Experimental Study Of Neural Stem Cell'S Migration Characteristic For Glioma Cell

Objective:To observe whether neural stem cells have migrat-ing characteristic for glioma cells in vitro. Methods:1. Neural stem cells isolated from brain of mouse were cultured suspensively. The additive of serum-free culture B27, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were added into the DMEM/F12 medium in vitro. Mechanical separation method was used to subculture continuously, subsequently cell morphology observed by inverted phase contrast microscope. The expression of neural stem cell marker nestin by immunofluorescence assay and differentiated neural stem cell marker neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) by immuriocyto-chemistry assay were detected respectively.2. Normal neural stem cell migration assay with a transwell cells chamber:The experimental groups were C6 cells. The control groups were 3T3 cell. Serum-free medium groups were the blank group. The above mentioned three cell suspension 600μl were added to the lower chamber of transwell respectively. Neural stem cells in the logarithmic phase of the cell cycle 200μl were added into the upper chamber respectively. The mixed cells were co-cultured in the transwell cells chamber, and neural stem cells migrated from the upper surface of chamber to the lower were counted under light micros-cope; 3. Neural stem cells pre-induced migration assay:In experimental group the neural stem cells were pre-induced for 5 hours by 10% fetal bovine serum and RA (0.5μg/ml) added to the neural stem cell suspension. The control group were not induced. The blank group in a transwell cells chamber without C6 cell were pre-induced samely. The three groups liquid were added to the upper chamber of cell culture chamber for 200μl respectively. The 600μl suspension of C6 cells were added to the lower chamber of experimental group and control group, and the 600μl of serum-free medium were added to the lower chamber of the blank group. The transwell cells chamber was co-cultured in the incubator and neural stem cells migrated from the upper surface to the lower were counted under light microscope. Results:1. Cells isolated from brain of mouse in neural stem cell culture medium containing serum-free culture additives B27, bFGF and EGF growth factor can form large floating balls of neural stem cells. Nestin antibody was positive by immunofluorescence. The differentiated cells expressed specific antigen of neuronal cells and glial cells by immunocytochmistry; 2. In normal neural stem cell migration experiments, the number of neural stem cells migrated from the upper chamber surface to the lower were 221.50±34.46 (the experimental group),86.50±23.09 (the control group) and 24.00±11.15 (the blank group) respectively,and were different among the three groups significantly (P＜0.05).3.In the migration experiments of pre-induced neural stem cells:The number of neural stem cells migrated from the upper chamber surface to the lower were:247.00±45.18 (the experimental group),212.50±34.46 (the control group) and 30.00±11.15 (the blank group) respectively, and were different among the three groups significantly (P＜0.05). Conclusion:1. Neural stem cells may be isolated and cultured with a simple and effective method.2. The migration trend of neural stem cells to glioma cell in vitro was observed. 3. The migration trend of the pre-induced neural stem cells may be stronger than the neural stem cell without induction to the glioma cell.