Differentiation-inducing And Protective Effects Of Adult Olfactory Ensheathing Cell Conditioned Medium (AOCM) On PC12 Cells

Olfactory system is the few parts of the adult mammalian central nerve system where neural regeneration occurs all through life. OEC is a kind of special glia located exclusively in the olfactory bulb, olfactory epithelium and olfactory nerve. It shares many properties of both astrocytes and Schwann cells. During development, it enters into the olfactory bulbs accompanying the growing olfactory axons, therefore, playing a supportive, neurotrophic and guidance role for the outgrowth of olfactory axons. Recently, transplantation of adult OECs into the injured sites of CNS has successfully enhanced nerve fiber regeneration. However, the underlining mechanism remains partly speculative. Transplanted adult OECs may exert their effects via cell-cell interaction as well as by releasing various factors, the latter, however, has not been fully substantiated by experimental data. The presentstudy was, therefore, taken to investigate whether AOCM is beneficial for the widely used neuronal cell model: PC 12 cells.We purified adult OECs from primary culture, and prepared conditioned medium as stimulating factor. In the first part of our studies, we observed the morphologic changes of PC12 cells respectively after 24h, 48h, 72h's culture in AOCM, the expression of neuronal specific protein (3-tubulinIII after 3d's culture, and the membrane excitability after 6d's culture. The results show that PC 12 cells quickly undergo morphologic changes in AOCM. At 24h, 48h, 72h' culture, the percentages of cells having process(es), long process(es) and branch(es) in experimental groups are significantly higher than those in controls. After 3d's culture, the expression of p-tubulinlll is significantly higher than that in controls. The membrane excitability of PC12 cells in experimental group is enhanced after 6d's culture as action potential can be elicited by depolarizing stimulus. The results suggest that AOCM can induce PC 12 cells to differentiate.In the second part of our studies, we first observed the effect of AOCM on the viability of PC 12 cells via MTT assay. Then we investigated the protective effect of AOCM on the Zn2+-induced cell mortality and ultrastructural changes of PC 12 cells, using Heochst33342 staining, typan blue staining, and transmission electron microscope. The results show AOCM can enhance the viability of PC 12 cells and reduce Zn2+ induced cell death. With zinc sulfate treatment, most of the PC 12 cells are necrotic in terms of the ultrastructure changes, some are degenerative, and few apoptotic. With AOCM treatment, the ultrastructures of most PC 12 cells remain normal. Few necrotic, degenerative and apoptotic cells can be seen. The results furtherprove that AOCM can protect PC12 cells against zinc injury.All the results of present study show that the factors secreted by the adult OECs can promote PC12 cell differentiation and protect it from Zn2+insult. Our results support the possibility that the adult OECs can release factors beneficial for neuronal survival and neurite outgrowth, as a part of the mechanism of enhancing CNS regeneration following their transplantation.