title: Human β-NGF gene transferred to cat corneal endothelial cells.
abstract: OBJECTIVE
  To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-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-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-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 β-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-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-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%.
  
  
  CONCLUSIONS
  Recombinant AAV-β-NGF promotes proliferation in cat CECs by expressing bioactive β-NGF protein in high efficiency and suggests that its modulation can be used to treat vision loss secondary to corneal endothelial dysfunction.
authors: Department of Ophthalmology, the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China.
authors: Wen-Juan
authors: WJ
authors: Luo
authors: Department of Ophthalmology, the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China.
authors: Min
authors: M
authors: Liu
authors: Department of Ophthalmology, the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China.
authors: Gui-Qiu
authors: GQ
authors: Zhao
authors: Department of Ophthalmology, the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China.
authors: Chuan-Fu
authors: CF
authors: Wang
authors: Department of Ophthalmology, the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China.
authors: Li-Ting
authors: LT
authors: Hu
authors: Central Laboratory of the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China.
authors: Xiang-Ping
authors: XP
authors: Liu
keywords: corneal endothelial cell; nerve growth factor; proliferation; transfect