Effects Of FK506, Through Which Promotes Schwann Cells Expressing Slit2, On Neuron Axons Growth

Objectives:To observe whether FK 506 was able to make schwann cell ,which originated from newborn SD rat DRG,synthesized and secreted nerve growth guidance factor Slit2.So that FK 506 could promote neuite branching and formate effective links between neurite.we hoped that the results could provide an experimental basis for studying the mechanism of FK 506 nerve regeneration.Methods:Firstly,we established and identified the co-culture system of DRG neurons and Schwann cells,and then added different concentrations of FK506 in cell culture medium. Secondly,using RT-PCR determined the expression of Slit2 mRNA in schwann cells,as well as GAP-43 mRNA in neurons.Thirdly,applicating fluorescence techniques observed neurons bran- ching and the link between neuritis.Forthly,using MTT method studied growth and vitality of neurons and schwann cells in the coculture sy- stem.Finally,using the Flow-cytometry determinated the number of survived neurons in the cell culture system.Results:Cells was cultured in different concentrations of FK506 for 48 hours.RT-PCR results showed that the expression of Slit2 mRNA in schwann cells and GAP-43 mRNA in neurons from FK506 1nM,10nM, 100nM group was significantly higher than the control group(P<0.05).Be- sides the expression of Slit2 mRNA in schwann cells and GAP-43 mRNA in neurons was the highest ,when the concentration of FK506 was 10nM.The results of immunoflurescence showed that Neurite length ,neurite bran- ches,as well as the network of inter-neurite from FK506 1nM,10nM,100nM group is better than control group.The best results came from 10nM group.MTT results showed that neurons and schwann cells got the best vitality(P<0.05) in FK506 10nM group,Fk506 1nM group seconded to it,and the other two groups is the last.And there was no statistically significant difference between control group and FK506 100nM group (P>0.05). Flow-cytometry results showed that neurons survival proportion in FK506 10nM group is the highest(P<0.05).Although FK506 1nM group seconded to it ,there was no statistically significant difference between the two groups(P>0.05). The other two groups is the last.And there was no statistically significant difference between control group and FK506 100nM group(P>0.05)Conclusion:1. FK 506 could make schwann cell synthesized and secreted nerve growth guidance factor Slit2.There was"dose-effect"trend ,when FK506 played the role of nerve regeneration. It was not conducive to the growth of Schwann cells and the secretion of nerve regeneration factor ,when the concentration of FK506 was too high or too low.2.The extension of neurite,branching of neurit and establishment of inter-linked between neurite could be promoted by FK506 through Slit2.3.The role of nerve regeneration,which was played by FK506,depended on the formation of renewable micro-environment that was created by Schwann cells.