Effect of ciliary neurotrophic factor on bcl-2 and c-jun gene and protein expression in cultured retinal nerve cells

摘要

BACKGROUND:In various retinal neurodegenerative animal models,ciliary neurotrophic factor (CNTF) exhibits prominent neuroprotective effects on retinal nerve cells.Bcl-2 is an anti-apoptotic protein.c-Jun is upregulated and phosphorylated in the activated c-Jun N-terminal kinase pathway,which subsequently mediates apoptosis.However,the effect of CNTF on Bcl-2 and c-Jun expression in retinal nerve cells remains unclear.OBJECTIVE:To determine the dynamic changes in retinal nerve cell apoptosis,as well as bcl-2 and c-jun gene and protein expression,following a single dose of CNTF in a short period of time.DESIGN,TIME AND SETTING:A single-blind,randomized,controlled,in vitro experiment was performed at the Central Laboratory of Beijing Tongren Hospital from May 2008 to April 2009.MATERIALS:Neonatal bovine retinal nerve cells (Chinese Holstein),recombinant human CNTF (PeproTech,Rocky Hill,NJ,USA),rabbit polyclonal anti-Bcl-2 and c-Jun antibodies (Abcam,Cambridge,UK),fluorescein isothiocyanate-conjugated annexin V/propidium iodide kit (BioVision,Mountain View,CA,USA),real time polymerase chain reaction instrument (ABI,Foster City,CA,USA),and flow cytometer (BD FACSCalibur,Franklin Lakes,NJ,USA).METHODS:Neonatal bovine retinal cells from passage 2 were cultured for 3 days and incubated with,or without,50 ng/mL CNTF (control).MAIN OUTCOME MEASURES:Cell apoptosis was detected via Annexin V-FITC/PI double-staining and flow cytometry.bcl-2 and c-jun mRNA and protein expression were detected by quantitative real time polymerase chain reaction and western blot analysis.RESULTS:The proportion of late-stage apoptotic cells was significantly decreased at 2,4,and 6 days after CNTF treatment compared with the control group (P < 0.01).CNTF did not alter bcl-2 mRNA expression at the three time points,but significantly increased Bcl-2 protein expression at 2 and 4 days (P < 0.01).c-jun mRNA expression was significantly decreased 4 days after CNTF treatment (P < 0.01).In addition,c-Jun protein expression was slightly increased at 4 days (P < 0.01),but decreased at 6 days,compared with the control group (P < 0.05).CONCLUSION:A single dose of CNTF (50 ng/mL) upregulated Bcl-2 protein and downregulated c-jun mRNA expression,followed by a parallel,but lagged,change in c-Jun protein production in cultured neonatal bovine retinal nerve cells.These results suggested that CNTF reduces retinal nerve cell apoptosis by modifying Bcl-2 and c-Jun expression.