Human ß-NGF gene transferred to cat corneal endothelial cells.

ABSTRACT

AIM: To transfect the cat corneal endothelial cells (CECs) with recombinant human ß-nerve growth factor gene adeno-associated virus (AAV-ß-NGF) and to observe the effect of the expressed ß-NGF protein on the proliferation activity of cat CECs. METHODS: The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco's modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-ß-NGF was constructed. The recombinant human AAV-ß-NGF was transferred into cat CECs directly. Three groups were as following normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-ß-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the ß-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. RESULTS: The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human ß-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-ß-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-ß-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result showed that the percentage of G1cells in CEC-AAV-NGF group was 76.8% while that in normal CEC control group and CEC-AAV control group was 46.6% and 49.8%. CONCLUSION: Recombinant AAV-ß-NGF promotes proliferation in cat CECs by expressing bioactive ß-NGF protein in high efficiency and suggests that its modulation can be used to treat vision loss secondary to corneal endothelial dysfunction.