The Experimental Research Of Tumstatin On Growth Of Monkey Choroid-retinal Endothelial Cells

Research Background Epidemiological studies show that ocular neovascular disease has become the leading cause of blindness in developed countries as controlling of infectious eye disease. Retinal neovascularization (RNV) disease is the main cause of blindness,but the treatment effect is not perfect. New angiogenesis is the process of keeping roliferation and migration of endothelial celols. At present, studies show that drugs used to treat these diseases are mainly the ones which can inhibit expression of angiogenesis factors such as anti VEGF antibody and endogenous angiogenesis inhibiting factors such as endothelial inhibition (Endostatin, ES) and so on, Tumstatin is the strongest endogenous angiogenesis inhibiting factor ever discovered and it has obvious inhibitory effect on pathological angiogenesis.Object To construct and identify eukaryotic expression vector of tumstatin active segment Tum5gene.To investigate the effect of anti-tumor angiogenesis inhibiting gene Tum5in rhesus choroidoretinal endothelial cell, RF/6A’s proliferation and apoptosis.Methods This research includes two parts:1.Design and build Tum5recombinant gene eukaryotic expression vector PCMV-Tum5-MCS, identified by enzyme digestion and sequencing Tum5gene construct;2.Transfect PCMV-Tum5-MCS plasmid vector to RF/6A cell by using of liposome Lipofectamin transfection system2000(experiment group), at the same time set up empty plasmid vector negative control group and no transfection group; Dot blot method with HA antibody were be used to test the expression of Tum5in cells; MTT method was used to detect cell proliferation rate after transfection24h,48h and72h; Respectively using in vitro cell scratch test to investigate the relationship between Tum5and retinal vascular endothelial cell migration, and invasion in0h,24h,48h three phase; Using flow cytometry to compare three groups of cells transfection cell cycle after48h.Results1. After enzyme digestion and sequencing appraisal identified, PCMV-Tum5-MCS eukaryotic expression vector was constructed successfully;2. The transient transfection Tum5carrier was build successfully;3. The Dot blot result to detect the Tum5genes expression in RF/6A protein in cell supernatant is positive;4. The results of MTT Experiment discovered that the proliferation inhibition rates of experimental group were significantly higher in control group and the negative control group(P<0.05),including each cell proliferation72h after carrying48h, inhibiting rates are higher than24h(P<0.05).5. Cell scratch experimental results showed that the Tum5genes in RF/6A cell migration rate compared with blank control group and the negative control group is significantly decreased (P<0.05);6. Flow cytometry results showed that normal cells group G1phase was72.32%G2phase was7.22%and S phase was20.46%, while in emty empty plasmid vector negative control group, G1phase was75.92%,G2phase was3.23%and S phase was20.84%, PCMV-Tum5-MCS plasmid vector transfection group G1phase was65.21%G2phase was4.79%S was30.00%, PCMV-Tum5-MCS plasmid vector transfection group Gl phase ratio decreased significantly, S phase proportion was rising significantly, normal cells and empty plasmid vector transfection group had no significant difference.Conclusion Successfully constructed Tum5gene eukaryotic expression vector and can express Tum5genetic stability after transfection into RF/6A cells. The expressed gene can inhibit the monkey choroid retinal vascular endothelial cell proliferation; promote the function of cell apoptosis, cell cycle arrest in S phase. And this research layed a foundation for further study on Tum5function for the treatment of ocular angiogenesis diseases.