Roles Of β-catenin Gene In The Proliferation And Differentiation Of Spinal Cord Derived Neural Stem Cells

Objective Separating the spinal cord derived neural stem cells(NSCs) from the CD1 fetal mouse and appraisal the cells; To investigate the role of β-catenin gene in the proliferation and differentiation of spinal cord derived neural stem cells,and to provide theoretical basis for the further vivo tests.Methods The E14 of CD1 pregnant rats were anesthetized and then the fetal rats were taken out, and the spinal cord tissue was separated under sterile conditions. Using single cell cloning technique and stem cell culture technique to culture the separation cells. The separation cells were identified by immunofluorescence. The experiment was divided into three groups: blank group(NSCs were separately cultured), GFP group(NSCs were infected with Ad-GFP adenovirus vector), β-catenin group(NSCs were infected with Ad-β-catenin adenovirus vector). The time of infection was 5 days.The morphological changes of three groups were observed by ordinary optical microscope. Cell infection efficiency was observed by fluorescence microscope and was also verified by flow cytometry. The protein expressions of β-catenin were measured by Western blot. The number of cells in three groups were measured by CCK8 detection and crystal violet staining. The cell survival rates of three groups were measured by blue dye. The m RNA expressions level of NSE and GFAP were measured by RT-PCR. The protein expressions and locations of Neu N and GFAP were detected by immunofluorescence. The protein expressions of Neu N and GFAP were also measured by Western blot.Results The cells derived from spinal cord tissue of fetal rats had the ability of continuous differentiation and cloning and started to form a small number of cell clusters. The cells were irregular in shape and had rich adhesion ability after mechanical separation. Nestin antigen of the majority of the separation cells was positive. After the infection of Ad-β-catenin adenovirus vector for 5 days, the shape was changed. The synapse grew longer, the cells were smaller,and the collection of synapse was more than the cells separately cultured. The refractive index was slao enhance. The results of fluorescence microscopy and flow cytometry showed that the infection rates were about 50% when the cells infected adenovirus for 3days, and the rates were about 80% for 5 days. The results of Western blot showed that β-catenin protein in β-catenin group was higher than blank group and GFP group(P<0.05).The results of CCK8 detection and crystal violet staining showed that the number of cells in β-catenin group was much higher than blank group and GFP group(P<0.05),and the cell survival rates in β-catenin group were also higher than other two groups by blue dye(P<0.05). The m RNA expression level of NSE in β-catenin group was significantly higher than those in blank group and GFP group(P<0.05).The m RNA of GFAP was lower than those in the other two groups(P<0.05). Neu N in β-catenin group was significantly higher than blank group and GFP group(P<0.05),and it was mainly situated in the nuclei,but the GFAP was lower than others(P<0.05). The results of Western blot showed that Neu N in β-catenin group was higher than blank group and GFP group, and the GFAP in β-catenin group was lower(P<0.05).Conclusions The cells separated from spinal cord tissue of fetal mouse can be cloned into nerve balls, and the cells are neural stem cells with self replication and multi-lineage differentiation potential. The adenovirus can be used as a vector to transfer the β-catenin gene into NSCs, and the rate of infection is high. β-catenin can promote the proliferation of spinal cord derived NSCs and improve the cell survival rate, and promote the NSCs into neuron.