The Expression Of Regeneration Gene Protein-2 (Reg-2) In Spinal Cord Traverse Injury Model

【Background and Objectives】Regenerating gene(Reg)protein is a inflammatory protein isolated from the pancreaticjuice of rats with pancreatitis and belongs to calcium-dependent phytohemagglutinin superfamily.It represents a group of secretory protein,which function is equivalent of acute reactive protein,anti-apoptotic factor,or pancreaitcβcell and nerve cell growth factor.Reg protein family was first identified in diabetes research,and the important role that the Reg protein played in nerve system injury have gotten more and more attention in recent years..Reg protein has four family members:Ⅰ,Ⅱ,Ⅲ,Ⅳ.Reg-2 of RegⅢprotein is a stress protein,previous studies in disease of digestive system have showed that Reg protein may have anti-apoptosis,promote mitosis,anti-inflammatory and protection organizations. Reg-2 did not express in normal adult rats central and peripheral nervous system,but in the development of the central nervous system and peripheral nervous system injuries,diseases and other conditions,Reg protein expression will increase.Current research on Reg-2 only focused on the development of the central nervous system and peripheral nerve injury,but not on the central nervous system following injury.The investigation of the distribution and expression of Reg-2 protein in the central nervous system during traumatic condition,and the relationship of Reg-2 protein and other nerve factors,may further open out the role that Reg-2 protein have played in the central nervous system regeneration.Our study used immunofluorescence staining,double immunofluorescence staining,and Western blotting semi-quantitative method to detect the expression of Reg-2 protein in the central nervous system after injury and the relationship of Reg-2 protein and other nerve factors,speculated Reg-2 protein role on the process of nerve regeneration.【Materials and Methods】1.Experimental animals and Model of spinal cord injuryFourty five female SD rats,weighing 250-300 g,were used in our study.The model of spinal cord injury was produced by complete transection of the cord at the 9th thoracic level. The spinal cords were taken out in 1h,1d,3d,7d,14d after spinal cord injury(SCI)and cuted in cryostat transverse sections.2.Immunofluorescence staining and double immunofluorescence staining after spinal cord injurySections were incubated with mouse-anti-Reg-2 antibody and FITC-conjugated goat-anti-mouse antibody,observed under fluorescence microscope.Sections were incubated with mouse-anti-Reg-2 antibody,rabbit-anti-GFAP, rabbit-anti-Olig2,rabbit-anti-NPY,rabbit-anti-CGRP and rabbit-anti-GAP-43 antibody,and then incubated with FITC-conjugated goat-anti-mouse antibody and TRITC-conjugated goat-anti-rabbit antibody,observed under fluorescence microscope.3.Western blottingThe spinal cords were taken out in 1h,1d,3d,7d,14d after SCI.After homogenation, they were taken to PAGE,and transferred to PVDF membrane.Membrances were incubated with mouse-anti-Reg-2 antibody,biotinylated goate-anti-mouse antibody and ABC complex, respectively.【Result】1.The results of immunofluorescence staining Reg-2 in the normal spinal cord did not express.The expression of Reg-2 were found at lh after SCI,reached peak on 3 days after SCI in neurons of dorsal horn and 7 days in neurons of ventral horn.The high expression persisted for 1 week,then decreases gradually.2.The results of double immunofluorescence staining after spinal cord injuryAfter SCI,the expression of Reg-2 with CGRP,NPY and GAP-43 existed co-localization;Olig2 appeared obvious expression in the myelin around Reg-2 postive cells; Reg-2 and GFAP did not exist obvious co-localization.3.The results of western blottingReg-2 in the normal spinal cord did not express.The expression of Reg-2 were found at 1h,reached peak on 3 days and to a minimum level on 7 days after SCI.The expression persisted for 2 weeks。【Conclusions】1.In the early stage,the expression of Reg-2 neurons mainly existed in the small cells, gradually extended to the large cells,and the position of expression of shifted from dorsal horn to ventral horn,from sensory neurons to the motor neurons.2.After SCI,the expression of Reg-2 with CGRP,NPY and GAP-43 existed co-localization;Olig2 appeared obvious expression in the myelin around Reg-2 postive cells; Reg-2 and GFAP did not exist obvious co-localization.