|
목적 : To establish an in vitro model of axonal regeneration from developing chick retinal cells and to evaluate the role of PKC isozymes in regulating such retinal axon regeneration.
방법 : At E6, developing chick retinas were carefully dissected, and dissociated retinal cells were cultured in F-12 media. After 3 hours of incubation for stabilization, the cells were cultured either in the presence or the absence of 0.5, 1, 2 μM rottlerin, PKC δ inhibitor, for 24 h. After fixation with 4% PFA, the cells were incubated with primary antibodies against Tuj-1, visinin and N-cadherin at RT for 1 h. Nuclei were counterstained with DAPI, and digital images were captured by confocal microscopy. Neurite outgrowth was measured by using ImageJ program.
결과 : After being stained with Tuj-1, which is a well known neuronal marker, and visinin, which is a cone cell photoreceptor marker, the cells were counted by only each positive cell. Tuj-1 and visinin- positive cells showed no change form treatment with rottlerin (P > 0.5). However, neurite length was dramatically reduced in dose-dependent rottlerin treatment (P < 0.001). Also, distribution of N-cadherin was different between non- and treatment condition of rottlerin. N-cadherin was not cleaved but aggregated in cell-cell contact areas.
결론 : We demonstrated that neurite outgrowth of retinal ganglion cell was decreased by rottlerin treatment rather than differentiation, and that this reduction could be caused by blockage of PKC δ. Thus, the activation of PKC δ may be a uesful procedure for the treatment of impaired nerve regeneration in such case as glaucoma. Also N-cadherin cleavage is essential for the extension of neurites outgrowth and the neuronal differentiation.
|