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Objective To investigate the effect ofstromal cell derived factor-1 (SDF-1) on the regulation of neural stem cells (NSCs) migration.Methods NSCs were obtained from the cerebral cortex of embryonic rats and cultured in serum-free medium,and their stem cell properties were assessed by means of induced differentiation in vitro into neurons and astrocytes.After in vitro cell culture,the purity of NSCs and the co-expression rate of CXCR4/nestin were detected by flow cytometry.Blind-well chambers were employed to detect the chemotactic effects of SDF-1 by counting the cells which had crossed a 8 μm pore membrane when confronted with varying concentrations of SDF-1 (0,1,10,50,100,500 and 1000 ng/mL),and the distribution of cells migrated out of the same neurosphere was overviewed by μ-slides in the persistent concentration gradient of SDF-1.Results Neurospheres were formed by persistent proliferation of NSCs, which were capable of differentiating into neurons (β-tubulin+) and astrocytes (GFAP+) in media without mitogens,and flow cytometry analyses showed that most of the cultured cells expressed nestin and the co-expression rate of CXCR4/nestin was nearly 80%.SDF-1 showed great chemotaxis to NSCs,and the amount of cells having migrated through the membrane in 500 ng/ml SDF-1 group was higher than that in other groups (P<0.05).When the cells were confronted with a linear concentration gradient (from 500 to 0 ng/mL),which was generated by diffusion and stable for at least 48 h,the cells migrated out ofa neruosphere could distribute irregularly with more cells locating in the region of higher concentration of SDF-1 and longer migration distance away from the center of the neurosphere than the opposite.Conclusion SDF-1 binding to its specific receptor CXCR4 was capable of inducing NSCs migrating directionally to the source of SDF-1.
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Objective To explore the expression of human brain-derived neurotrophic factor (hBDNF) and green fluorescent protein (GFP) in hBDNF-GFP gene-transfected rat neural stem cells (NSCs) and the changes in the biological characteristics of the transfected cells. Methods NSCs were transfected with a lentiviral vector carrying hBDNF and GFP genes (hBDNF-GFP-NSCs) or GFP gene only (GFP-NSCs), with normal NSCs as the control. The expression levels of hBDNF mRNA and hBDNF protein in all the 3 groups were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. Enzyme-linked immunosorbent assay (ELISA) was performed to detect hBDNF level in the cell culture medium before and after hBDNF-GFP gene transfection. Dorsal root ganglion (DRG) neurons and NSCs were cultured with the supernatants of the transfected NSCs and normal NSCs, and the growth status of the DRG neurons was observed and the proportion of NSCs differentiating into neurons determined. Results Compared with GFP-NSCs and normal NSCs, hBDNF-GFP-NSCs showed obvious hBDNF overexpression at both rnRNA and protein levels 7 days after the transfection, hBDNF content in the supematant of hBDNF-GFP-NSCs culture increased significantly with time and peaked 5 days after the transfecfion (P<0.05). Four days after culture in hBDNF-GFP-NSCs supernatant, the DRG neurons and adherent NSCs extended cells processes, and the ratio of the NSCs differentiating into neurons was higher in cells cultured in hBDNF-GFP-NSCs supematant than in those culture in GFP-NSCs and normal NSCs supematants. Conclusion Lentivitus can be used as the vector for hBDNF and GFP gene transfection into NSCs, and hBDNF-GFP gene-transfected NSCs maintain the basic characteristics of NSCs and are capable of stable expression and secretion of hBDNF and GFP.
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Objective To investigate the effects of different medium and rat age on optic nerve tissue culture of rats.Methods Theoptic nerves from newborn rats(4dpostbirth)or adult rats(3-month old)were cultured on the rat-tailed collagen slide,pely-L-Lycine(PEL)slide,and Biocoat culture inserts,respectively.Their growth status was dynamically observed under a phase contrast microscope every day.The adherence rate of explant was recorded 48 h after culture.The maximum migration distance Was measured by an image analysis system on the 5th day after culture.The activity of lactic dehydrogenase (LDH)in the tissue culture medium Was measured dynamically.Morphological observance Was carried out by routine HE staining and the ultrastmcture of the tissue explants Were observed by a transmission electronmicroscope. Results The tissue adherence rate was higher in the Bioeoat insert group than in the rat-tailed collagen slide group or PLL slide group.The magnum migration distance of the tissue explants cultured in the Bioeoat insert group was longer than that in the rat-tailed collagen slide group or the PLL slidegroup.The maximum migration distance of the newborn rats Was longer than that of the adult rats under same culture condition(P<0.05).The LDH activity in the tissue culture medium began to descend 3 d after culture.The LDH activity in the adult rat group increased again on the 9th day since culturewhile it remained low level in the newborn rat group even on 12th day since culture.The cell processes showed up from the edge of explants and neuralgia cell migration was observed at the early stage,especially in newborn rats.The optic nerve structure gradually died out with the increase of culture time.The survival timeofopticnerve explant from newborn rats was longer than that of adult rats. Conclusion The optic nerve tissue can be cultured for a long time under suitable culture conditions.