cells increased significantly (t=14.190, 22.630; P＜0.05), but contractility decreased remarkably (t=6.221, P＜0.05) in group B. Compared to group A, the expression level of Snail1 mRNA increased significantly (t=3.218, P=0.032), but the expression level of alpha;-SMA mRNA decreased (t=3.990, P=0.016). Conclusions Vitreous humor can induce the EMT of RPE cells. Increasing cell migration, cell invasion, and expression of Snail1 mRNA as well as up-regulated cellsprime; contractility and expression of alpha;-SMA mRNA may be the mechanism.
Objective To observe the effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell (RGC). Methods Four short hairpin (sh) RNA interference sequences targeting rat Sirt1 gene were designed. The target sequences of Oligo DNA were synthesized and annealed to double strand DNA, which was subsequently connected with pGLV3 lentivirus vector to build the lentiviral vector. The positive clones were identified by polymerase chain reaction (PCR) and DNA sequencing. The lentiviral vector construct and lentiviral packaging plasmids were co-transfected into 293T cells, then the titer of lentivirus were determined. The RGC were divided into 6 groups including blank group, negative control group and si-Sirt1-1, si-Sirt1-2, si-Sirt1-3, si-Sirt1-4 groups. Real-time PCR and Western blotting were used to detect the expression of Sirt1 mRNA and protein in the RGC cells. Results PCR and DNA sequencing analysis confirmed that the shRNA sequence was successfully inserted into the lentivirus vector. The concentrated titer of virus suspension was 8×108 TU/ml after the recombinant lentiviral vector successfully transfected and harvested in 293T cells. Comparing with NC group, the expression of Sirt1 mRNA and protein were significantly decreased in the si-Sirt1-1, si-Sirt1-2, si-Sirt1-3 and si-Sirt1-4 groups (F=27.682, 1 185.206; P=0.000, 0.000). The si-Sirt1-2 group had the strongest effect in reducing the expression of Sirt1 mRNA and protein. Conclusion The 4 lentiviral vectors harboring RNAi targeting rat Sirt1 gene can effectively down regulate the expression of Sirt1 mRNA and protein in RGC cells.