Inhibition Of Cornea Neovascularization By Adenovirus Mediated Tum5 Recombination Gene

Purpose The recombinant Tum5 gene adenovirus mediated overexpression on the physiological state of human umbilical vein endothelial cells the influence of proliferation,migration and tube formation;inhibitory effect on corneal neovascularization induced by alkali burn model,and to explore the possible mechanism.Methods Construction and expression of green fluorescent protein(r Ad-GFP)recombinant adenovirus vector carrying recombinant adenovirus with Tum5 gene(r Ad-Tum5),to the transfection of HUVEC cells under the inverted fluorescence microscope to observe and calculate the transfection efficiency with the expression of Western blot detection Tum5.HUVEC was divided into normal,control group,r Ad-GFP group and r Ad-Tum5 group;r Ad-GFP and r Ad-Tum5 virus particles(1×1010/ml)were transfected into corresponding groups of cells after 48 h using CCK-8,Transwell experiment and Matrigel were detected in cell proliferation,migration and lumen formation.In addition,VEGF ELISA kit was used to detect the content of VEGF in the supernatant of 24,48 and 72 h after transfection.Corneal neovascularization was studied.The rats were divided into four groups: normal group;normal saline group,r Ad-Tum5 group,r Ad-GFP group;In first days after alkali burn,20 μl r Ad-Tum5,r Ad-GFP virus in the conjunctival injection,sterile saline were injected,normal control group without treatment.In the seventh day after alkali burn,fourteenth days from rat eyeball,paraffin section,observe the growth of corneal neovascularization by HE staining,and the number of inflammatory cells were statistically analyzed: in first days,seventh days after burn,fourteenth days in the slit lamp microscope observation of rat cornea the edema and neovascularization,and neovascularization area measurement,statistical analysis.To study the inhibitory effect of Tum5 on corneal neovascularization.Result Fluorescence microscopy showed that r Ad-GFP group and r Ad-Tum5 group showed clear green fluorescence,suggesting that the expression of adenovirus transfection cells were successful.the transfection efficiency of r Ad-GFP group and r Ad-Tum5 group were 55.13% and 50.31%.Western blot showed that Tum5 was successfully expressed in vitro.CCK-8,Transwell and Matrigel results showed that the differences were statistically significant(F = 78.914,8.524,226.498,P are namely the above 0.05).The number of cell proliferation,migration and tube formation number comparison between the groups showed no significant between normal control group and r Ad-GFP group(P 0.05),and r Ad-Tum5 group compared to the others significantly decreased,the difference was statistically significant(P < 0.05).The results of ELISA showed that the adenovirus 24 h after transfection,there was no significant difference in the content of VEGF in the supernatant of transfected group;in 48 h and 72 h,Tum5 content in VEGF group than in the others were significantly reduced(P< 0.05)and the two others compared the content of VEGF,there were no significant differences(P> 0.05).The successful construction of rat corneal alkali burn model,HE results showed: in seventh days after burn r Ad-Tum5 grou compared with the others,The inflammatory cells were incresed,as is angiogenesis.The CNV area comparison results show: comparison of corneal neovascularization area,with statistically and time difference group(F group = 2032,P < 0.01;F =1023,P < 0.01),the first day of each see the area of neovascularization was not statistically significant(P > 0.05),seventh days r Ad-Tum5 group compared with the saline group and r Ad-GFP group,the vascular area was significantly reduced,with statistical significance(P < 0.05),fourteenth days r Ad-Tum5 group compared with the saline group and r Ad-GFP group,the vascular area was significantly reduced,was statistically significant(P < 0.05).Conclusion Adenovirus mediated Tum5 can efficiently transfect cells in vitro and can be successfully expressed.It can inhibit the proliferation,migration and lumen formation of HUVEC cells.In addition,Tum5 can inhibit corneal neovascularization induced by alkali burn,which may be related to the down-regulation of VEGF.