ObjectiveTo observe the longterm effect of suramin on the inhibition of proliferation of human retinal pigment epithelial (RPE) cells in vitro. MethodsRPE cells grown in 9 pieces of 96well plate (12 wells each plate) were divided into experimental and control group, with 6 wells in each group. The concentration of 0.1 ml RPE cells in each well is 5×104 cells/ml. After the change of the medium, RPE cells were treated with suramin (250 μg/ml) in experimental group while treated with nothing in the control group. The medium of the 2 groups were changed to the normal medium after 4 days. At the 1st, 2nd, and 4thday after the addition of suramin and at the 1st, 2nd, 3rd, 5th, 6th, 7th, 9th , 11th and 13th day after removing suramin, 1 plate was randomly selected to stop culturing, and the proliferation of RPE cells were detected by methyl thiazolyl tetrazolium (MTT) assay. ResultsUnder reversed microscope, RPE cells in control group were fused completely at the 7th day after inoculation. The extracellular space of RPE cells in experimental groups was larger than that in the control group, and remained unfused at the 13th day after inoculation. The inhibitory rate of proliferation of RPE cells at the first day after treated with suramin was 14.85% and increased to the highest 25.79% at the 4th day. The first day after the suramincontaining media was removed, the inhibitory rate decreased to 12.35%, and then raised gradually to over 20% at the 3rd to 5th day. Finally, the rate drop to 14.71%. ConclusionSuramin has the long-term effect on the inhibition of RPE cells induced by serum, especially the inhibitive effect after the remove of suramin, which indicates the specific double-peak inhibition during the whole process.(Chin J Ocul Fundus Dis, 2005,21:25-27)
Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells.Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamineTM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 μmol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcripti on factor E2F,and inhibit the proliferation of HRPE cells.(Chin J Ocul Fundus Dis，2004，20：182-185)
Objective To explore the expression characteristics of chaperone interacting protein (CHIP) in normal, scar and chronic ulcer tissues and its relationship with wound healing. Methods Twenty biopsies including scar tissues(n=8), chronic ulcer tissues(n=4) and normal tissues(n=8)were used in this study. The immunohistochemical staining (power visionTMtwo-step histostaining reagent) was used to explore the amount and expression characteristics of such protein.Results The positive expression of CHIP was observed in fibroblasts, endothelial cells and epidermal cells in dermis and epidermis. It was not seen ininflammatory cells. The expression amount of CHIP in scar tissues, chronic ulcer tissues and normal tissues was 89%, 83% and 17% respectively. Conclusion Although the function of CHIP is not fully understood at present, the fact that this protein is expressed only at the mitogenic cells indicates that it may be involved in mitogenic regulation during wound healing.
To investigate the inhibitory effect of Col I A1 antisense ol igodeoxyneucleotide (ASODN) transfection mediated by cationic l iposome on Col I A1 expression in human hypertrophic scar fibroblasts. Methods Scar tissue was obtained from volunteer donor. Human hypertrophic scar fibroblasts were cultured by tissue block method. The cells at passage 4 were seeded in a 6 well cell culture plate at 32.25 × 104 cells/well, and then divided into 4 groups: group A, l iposomeand Col I A1 ASODN; group B, Col I A1 ASODN; group C, l iposome; group D, blank control. At 8 hours, 1, 2, 3 and 4 days after transfection, total RNA of the cells were extracted, the expression level of Col I A1 mRNA was detected by RT-PCR, the Col I A1 protein in ECM was extracted by pepsin-digestion method, its concentration was detected by ELISA method. Results Agarose gel electrophoresis detection of ampl ified products showed clear bands without occurrence of indistinct band, obvious primer dimmer and tailing phenomenon. Relative expression level of Col I A1 mRNA: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), and groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (Pgt; 0.05); at 1 day after transfection, groups A and B were less than groups C and D (P lt; 0.05), and there was no significant difference between group A and group B, and between group C and group D (P gt; 0.05 ); at 2 days after transfection, there were significant differences among four groups (P lt; 0.05); at 3 and 4 days after transfection, group A was less than groups B, C and D (P lt; 0.05), group B was less than groups C and D (P lt; 0.05), and no significant difference was evident between group C and group D (P gt; 0.05). Concentration of Col I protein: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (P gt; 0.05); at 1 day after transfection, significant differences were evident among four groups (P lt; 0.05); at 2, 3 and 4 days after tranfection, groups A and B were less than groups C and D (P lt; 0.05), and no significant difference was evident between group A and group B (P gt; 0.05). Conclusion Col I A1 ASODN can inhibit mRNA and protein expression level of Col I A1. Cationic l iposome, as the carrier, can enhance the inhibition by facil itating the entry of ASODN into cells and introducing ASODN into cell nucleus.
Purpose To study the possibility of prevention of proliferative vitreoretinopathy（PVR） by transduction of exogenous gene in vivo． Methods PVR model of rabbits was induced by intravitreal injection of fibroblasts．beta;-galactosidase (lacZ) gene as a reporter gene was transfered into the vitreous of PVR model eyes mediated by retroviral vector, and the expression of the gene in eye tissues was determined ． Gene transfection was done on the 6th day after fibroblasts injection,and the dosage of intravitreal injection of reporter gene was 0．1ml PLXSN/lacZ serum-free supernatant (1．1times;106 cfu/ml)． Results lacZ gene expression was seen in proliferative membranes after gene transfection, and the expression was located maily at the surface of PVR membrane．The reporter gene expression lasted at least more than 30 days．No expression was found in retinal tissues． Conclusions Retrovirus mediated gene can be directionally transducted in PVR membrane,and might possess the feasibility of gene therapy for PVR． (Chin J Ocul Fundus Dis, 2001,17:224-226)
Objective To investigate the effects of asiaticoside onthe proliferation and the Smad signal pathway of the hypertrophic scar fibroblasts.Methods The hypertrophic scar fibroblasts were cultured with tissue culture method. The expressions of Smad2 and Smad7 mRNA after asiaticoside treatment were determined by reverse transcriptionpolymerase chain reaction 48 hours later. Thecell cycle, the cell proliferation, the cell apoptosis and the expression of phosphorylated Smad2 and Smad7 with(experimental group) or without(control group) asiaticoside were detected with flow cytometry, immunocytochemistry and Western blot. Results Asiaticoside inhibited the hypertrophic scar fibroblasts from phase S to phase M. The Smad7 content and the expression of Smad7 mRNA were (1.33±1.26)% and (50.80±22.40)% in experimental group, and (9.15±3.36)% and (32.18±17.84)% in control group; there were significant differences between two groups (P＜0.05). While the content and the mRNA expression of Smad2 had no significant difference between two groups. Conclusion Asiaticoside inhibits the scar formation through Smad signal pathway.
Proliferative vitreoretinopathy (PVR) is a common complication and major cause of blindness of ocular trauma. Many cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), participate in the process of the pathogenesis of traumatic PVR. VEGF competitively inhibits binding of PDGF to its receptor (PDGFRα), enables indirect activation of PDGFRα by non-PDGF ligands, resulting in reduced p53 expression, cell proliferation and migration, which is a key point in the pathogenesis of traumatic PVR.