Proliferation andregeneration effects of constructed human β -nerve growth factor gene ontransfected cat corneal endothelial cells in vitro

：Objective To explore themechanisms of proliferation and regeneration effects of a human nerve growthfactor(p-NGF)expression vector(pcDNA4-β-NGF)on the transfected catcorneal endothelial cells in vitro.To provide a new method for long term cultivation ofhuman corneal endothelial cells in vitro and to establish theoretical basis of genetherapy for corneal endothelial defects.Methods It was a experimental study.The humanpcDNA4-B-NGF expression vector was constructed and transfected into cultured cat cornealendothelial cells bv EffecteneTM lipofectine transfection technique.The expression of thereporter gene pcDNA4-β-LacZ expression Was used to determine thetransfection efficiency 48 hours after the transfection．RT-PCR andimmunohistochemistry techniques were used to check the transient expression status at mRNAand protein levels in cat comeal endothelial cells.Mitotic index and methyl thiazolyltetrazolium(MTT) value were measured and cell numbers at different stages of cell cycleswere deterd by flow cytometer 96 hours after transfection.An in vitro quantitative catcorneal endothelial cell traumatic model was established which was used for observing theeffect of human B-NGF expression product on the DNA synthesis of cat endothelial cells andheMing process of traumatized endothelial cells.Results Ahuman nerve growthfactor(B-NGF)expression vector(pcDNA4-13-NGF)was successfully constructed and confirmed bysequence analysis.Single layered pure cat corneal endothelial cells were obtained by amodified sliced tissue culture technique and confirmed by morphological analysis,neuronespecific enolase immunohistoehemistry study and transmission electronicmicroscope.EffecteneTM lipefectine mediated transfection efficiency of pcDNA4-β-NGFinto cat Corueal endothelial cells in vitro was 11.3%.The human β-NGFcould be highly expressed in the transfected comeal endothelial cells at mRNA and proteinlevels.Mitotic index.MTT valae and G1 stage cell numbers,as well as traumatically defectedendothelial cells numbers during the healing process of human β-NGF transfected comeal endothelial cells werestatistically differed from the pre-transfected cells and control groups.ConclusionsEffeeteneTM lipefectine transfection technique could be effectively used for transfectingpcDNA4-B-NGF into cat comeal enl cells in vitro with good efficacy and the gene couldstably express to improve the proliferation and regeneration of the cat comeal endothelialcells.This method could be managed as an experimental basis to be applied in theexperimental study for transfecting the human B-NGF gene into human corneal endothelialcells.Therefore a new method for resolving the problem of impossible regeneration ofcorneal endothelial cells could become possible. Key words: Nerve growthfactor; Transfection; Endothelium,corneal; Endothelial cells; Regeneration; Cells,cultured