Alpha-actinin expression at different differentiating time points from temporal lobe cerebral cortex neural stem cells to neuron-like cells using energy dispersive X-ray analysis

摘要

BACKGROUND: Alpha-actinin (a-actinin) plays a key role in neuronal growth cone migration during directional differentiation from neural stem cells (NSCs) to neurons.OBJECTIVE: To detect in situ microdistribution and quantitative expression of a-actinin during directional differentiation of NSCs to neurons in the temporal lobe cerebral cortex of neonatal rats.DESIGN, TIME AND SETTING: Between January 2006 and December 2008, culture and directional differentiation of NSCs were performed at Department of Histology and Embryology, Preclinical Medical College, China Medical University. Immune electron microscopy was performed at Department of Histology and Embryology and Department of Electron Micrology, Preclinical Medical College, China Medical University. Spectrum analysis was performed at Laboratory of Electron Microscopy, Mental Research Institute, Chinese Academy of Sciences.MATERIALS: Basic fibroblast growth factor, epidermal growth factor, brain-derived nerve growth factor, type-1 insulin like growth factor, and a-actinin antibody were provided by Gibco BRL, USA; rabbit-anti-rat nestin monoclonal antibody, rabbit-anti-rat neuron specific enolase polyclonal antibody, and EDAX-9100 energy dispersive X-ray analysis were provided by PHILIPS Company, Netherlands.METHODS: NSCs, following primary and passage culture, were differentiated with serum culture medium (DMEM/F12+10% fetal bovine serum+2 ng/mL brain-derived nerve growth factor+2 ng/mL type-1 insulin like growth factor).MAIN OUTCOME MEASURES: Expression of a-actinin in neuron-like cells was quantitatively and qualitatively detected with immunocytochemistry using energy dispersive X-ray analysis. RESULTS: Immunocytochemistry, combined with electron microscopy, indicated that positive a-actinin expression was like a spheroid particle with high electron density. In addition, the expression was gradually concentrated from the nuclear edge to the cytoplasm and expanded into developing neurites, during differentiation of neural stem cells to neurons. Conversely, energy dispersive X-ray analysis indicated that the more mature the neural differentiation was, and the greater the expression of a-actinin.CONCLUSION: The gradual increase of a-actinin expression is related to growth, development, and maturity of differentiated neuron-like cells, in neonatal rat frontal lobe cortex, at different differentiating time points of NSCs to neurons.