Experimental Studies Of Effects Of NCAMs On Recovery Of SCI,Construct And Transfect PcDNA4/myc-NCAM140 Into BMSCs

This study firstly focuses on the location and regularity of neural cell adhesion molecules' (NCAMs) expression after spinal cord contusion injury by the methods of an improved version of NYU Weight-Drop Impactor (NYU Spinal Cord Contusion System), which created a spinal cord injury (SCI) model. Sections from the spinal cord were performed to investigate the changes of neural cell adhesion molecules (NCAMs) at different time points by immunofluorescence staining and western blotting after SCI; rhNCAMl/CD56 protein was immediately being administrated into the subarachnoid space after SCI, and through measurements of the spinal cord area, BBB scores, HE staining and NF200, GFAP immunofluorescence staining, and other means to study the effects of NCAM on recovery of SCI. The results showed that NCAMs were located in the lamina I and the surface of lamina II in spinal cord's posterior horn, and the membrane of the central cannal epithelial cells; Furthermore, the strong positive expression of NCAMs can be found in the most of neurons' membrane of dorsal root ganglion (DRG) and dorsal root nerve fibers, but ventral root nerve fibers were negative. The expression of NCAMs was obviously changed after SCI, suggesting that NCAM may be relative to the plasticity of spinal cord; after administrating rhNCAM1 into the subarachnoid space following SCI, we found that NCAM could increase the residual area of injuried spinal cord, increase the expression of NF200 and GFAP, and improve the recovery of locomotive of hindlimbs, which indicated NCAM may play a role in the recovery of SCI. On the basis of NCAM's promoting the recovery of SCI, we also constructed the transgenic bone marrow storm cells (BMSCs) of NCAM gene in order to observe the effects of NCAM140 on SCI. The taken measures were following: mouse NCAM140 cDNA was amplified from total RNA of mouse brain tissue by RT-PCR and cloned into the plasmid pcDNA4/myc, which was simultaneously digested by HindIII and XbaI, then purified, ligased, and transformated to construct the recombinant vector, pcDNA4/myc-NCAM140. After then, the pcDNA4/myc-NCAM140 was transfected into cultured BMSCs in vitro. The BMSCs highly expressing NCAM140 were selected by Zeocin, and the expression of NCAMs and gene marker myc were confirmed by western blot. The results showed that RT-PCR products were matched the expected size by digesting and sequencing; pcDNA4/myc-NCAM140 has been successfully constructed and the inserted DNA sequence was the gene of premature gene of NCAM140 cDNA; NCAM140 and the marker gene, myc, were made to express stably in BMSCs, transfected the pcDNA4/myc-NCAM140. Taken together,the present studuy, on the one hand, explored the regularity of NCAM expression and the effects of NCAMs on the recovery of SCI, and on the other han, successfully constructed transgenic BMSCs of highly expressig NCAM. Thereby, the present investigation provided a new thinking of therapying SCI using transgenic BMSCs implantation for going step further.