The Study Of Expression Change And Functuon Of Spy1in Rat Sciatic Nerve After Injury

ObjectiveTo investigate the temporal-spatial expression of Spy1in in rat sciatic nerve after crush, explore the relationship of Spy1expression and neurites growth and discuss the potential biological role of Spy1in the peripheral nerves injury and regeneration.Methods1. Sprague-Dawley (SD) rats were subjected to sciatic nerve crush (SNC) model. The expression of Spyl mRNA and protein in sciatic nerve were assayed by reverse transcription polymerase chain reaction and Western blot. The location of Spyl in sciatic nerve was detected by immunohistochemistry and double immumofluorescence. The relationship between the expression of Spyl and peripheral nerve injury was preliminary explored.2. We performed co-immunoprecipitation and GST pull-down experiments to understand whether Spyl interacts with SCG10(Superior Cervical Ganglion-10) in vivo and in vitro. Spyl and SCG10truncated mutation plasmids were constructed and transfected to tool cells, immune coprecipitation was performed to detect their interaction domain. The subcellular location of Spyl and SCG10in neuron was analysed by double immunofluorescence staining. We also used Western blot to detect changes of Spyl and SCG10expression in the process of neurites extension. To analyse the effects of the interaction of Spyl and SCG10in the neurites elongation, Spyl was overexpressed in undifferentiated PC12cells and induced by NGF.Results1. RT-PCR results showed that mRNA level of Spyl in rats sciatic nerve increased1d after crush, reached the peak at day3, then gradually reduced to normal level. Western blot results showed Spy1protein levels began to rise1d after injury, peaking in the3d, which was similar to mRNA level. Immunohistochemical results revealed that Spyl expression in crushed and distal segments of sciatic nerve was significantly enhanced. Double Immunofluorescence observed Spy1was mainly collocated with axons marker NF200and axonal regeneration marker GAP43in the crushed segment, mainly collocated with S100marked schwann cells in distal segment.2. Immune co-precipitation and GST pull-down experiments found Spy1can interact with SCG10, Spy1through its Speedy/RINGO box combined with SLD domain of SCG10. Western blot results showed that Spyl expression gradually reduced in the in the process of neurites extension, while SCG10protein expression gradually increased. Overexpression of Spy1decrased the protein level of SCG10and regulated neurites extension.Conclusions1. Expression of Spyl in rat sciatic nerve was upregulated following crush as compared with the normal nerves. These alterations were due to the expression of Spyl increased in axons in crushed segment and SCs in distal segment.2. During the neurites elongation progress, the expression of Spyl was decreased. Spyl participated in axon growth extension by interacting with SCG10.