Inhibition Of Corneal Neovascularization By Liposomes Mediated Plasmid Encoding Human Vascular Endothelial Growth Inhibitor Gene

Object:To evaluate the effect of Effectene^TM lipofectine mediated plasmids encoding human pcDNA₄-Vascular Endothelia Growth Inhibitor (pcDNA₄-VEGI) gene on Corneal Neovascularization (CNV).Methods:1. Human VEGI gene fragment was connected with expressional vector plasmid pcDNA₄ to construct the pcDNA4-VEGI 151 gene expression vector. Computer automatic sequence analysis was used to identify the gene.2. pcDNA₄-VEGI151 gene was transfected into CRL-2480 cells cultured in vitro by Effectene^TM lipofectine transfection technique. Plasmid pcDNA₄, Effectene^TM , and 1640 solution as the control. 24h, 48h and 72h after transfection, cell status was observed, expressed protein was tested by Western-blotting, cellular inhibition was determined by MTT method.3. Forty New Zealand albino rabbits were sutured by 5-0 silk on the superior cornea to establish the animal model and divided into 4 random teams, ten per team: A transfected by pcDNA4-VEGI151 gene mediated by Effectene^TM lipofectine transfection, B by Plasmid pcDNA₄, C by Effectene^TM, and D by Normal Saline. Length and area of CNV were measured by slit lamp every day after transfection, Immunohistochemistry was used to detected the expression of VEGI151 protein in cornea at the time 3d, 7d, 14d and 21d.Results:1. Computer automatic sequence analysis and Western-blotting analysis confirmed the correct recombination of pcDNA4-VEGI151 gene.2. Cells experiment:2.1 Status of cells: Cells transfected by Effectene1M lipofectine mediated pcDNA4-VEGI151 gene stopped growing and many of them apoptosised while cells of control teams continued proliferating.2.2 MTT value showed the obvious difference in the pcDNA4-VEGI151 team and control teams on inhibition of cell proliferation and the difference of B, C, and D was meaningless .3. Animal experiment:3.1 Average occurrence of CNV in the pcDNA4-VEGI151 gene transfected team (team A) was 6.3 days, in plasmid pcDNA4 control team (team B) was 3.1 days, in Effectene? lipofectine control team (team C) was 3.2 days, in Normal Saline control team (team D) was 3.2 days. Difference between A and B. C, D was meaning (P<0.01), while difference in B, C and D was meaningless (P>0.05).3.2 Lenth and average area of CNV in each period in team A was mengingful different from that in team B, C, D (P<0.01) , while difference in team B, C and D was meaningless (P>0.05).3.3 Cornea sections observed morphologically, team A showed an integrated epithelium, light stroma edema and less CNV and inflammation while more CNV, inflammation and stroma edema were observed in the control teams (team B, C and D).3.4 Immunohistochemistry result: VEGI positive cells could be seen in epithelium, stroma, endothelium and the cliff of CNV in team A at the time 3 days after transfection. VEGI cells changed with the decrease of CNV. None positive cells in the control teams (team B, C and D) all the time.Conclusion:RT-PCR, enzyme cutting, recombination and other genetic techniques could beused to construct expressional pcDNA4-VEGI 151 gene;Effectene? lipofectine transfection technique could be effectively used in transfecting pcDNA4-VEGI151 gene into CRL-2480 cells in vitro to inhibit cell proliferation and into rabbit cornea to inhibit CNV.