| Objective: A human nerve growth factor (β-NGF) expression vector (pcDNA4-β-NGF) was constructed and transfected into cat corneal endothelia in vitro by EffecteneTM lipofectine transfection technique in order to confirm that the P-NGF gene not only can be expressed in vitro but also had the ability of stimulating mitotic effect on cat corneal endothelia. This primary results might provide a new way for long term cultivation of human corneal endothelium in vitro and the theoretical basis of gene therapy for the blindness caused by endothelial defects. Methods: 1.PCR technique was applied to clone the human β-NGF gene which is a single exon structure from human white blood cell genomic DNA. The human β-NGF gene fragment was cloned into pcDNA4B expression vector to form the pcDNA4-β-NGF expression vector. The clone was confirmed to be the human β-NGF gene by Ab screening,enzyme cutting identification and computer automatic sequence analysis.2.Modified sliced tissue culture technique could rapidly cultivate to form pure single layer cat corneal endothelia .The cell type and purity of the endothelial cells were further confirmed by morphological analysis, NSE immunohistochemistry study and transmission electronic microscope. 3.The human β-NGF gene expression vector pcDNA4-β-NGF was constructed and transfected into cat corneal endothelia in vitro by Effectene?lipofectine transfection technique. The expresion of the reporter gene pcDNA4-β-lacZ expression was used to determine the transfection efficiency 48 hours after the transfection. RT-PCR and β-NGF monoclonal antibody were used to check the transient expression status at mRNA and protein level in cat corneal endothelia . 4.Mitotic index, MTT value were measured and cell numbers at different stages of cell cycles were determined by flow cytometer 48 hours after transfection. An in vitro quantitative cat corneal endothelial cell traumatic model was established which was used for observing the effect of human P-NGF expression product on the DNA synthesis of cat endothelial cells and healing process of traumatized endothelia. 5.By using the characteristics of anti-neomycin effect of the cloning expression vector, a minimal mortal dosage G418 was applied for obtaining the neomycin resistant cell clone and the clone was shown human P-NGF gene highly expressed. Results: 1.The human β-NGF gene was cloned which is a single exon structure from normal human white blood cell DNA. Computer automatic sequence analysis showed the identical sequence in gene bank. 2.Modified sliced tissue culture technique could rapidly cultivate to form pure single layer cat corneal endothelia .The cell type and purity of theendothelial cells were further confirmed by morphological analysis, NSE immunohistochemistry study and transmission electronic microscope. 3.Effectene?lipofectine transfection technique could be effective used in transfecting pcDNA4-β-NGF into cat corneal endothelia in vitro with efficacy of 11.3% transfection rate. RT-PCR results showed that human β-NGF was expressed in cat corneal endothelia and significantly highly expressed in the human β-NGF transfected corneal endothelia. The expression of human β-NGF in Effectene?transfected control group and transient transfection group with pcDNA4Bplasmid showed no Statistical increase. β-NGF monoclonal antibody staining was marked seen in humanβ-NGF transfected cat corneal endothelium. 4.Mitotic index, MTT value,G1 staged cell numbers, as well as the healing process of traumatically defected endothelial cell numbers in human β-NGF transfected corneal endothelia were statistically differed from pre-transfected cells and control groups.The result stated that human β-NGF could increase the DNA synthesis in cat corneal endothelial cells and improve the mitotic effect of the cat corneal endothelial cells . 5.G418 screened neomycin resistant human β-NGF transfected corneal endothelial clone could highly express the human β-NGF gene product and morphologically different from pre-transfected cells. Conclusion : Ef... |
