Experiment Study On Purification And Identification Of The Neural Stem Cells From Rats' Brain

Objective To create a method of separating neural stem cells (NSCs) from rat's brain in vitro using immunomagnetic beads. To investigate the possibilities of NSCs proliferating and differentiate by cell culture and to observe the cells' form in different growing phases, and to analyze the products of the cells. Methods Cell suspension of adult rat's corpus striatum or fetal rat's cerebral cortex was incubated with antibody of Nestin. The labeled cells were separated from the suspension through the magnetic field after combined with immunomagnetic beads coated with the second antibody. The purity and the activity of the separated cells were determined through the light microscope, and the ratio of nestin positive cells among the separated cells was determined by the flow cytometer. These cells were cultured and dissociated with serum of fetal bovine for 3 days. Immunocytochemistry and immunofluorescence were performed to identify the properties of the differentiated cells. The expression of neural associated genes were detected by RT-PCR assays. The morphology of NSCs was studied by the scanning electron microscope. Results 1. The purity of the separated cells from adult rat's corpus striatum and fetal rat's cerebral cortex was (89.8?.7)% and (89.2?.8)% respectively, and nestin expression ratio of the cells was (88.3?.4)% and (89.5?.8)%. 2. The activity of the cells was (96.0?.4)% and (94.9?.0)% respectively after purification, which was not different significantly with that before purification. 3. The combination of the cells with immunomagnetic beads was shown under the scanning electron microscope. 4. The sorted cells treated by the fluorescence staining were showed green fluorescence through the fluorescence microscope. The different kinds of cells were observed at 3 days after the adding of fetal bovine serum. The differentiated cells contains Nestin+, NSE+, or GFAP+ cells. 5. RT-PCR assay identified that the induced cells expressed transcripts for neural associated genes including brain factor-l(BF-l), gamma-aminobutyric acid (GABA) α-receptor y-subunit, tyrosinehydroxylase (TH) a nd tryptophan hydroxylase (TPH). Conclusion 1. The magnetic cell sorting system we had used could effectively separate neural stem cells from both the fetal rat's cerebral cortex and the adult rat's corpus striatum .The activities of the sorted cells were not effected. 2. The magnetic cell sorting system are compatible with both flow cytometer and electron microscope. The sorted c ells could be used in the subsequent e xperiments. 3.The cells separated form adult rat's corpus striatum and fetal rat's cerebral cortex had the abilities of proliferation and multi-differentiation. The induced cells might express some neural associated genes such as BF-1, GABA-receptor y-subunit, TH and TPH genes, etc. which implied the separated cells belonged to the NSCs of central nervous system.