| Part 1 Expression of cell factors by human retinal pigment epithelial(RPE) cell(APRE-19) under hypoxic conditionPurpose:To Investigate the production and relaease of transforming growth factor-β2(TGF-β2),vascular endothelial growth factor(VEGF) pigment epithelium-derived factor(PEDF)and thrombospondin-1(TSP-1) by human retinal pigment epithe(?)(RPE) cell(APRE-19) under hypoxic condition.Methods:APRE-19 cells were cultured in DMEM/F12 medium with 10%fetal calf serum.Using 150μmol/ml CoCl2 to simulate the hypoxic condition.After 0,4,8,12,24 and 48 hours of hypoxia,TSP-1, TGF-β2,VEGF and PEDF peptides were detected by immunofluorescent staining and Western Blot.Results:1.Immunostaining for VEGF was observed in the cytoplasm of ARPE-19 cells,and it showed a time-dependent increase by hypoxia.The most remarkable expression was at 24 hour of hypoxia. Immunostaining of PEDF was observed in the cytoplasm of the ARPE-19 cells,and it showed a time-dependent derease by hypoxia. Immunostaining of TGF-β2 and TSP-1 was observed in the cytoplasm, entoblast,epicyte,and karyotheca of the ARPE-19 cells,and they showed a time-dependent derease by hypoxia.2 Western Blot identified a time-dependent increase of VEGF peptides in the media of hypoxic ARPE-19 cells compared to normoxic cells.At 24 hour of hypoxia,the expression of VEGF reached the peak,it was 2.6 times more than that of normoxic condition,and then decresed. There were signifieanfly statistical differences between hypoxic group and normoxic group at each time(P=0.000-0.002) excluding at 0 hour (P=0.935).Western Blot identified a time-dependent decrease of PEDF, TSP-1 and TGF-β2 peptides in the media of hypoxic ARPE-19 cells compared to normoxic cells.At 48 hour,the expression of PEDF,TSP-1 and TGF-β2 peptides in hypoxic groups were normoxic groups' 0.30 times,0.35 times and 0.28 times respectively.There were signifieantly statistical differences between hypoxic group and normoxic group at each time(P=0.000-0.014),(P=0.000-0.003),(P=0.000-0.001) excluding at 0 hour(P=0.125),(P=0.803),(P=0.086)3.Positive correlations(r=0.902,P=0.000),(r=0.990,P=0.000) were observed between the expression of PEDF,TGF-β2 and TSP-1.A negative correlation(r=-0.853,P=0.000) was observed between VEGF and TSP-1.Conelutions:1.Hypoxia boot up neovascularization by the increase of angiogenic growth factor VEGF and the decrease of anti-angiogenic facors PEDF,TGF-β2 and TSP-1.2.TSP-1 was different from positive role of VEGF,it might contribute to the happening and development of the disease by the negative role similar with PEDF and TGF-β2. Part 2 Role of synthetical peptide VR-10 on rhesus choroidal -retinal endothelial(RF/6A) cell.Purpose:To investigate the effects of synthetical peptide VR-10 on proliferation and migration of rhesus choroidal -retinal endothelial (RF/6A) cell and the expressions of TGF-β2,VEGF and PEDF in RF/6A cell.Methods:Proliferation of RF/6A cell after exposure to 1μg/ml TSP-1 and synthetical peptide VR-10(0.1μg/ml,1μg/ml and 10μg/ml) after 6h,12h,24h and 48h were detected by the tetrazolium dye-reduction assay(MTT).Transwell chamber was used to investigate the migration of RF/6A cell after exposure to 1μg/ml TSP-1 and synthetical peptide VR-10(0.1μg/ml,1μg/ml and 10μg/ml) after 24h.The expression of mRNA of TGF-β2,VEGF and PEDF in RF/6A cell after exposure to 1μg/ml TSP-1 and synthetical peptide VR-10(0.1μg/ml,1μg/ml and 10μg/ml) were detected by immunofluorescent staining and reverse transcription -polymerase chain reaction(RT-PCR) analysis.Results:1.TSP-1(1μg/ml) and synthetical peptide VR-10 (0.1μg/ml,1μg/ml and 10μg/ml) inhibited proliefration of RF/6A cells in a time and dose-dependent way.Survival ratio of RF/6A was decrease with the increase of time and concentration.The lowest survival ratio of RF/6A was 78%(P＜0.001) by the treatment of 10μg/ml synthetical peptide VR- 10 after 48h.2.TSP-1 and synthetical peptide VR-10 could inhibite migration of RF/6A cells in transwell chamber(P＜0.001 ).10μg/ml synthetical peptide VR-10 had the strongest effect,1μg/ml TSP-1 was the next.Migration inhibition rate was increase with the increase of the concentration of synthetical peptide VR-10(P＜0.001).There was no signifieantly statistical differences between 0.1μug/ml synthetical peptide VR-10 and 1μg/ml synthetical peptide VR-10(P=0.114 ).3.Immunostaining for TGF-132 was observed in the cytoplasm of RF/6A cells,the expression was increase after exposure to 1μg/ml TSP-1 compared with control group.There were no impact on the expression of TGF-β2 after exposure to synthetical peptide VR-10.Immunostaining of PEDF was observed in the cytoplasm of the RF/6A cells,and the expressions were increased aider exposure to 1μg/ml TSP-1 and synthetical peptide VR-10 compared with control group.10μg/ml synthetical peptide VR-10 had the strongest effect.Immunostaining of VEGF was observed in the cytoplasm of the RF/6A cells,and it decrease after exposure to 1μg/ml TSP-1 and synthetical peptide VR-10 compared with control group.10μg/ml synthetical peptide VR-10 had the strongest effect.4 Expression of TGF-β2 mRNA in RF/6A cell was increased after treatment of 1μg/ml TSP-1(P=0.000).There were no signifieantly statistical differences between synthetical peptide VR-10 and control group(P＞0.05).Expression of PEDF mRNA in RF/6A cell was increased after treatment of 1μg/ml TSP-1 and synthetical peptide VR-10, and 10μg/ml synthetical peptide VR-10 had the strongest effect(P＜0.001). There were significantly statistical differences between groups(P＜0.001). Expression of TGF-β2 mRNA in RF/6A cell was increased after treatment of 1μg/ml TSP-1(P=0.000).There were no significantly statistical differences between synthetical peptide VR-10 and control group(P＞0.05).Expression of PEDF mRNA in RF/6A cell was decreased after treatment of 1μg/ml TSP-1 and synthetical peptide VR-10, and 10μg/ml synthetical peptide VR-10 had the strongest effect(P＜0.001). There were significantly statistical differences between groups(P＜0.001), excluding 1μg/ml synthetical peptide VR-10 and 1μg/ml synthetical peptide VR-10(P=0.615).Conclusions:1.Synthetical peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell.2.Synthetical peptide VR-10 had anti-angiogenic ability by up-regulation of anti-angiogenic factor PEDF,down-regulation of angiogenic factor VEGF and TGF-β2 independent mechanism. Part 3 Role of Synthetical Peptide VR-10 on the Expression of Fas/FasL,Caspase-3 and Bcl-2 by RF/6A CellPurpose:To investigat the influence of synthetical peptide VR-10 on the expressions of apoptosis relative genes in RF/6A cell.Methods:The expression of Bcl-2 and FasL mRNA in RF/6A cell after exposure to 10μg/ml synthetical peptide VR-10 were detected by RT-PCR analysis.The expression of Fas and Caspase-3 peptides in RF/6A cell after exposure to 10μg/ml synthetical peptide VR-10 were detected by Western Blot.Results:1.Compared with control group,expression of FasL mRNA were significantly increased in 10μg/ml synthetical peptide VR-10 treated group,but the expression of Bcl-2 mRNA was decreased.2.Western bolt showed that RF/6A cell in control group mainly expressed the 32-kD procaspase-3 forms.To 10μg/ml synthetical peptide VR-10 treated group,it showed decreased expression of procaspase-3(32 KD) and concomitant increased expression of its shorter proapoptotic forms(20 KD).Compared with control group,expression of Fas peptides were significantly increase in 10μg/ml synthetical peptide VR-10 treated group.Conclusions:Synthetical peptide VR-10 mediated endothelial cell apoptosis and inhibited angiogenesis in association with increased expression of Fas/FasL,decreased expression of Bcl-2,and processing of caspase-3 into smaller proapoptotic forms.
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