| PurposeThe most common complication of cataract surgery is the development of posterior capsule opacification (PCO), which is a problem that has not been well solved. Generally, the occurrence rate of PCO 2-5 years after surgery is as high as 50% in adults and 100% in children [1]. Hyperplasia of the lens epithelial cells is one of the main cellular events following phacoemulsification and is found to be an important feature contributing to PCO [2].Attempts have been made to prevent PCO by modifying the designs of intraocular lenses and the surgical procedures, but PCO still occurs. Chemicals and antimitotics (such as 5-FU, MMC) have been used to inhibit the proliferation of lens epithelial cells, but the efficiency and the tolerance are not satisfying especially because of their toxic effects [3,4]. Nd:YAG laser capsulotomy has also been used. However, it can lead to severe complications such as macular edema and retinal detachment [2,5].Recent advances in science and technology have provided opportunities to develop new treatment strategies using gene therapy. Among them, antisense strategies have proved efficient in numerous studies with different kinds of cells [12-15].In this study, we explored the feasibility of adenovirus-mediated transfer of an antisensec-myc protooncogene into human lens epithelial cells in vitro and in a rabbit model of PCO after phacoemulsification in vivo.MethodsI. In vitro experimentsRecombinant adenovirusesAd-LacZ and Ad-AS-myc were kindly provided by Professor Chen Lin (Cancer Institute, Chinese Academy of Medical Science, Peking Union Medical College). These two replication-defective recombinant adenoviruses (E1-E3 deleted) were expanded in the 293 cell line (Fig. 2) and purified by centrifugation. Virus titer was determined by standard assay in 293 cells.Cell cultureImmortalized human lens epithelial cell (HLEC) line (HLE-B3, ATCC, USA) was kindly provided by Dr. Youhai Chen (Department of Molecular and Cellular Engineering, University of Pennsylvania, School of Medicine)(Fig.3). The cells were cultured in Eagle's minimum essential medium (MEM, Sigma, USA) containing 10% fetal bovine serum (FBS, Hyclone, USA), 1.0mM sodium pyruvate (Hyclone, USA), 100IU/ml penicillin G and 100 u g/ml streptomycin sulphate. The incubation condition was 37C in a humidified atmosphere of 95% air and 5% carbon dioxide. The cells were subcultured at a ratio of 1: 3.Determination of transduction efficiencyHLECs were seeded at a density of 2x104 cells/well in six-well plates. After overnight attachment, the cells were transduced with 1ml of Ad-LacZ suspension (MOI of 100 or 200) and incubated for 1hr at 37C in 5% CO2. Then 3ml of fresh MEM containing 2% FBS was added to each well. Forty-eight hours after transduction, the cells were washedtwice with warm PBS and fixed for 10min in 1% formaldehyde-0.05% glutaraldehyde-PBS. After fixation, the cells were washed twice again with PBS and stained with X-Gal (5-bromo-4-chloro-3-indolyl-B-D-galactosidase, Shanghai Sangon Biological Engineering Technology and Service Co., China).X-Gal-positive cells, as well as total cells were counted in the microscopic field at a magnification of x20 using a phage-contrast microscope (IMT-2, Olympus, Japan). This procedure was performed for five random fields and the transduction efficiency was calculated.Morphological changes of transduced cellsThe transduced cells were pictured at daily intervals (0, 24, 48, 72 and 96 hrs) afterAd-AS-myc transduction (MOI of 100).HLECs proliferation of transduced cellsThe inhibition of Ad-AS-myc transduction on HLECs proliferation was evaluated bymaking cell growth curve and MTT colorimetric assay.i. Cell growth curveHLECs were seeded at a density of 5x103 cells/well in six-well plates. The next day, thecells were transduced with Ad-AS-myc (MOI of 100). Then the cells were counted atdaily intervals (0, 24, 48, 72 and 96 hrs) and the cell growth curve was made.Non-transduced HLECs were taken as control group. The procedures were performed... |
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