ABSTRACT
The changes in the tau protein phosphorylation and expression of bcl-2,and bax in rat parietal cortex neurons after focal cerebral ischemia-reperfusion(I/R)were explored,and the relationship between the tau protein phosphorylation and the expression of bax or apoptosis was clarified in order to elucidate the relationship between cerebral infarction and Alzheimer's disease.The rat focal cerebral I/R model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method.The level of tau protein phosphorylation at Ser396,Ser404,Tyr231,Ser199/202 sites and the expression of bcl-2,bax and total tau 5 in rat parietal cortex during focal cerebral ischemia/reperfusion were detected by Western blot.The relationship between the tau protein phosphorylation and the expression of bax,or apoptosis was examined by TUNEL method and double-labeling immunofluorenscence method.The results showed that the level of tau hyperphosphorylation at Ser199/202,Ser396,Ser404,Tyr231 sites and the expression levels of bcl-2,and bax were significantly higher in I/R group than in the sham group,bat the ratio of bcl-2/bax was decreased.Neuronal apoptosis,bax expression and the tau protein hyperphosphorylation were co-localized.It is suggested that Alzheimer's disease-like pathological changes occur after cerebral I/R.The highly abnormal phosphorylation of tau protein plays a key role in cerebral I/R-induced apoptosis.The cerebral infarction may contribute to Alzheimer's disease occurrence and development.
ABSTRACT
This study established superparamagnetic iron oxide (SPIO)-labeled nerve growth factor-β (NGF-β) gene-modified spinal cord-derived neural stem cells (NSCs).The E14 rat embryonic spinal cord-derived NSCs were isolated and cultured.The cells of the third passage were transfected with plasmid pcDNA3-hNGFβ by using FuGENE HD transfection reagent.The expression of NGF-β was measured by immunocytochemistry and Western blotting.The positive clones were selected,allowed to proliferate and then labeled with SPIO,which was mediated by FuGENE HD transfection reagent.Prussian blue staining and transmission electron microscopy (TEM) were used to identify the SPIO particles in the cells.The distinctive markers for stem cells (nestin),neuron (β-Ⅲ-tubulin),oligodendrocyte (CNPase) and astrocyte (GFAP) were employed to evaluate the differentiation ability of the labeled cells.The immunocytochemistry and western blotting showed that NGF-β was expressed in spinal cord-derived NSCs.Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells.TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma.The immunocytochemistry demonstrated that the labeled cells were nestin-positive.After differentiation,the cells expressed β-Ⅲ-tubulin,CNPase and GFAE It was concluded that the SPIO-labeled NGF-β gene-modified spinal cord-derived NSC were successfully established,which are multipotent and capable of self-renewal.