Effect Of Neural Stem Cells On Apoptosis Of PC12 Cells Induced By Serum Deprivation

Neural stem cells (NSCs) have been proposed as tools for treating neurodegeneration because of their capacity to give rise to cell types appropriate to the structure in which they are grafted. In the Pheochromocytoma cell line PC12, which has been used for studying the mechanism of Parkinson diseases, apoptosis can be induced by serum deprivation. The potential cytoprotective effects of NSCs on the apoptosis of PC12 cells induced by serum deprivation are of considerable interest of our study. In the study, the NSCs encapsulated in Calcium-Alginate Beads (Ca-Alg Bs) were cocutured with apoptotic PC12 induced by serum deprivation.We assessed the effects of NSCs on PC12 cells viability in serum-deprivation medium by CCK-8 assays to determine whether NSCs can prevent apoptosis of PC12 cells induced by serum-deprivated. The morphological changes of apoptotic PC12 cells were examined by Hoechst 33342 fluorescence assay. The differences of apoptotic cell percentage and cell cycle among the experimental and control groups were detected with AnnexinV/PI and PI analysis by flow cytometry. Eventually, the GDNF concentrations in medium were examined with GDNF ELLIS A kit. At the same time, the process of Ca-Alg Bs dissociated by sodium citrate was optimized.The results are as follows: (1)NSCs can significantly enhance the survival of PC12 cells from serum deprivation. (2)Chromatin condensation and apoptotic body were reduced in NSCs-coculture groups. (3)The majority of cells were detained in Gl phase in serum-deprived groups obviously. The advance of Gl to S phase was obstructed in cell cycle. Whereas in NSCs-coculture groups, the apoptotic cell percentage significantly decreased. The advance of G1 to S and G2 phase was not nearly obstructed in cell cycle. (4)There was GDNF in medium. The secretion capacity of NSCs cocultured with apoptotic PC12 cells induced by serum deprivation was greater than that cultured in NSCs' medium. (5)The optimum conditions of Ca-Alg Bs gelated and dissociated by sodium citrate were determined. On condition of 1.5% (wt%) sodium alginate, 1.5% (wt%) CaCl₂, the gelation time of 10 min and the volume ratio of 55 mmol.L^-1 sodium citrate and solution mixed with 1.5% (wt%) sodium alginate and NSCs' medium of 2:1, the time of dissociation was 7min.These results suggest that NSCs can protect apoptosis of PC 12 cells induced by serum deprivation through secreting nutrition factor such as GDNF. Most likely in the near future the NSCs grafts for PD treatment will be made available.