Objective: To evaluate the effect of Effectene lipofectene mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA4-VEGI) gene on corneal neovascularization (CNV).
Methods: It was an experimental study. Forty healthy New Zealand albino rabbits (40 eyes) were divided into 4 teams according to the random digits table: team A (10 rabbits) was the team which transfected by lipofectine mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA4-VEGI) gene; team B (10 rabbits) was the team transfected by empty vector; team C (10 rabbits) was the team transfected by lipofectine; team D (10 rabbits) was the empty control group which was transfected by saline. (1) Human VEGI gene fragment was connected with expressional vector plasmid pcDNA4 to construct the pcDNA4-VEGI gene expression vector. Computer automatic sequence analysis was used to identify the gene. (2) New Zealand albino rabbits were sutured by 5 - 0 silk in the superior cornea to establish the animal model and were transfected by pcDNA4-VEGI gene mediated by Effectene lipofectene transfection. Length and area of CNV were measured by slit lamp every day after transfection, immunohistochemistry was used to detected the expression of VEGI protein in cornea at the time 3, 7, 14 and 21 d.
Results: (1) Computer automatic sequence analysis confirmed the correct recombination of pcDNA4-VEGI gene. (2) Average occurrence of CNV in the pcDNA4-VEGI gene transfected team was 6.3 days while that in the control teams were 3.1, 3.2, 3.2 days (F = 39.838, P < 0.01). Average length and area of CNV were also different in the VEGI team and the control teams (q between team A and B on average length of CNV was 17.386, 20.944 and 8.892 on 7, 14 and 21 d; q between team A and C on average length of CNV was 19.488, 19.795 and 7.483 on 7, 14 and 21 d; q between team A and C on average length of CNV was 19.583, 20.413 and 8.941 on 7, 14 and 21 d, and P were all < 0.01. And q between team A and B on average area of CNV was 30.238, 57.820 and 35.543 on 7, 14 and 21 d; q between team A and C on average area of CNV was 32.607, 57.843 and 36.653 on 7, 14 and 21 d; q between team A and C on average area of CNV was 33.873, 57.590 and 34.724 on 7, 14 and 21 d, and P were all < 0.01). Integrated epithelium, light stroma edema, less CNV and less inflammation could be seen in the gene-therapy team. And immunohistochemistry results showed VEGI positive cells in epithelium, stroma, endothelium and the cliff of CNV in the gene therapy team.
Conclusions: PCR, enzyme cutting, recombination and other genetic techniques could be used to construct expressional pcDNA4-VEGI gene. Effectene lipofectene transfection technique could be effectively used in transfecting pcDNA4-VEGI gene into rabbit cornea to inhibit CNV.