ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of nucleolin silencing on the differentiation of rat neural stem cells (NSCs) and the role of Wnt signaling pathway in mediating such effect.</p><p><b>METHODS</b>Adenovirus vectors expressing small interfering RNA (siRNA) against nucleolin were constructed, verified, and packaged in HEK293A cells. The adenovirus was then transfected into NSCs isolated from neonatal SD rats and the differentiation of the NSCs was examined by detecting the expressions of neuron specific encloase (NSE) and glial fibrillary acidic protein (GFAP) using immunocytochemistry. The expressions of nucleolin, nestin, Wnt3, and β-catenin in the cells were determined with Western blotting.</p><p><b>RESULTS</b>Restriction endonuclease and sequencing analysis verified successful construction of the adenoviral vector expressing nucleolin siRNA (nucleolin-siRNA2). Infection of rat NSCs with nucleolin-siRNA2 significantly lowered nucleolin protein expression as compared with that in negative and blank control groups (P<0.05). The percentages of NSE-positive cells and GFAP-positive cells were significantly higher in NSCs infected with nucleolin-siRNA (P<0.01); the infection also resulted in obviously lowered expression of nestin protein and increased expressions of Wnt3 protein and β-catenin nucleoprotein in the cells.</p><p><b>CONCLUSIONS</b>Nucleolin silencing by adenovirus-mediated RNA interference induces the differentiation of NSCs into neurons and astrocytes, which is related with the activation of Wnt signaling pathway.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To study the effect of hyperbaric oxygen (HBO) administered at different pressures and different exposure time on the differentiation of neural stem cells (NSCs) in vitro.</p><p><b>METHODS</b>The cerebral cortices from newborn rats (0-3 days old) were sterilely collected, digested, and centrifuged. After removal of the supernatant, the cells were re-suspended with DMEM/F12 medium containing B27, bFGF and EGF. The NSCs of 2-3 passages were randomly divided into seven groups: a control (untreated) and 6 HBO treatment groups that NSCs were subjected to HBO treatment of different pressures (1, 2 or 3 ATA) and different exposure time (30 or 60 minutes). The differentiated NSCs were examined by neuron-specific enolase (NSE) immunocytochemistry 24 hrs later. Percentage of NSE positive cells differentiated from NSCs was assessed by fluorescent microscopy.</p><p><b>RESULTS</b>The percentage of NSE positive cells differentiated from NSCs was the highest in the HBO 2ATA-60 min group (9.17+/-0.50%) (P<0.01), followed by the HBO 3ATA-60 min (7.89+/-0.62%), HBO 2ATA-30 min (6.72+/-0.76%), HBO 3ATA-30 min (6.08+/-0.57%), HBO 1ATA-60 min (5.45+/-0.52%), HBO 1ATA 30 min (3.85+/-0.44%) and control groups (3.72+/-0.88%). In addition to the HBO 1ATA-30 min group, the other HBO treatment groups had increased significantly percentage of NSE positive cells compared with the control group (P<0.01). Under the same pressure, the 60 min treatment groups had increased significantly percentage of NSE positive cells compared with the 30 min treatment groups (P<0.01).</p><p><b>CONCLUSIONS</b>HBO treatment (2 ATA, 60 minutes) produces a best effect in the differentiation of NSCs into neurons.</p>
Subject(s)
Animals , Rats , Animals, Newborn , Cell Differentiation , Hyperbaric Oxygenation , Neurons , Cell Biology , Pressure , Stem Cells , Cell Biology , Time FactorsABSTRACT
<p><b>OBJECTIVE</b>To study the protective effects of multiple course hyperbaric oxygen (HBO) treatment against hypoxic-ischemic brain damage (HIBD) in neonatal rats when HBO treatment is delayed (96 hrs after the HIBD event).</p><p><b>METHODS</b>Eighty-eight 7-day-old Sprague-Dawley rat pups were randomly assigned to control, HIBD and HBO groups. The HBO group was subdivided into cohorts receiving treatment 2 h, 48 h and 96 h, respectively, after HIBD was induced. The three subgroups comprising different therapeutic windows were further randomly assigned to receive 1, 2 or 3 courses of HBO treatment ("HBO-1, -2 and -3 sub-groups"). HBO was administered once daily (2 ATA), a course lasting for seven days. There was an interval of three days between the courses. All pups were sacrificed at the end of HBO treatment (31 days after HIBD). TUNEL staining was used for testing neuronal apoptosis in the cortex and the CA1 of the hippocampus, and NSE staining was used to ascertain cortical neuronal population.</p><p><b>RESULTS</b>1.There were significantly more TUNEL positive cells in the HIBD group than in the control group; NSE positive cells were significantly lower than in controls (P<0.01). 2. With the more delayed therapeutic window, the effects of apoptosis inhibition and neuronal protection of a single course of HBO were gradually reduced. 3. With increasing courses of HBO treatment, the effects of apoptosis inhibition and neuronal protection of HBO increased gradually in rats receiving treatment 48 and 96 hrs after HIBD. In the HBO group receiving treatment 2 hrs after HIBD, the number of apoptotic cells and NSE positive cells were close to that of the control group after one course of HBO treatment.</p><p><b>CONCLUSIONS</b>One course of HBO administered within 2 hrs after HIBD can effectively inhibit neuron apoptosis and protect neurons. The effects of apoptosis inhibition and neuron protection of HBO can be increased through increasing the number of HBO treatment courses in neonatal rats with HIBD even if initiation of treatment is delayed after HIBD.</p>