Controllable Urokinase Gene Expression Into Cultured Porcine Trabecular Meshwork Cells

Abstract

Purpose: : We reported previously (ARVO 2005) successful urokinase (uPA) gene transfer into porcine TM cells. In the present study, we tried to establish a controllable in vitro uPA gene transfer model into cultured porcine TM cells using a Tet-ON system.

Methods: : Human TM cells were obtained from POAG patients undergoing a trabeculectomy and porcine TM cells were freshly isolated from eye specimens. Both types of cells were cultured in 5% fetal bovine serum added to F12/DME medium and used at the second or third passage. Total RNA was extracted from human TM cells and reverse transcribed into cDNA. A reverse-transcribed polymerase chain reaction method was performed to detect the gene expression of uPA using specific primers for human cDNA sequences. The complete sequenced cDNA of human uPA was then sub-cloned into an expression vector (Tet-On system, Clontech) and transfected into the cultured porcine TM cells using lipofectamine. As a control, the expression vector alone was also transfected into cultured cells. After addition of doxycycline (1 µg/ml), expression of the transferred human uPA was examined using an enzyme linked immunosorbent assay (ELISA).

Results: : ELISA revealed expression of the transferred human uPA almost equivalent to previous experiment (3.5 ng/ml) in the media of cultured porcine TM cells that had been transfected with the human uPA gene under existence of doxycycline, whereas no detectable uPA was found in the control cells.

Conclusions: : Controllable gene transfer of uPA, which may degrade the extracellular matrix in juxta-canalicular connective tissue, into TM cells was possible using Tet-On system. The present method may be applicable for advanced trabeculotomy procedures and may lead to development of a new therapy for open angle glaucoma.

Keywords: trabecular meshwork • extracellular matrix • gene transfer/gene therapy