Abstract
Abstract: :
Purpose:To determine whether siRNA down–regulation of p27kip1 will overcome G1–phase arrest and promote cell cycle progression in human corneal endothelial cells (HCEC). Methods: Confluent cultures of HCEC were incubated for 24 hrs in the presence of p27kip1 siRNA (2.5, 5, 25, 100nM) using lipid transfection. Control cultures were incubated with non–silencing siRNA. After 0, 24, 48, 72, and 96hrs of incubation in medium containing 8% FBS, cultures were fixed and immunostained for Ki67 and BrdU to detect actively cycling cells or prepared for western blotting. Staining was visualized using by fluorescence microscopy. Cell numbers were also counted by Coulter counter after 0, 48, 96, 144, and 192hrs. Viability was tested by a Live/Dead assay after 24 hrs. Results:A dose–dependent decrease of the p27kip1protein was observed after transfection. The number of Ki67 and BrdU positive cells was increased by 72hrs post–transfection in HCEC from young donors (<30 years old). Cell numbers were also increased in HCEC from young donors after 144hrs of incubation. In contrast, no change was observed in controls or in HCEC from older donors (>60 years old) at any time–point tested. Viability was not significantly affected by transfection. Conclusions:The transfection of p27kip1 siRNA was sufficient to promote proliferation in HCEC from younger donors, but not older donors. It is considered that inhibition of proliferation in older donors is regulated by other mechanisms in addition to p27kip1.
Keywords: cornea: endothelium