Abstract:

Abstract:Objective
To establish a nerve stem cell model possessing the potentiality of   differentiating into γ-aminobutyric acid neuron-like cells(GABAergic-like cells)， and provide eligible investigative vector for study on  GABAergic neurons degenerative diseases. Methods Dibutyryl cyclic adenosine monophosphate (dbcAMP) was applied to induce human neuroblastoma SH-SY5Y cells,and the SH-SY5Y cells  were  divided into control group(0 mmol?L-1 dbcAMP),0.3 mmol?L-1 dbcAMP group,0.6 mmol?L-1 dbcAMP group,1.0 mmol?L-1 dbcAMP group and 2.0 mmol?L-1 dbcAMP group;the morphological  changes of SH-SY5Y cells were observed.The Image-Pro Plus 5.0 software was used to measure the length of neurites of the neuron-like cells,and the  percentage of the SH-SY5Y cells with neurites longer than 30  μm  was calculated.The immunofluorescence cytochemistry technique was utilized to test GAD65 positive cells,and the positive rate of immune response was calculated.Results The results of light microscope observation showed that the cells in control group were polygonal,circular or shuttle type with smooth membrane and clear boundaries.With the increasing of the  concentrations  of dbcAMP and the prolongation of time,the morphology of SH-SY5Y cells’ bodies became smaller with longer processes in dbcAMP groups.The cells interwined each other and showed mature neuron phenotype in 1.0 mmol?L-1 dbcAMP group.Compared with control group(31.4%±4.2%)，the  percentages of the cell with  neurites longer than 30  μm in 0.3，0.6，1.0,and 2.0 dbcAMP groups  (40.1%±5.7%,47.5%±6.2%,73.1%±3.2%,and 74.3%±6.1%) 72 h after induction were significantly increased(P<0.05 or P<0.01).Compared with control group(10.2%±2.1%)，the GAD65 positive expression rates in 0.3,0.6,1.0, and  2.0 mmol?L-1, dbcAMP groups(22.1%±2.4%,46.9%±3.2%,70.7%±3.4%,and 72.3%±3.7%)72 h after induction  were significantly increased(P<0.05 or P<0.01).Conclusion The SH-SY5Y cells have the potentiality of differentiating into  GABAergic-like cells,and 1.0 mmol?L-1  is the optimal concentration of dbcAMP.

Key words: dibutyryl cyclic adenosine monophosphate, human neuroblastoma cells, induction, differentiation