The Biologic Effects Of Human Platelet Derived Growth Factor B (PDGF-B) Encoding Gene Transfered Cat Corneal Endothelial Cells

Objective: A human platelet-derived growth factor-B (PDGF-B) expression vector (pcDNA4-PDGF-B) was constructed and transferred into cat corneal endothelia in vitro by Effectene? lipofectine transfection technique in order to confirm that the PDGF-B 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.RT-PCR technique was applied to clone the human PDGF-B gene . The human PDGF-B gene fragment was cloned into PCDNA4B expression vector to form the pcDNA4-PDGF-B expression vector. The clone was confirmed to be the human PDGF-B 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 and scanning microscope. 3.The human PDGF-B gene expression vector pcDNA4-PDGF-B 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 , Western blot and PDGF-B monoclonal antibody staining were used to check the transient expression status at mRNA and protein level in cat corneal endothelia . 4. MTT value was measured and cell numbers at different stages of cell cycles were determined by flow cytometer 48—96 hours after transfection. An in vitro quantitative cat corneal endothelial cell traumatic model was established which was used to observe the effect of human PDGF-B expression product working 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 to obtain the neomycin resistant cell clone which showing human PDGF-B gene highly expressed. Results: 1. The human PDGF-B gene was cloned from heathy parturien placent tissue. 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...