| ObjectiveComplement cascade is the body's first line of defense,but if overactive,it can peoduce tissue damaging,which is concerned in pathogenesis of choroidal neovascularization(CNV)in age-related macular degeneration(AMD).Complement 5b-9 is the end of complement cascade,which is found on retinal pigment epithelium (RPE)in the area with CNV.The aim of these experiments is to observe the effect of complement 5b-9(C5b-9)complex on membrane permeability,the dynamica of calcium ion change and molecular biology behavior in cultured human retinal pigment epithelium(RPE)cells,and to consider the role of C5b-9 in choroidal neovascularization(CNV).Methods1.Primary RPE cells were harvsted with trypsin digerstion,and were identified with immunohistochemistry.Passage 2-3 cells were used for experiments.2.C5b-9 complex was deposited on rabbit erythrocyte by incubating rat fresh sera and rabbit erythrocyte together.The extract C5b-9 complex was purified, quantified and identification.3.Human RPE ceils were exposed in different concentration of C5b-9 complex for 24 hours,then be observed through light microscope.The stimulating dose to RPE cells in the following experiments was chosen.4.Human RPE cells were exposed in C5b-9 complex for 1 hour.The change of the ultrastructure in RPE cells was observed through electron microscope.5.After deposition of C5b-9 complex on flou-3 loaded RPE cells,the dynamica of calcium ion change was analysed with cofocal laser scanning microscope.6.Human RPE cells were exposed in C5b-9 complex for 4h and 24h Respectively, then the amount of vascular endothelial growth factor(VEGF)and transforming growth factor(TGF)-β2 mRNA in each group and normal RPE cells were determined by RT-PCR.All date were dealt by SPSS13.0.Analysis of variance was used,followed by multiple comparisons.Results 1.Primary RPE cells can adherent and grow 48 hours later,after cells were isolated.5-10 days later,cells attained 80%or 100%confluence and were passaged. The passaged cells were vigorous.The result of immunohistochemistry was positive of the antigen of anti-keratin.2.The extracted C5b-9 complex were dialyzed,concentrated and quantified.The speciments were examined after SDS-PAGE electrophoresis.It was noted that the conformation of the 7 bands from SDS-PAGE were all the same as that had been described in literature.3.Through light microscope,RPE cells were destroyed exposed in 80μg/ml and 40μg/ml C5b-9 complex.The structure of RPE cells did not be changed obviously exposed in 20μg/ml or less C5b-9 complex.20μg/ml was chosen as the stimulating dose to RPE cells in the following experiments.4.Through electron microscope,cell membrane was well,and pigment granule lied in cytoplasm in normal RPE cells.In RPE cells exposed 20μg/ml C5b-9 complex,pigment granule could be seen released from cell membrane through electron microscope,however,there is no defect obviously in cell membrane.5.In most of the cells with 20μg/ml C5b-9 complex,calcium fluorescence intensity increased rapidly upon C5b-9 deposition,to the peak in 4 min.Calcium fluorescence intensity retain in the peak for about 6min,then began to decrease mostly,even to the resting level after a while.6.At 4h and 24h after cells were exposed 20μg/ml C5b-9 complex,the amount of VEGF mRNA was significantly increased(1.53±0.49 and 1.32±0.39)than in control group(1.00±0.15)(P＜0.01).There was no sigmificant difference between groups at 4h and 24h.Similarly,the amount of TGF-β2 mRNA was significantly increased in 4h group(0.72±0.13)and in 24 group(0.46±0.22)than in control group (0.24±0.076)(P＜0.01).Furthermore,the amount of TGF-β2 mRNA in 24h group had begun to reduced,which was less than in 4h group(P＜0.01).Conclusion1.We can get sufficient human RPE cells by culturing primary cells.2.C5b-9 complex can change membrane permeability of RPE cells,which depends on the concentration of C5b-9 complex. 3.C5b-9 complex can induce calcium ion concentration in cytoplasm increased, and angiogenesis factors significantly up-regulated in cultured RPE cells,which may be the pathogenesis of CNV.4.In RPE cells with C5b-9 complex stimulated,the change of VEGF and TGF-β2 mRNA was consistent with calcium ion concentration in cytoplasm. Up-regulation of VEGF and TGF-β2 mRNA may be related to incresion of calcium ion concentration in cytoplasm.On the basis of the results presented here,we propose the following potential mechanism defining the role of C5b-9 complex in CNV:Complement activation in the posterior segment of the eyes leads to increased formation and deposition of C5b-9 complex on RPE.This results in transient changes in the membrane permeability followed increasion of calcium ion concentration in cytoplasm,then growth factors are released.Released growth factors cause abnormal proliferation of choroidal endothelial cells leading to the development of CNV.Thus,complement inhibition may be used as a suitable therapeutic tool in the treatment of CNV.
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