The Study Of Inhibiting Of NOGO Gene Expression By Short Interfering RNA In Repair Of Spinal Cord Injury

BackgroundThe inability of adult mammalian central nervous system (CNS) axons to regenerate can result in the loss of some important functions permanently, while in the peripheral nervous system (PNS), not only axons can regenerate but lost functions resume generally.Interestingly, axonal regeneration will be observed if embryonic peripheral nervous tissue be transplanted to the region of injury, which indicates that the lack of regeneration of CNS is partly attributed to growth-inhibitory molecules present in local environment but not the absence of regrowth.More and more researchers are focusing their eyes on the study on spinal cord injury (SCI) ,because SCI has a high disability rate and treatment of SCI has puzzled researchers for a long time. It is very important to find a available treatment of SCI for society and family. Howere, the restoration of SCI has been a huge task for the human being and no available therapeutic methods has been found till now so that trying to find an ideal method is the common goal for medical staffs. One of the obvious physiological factors for promoting axon regrowth is the presence of growth inhibitors present in CNS myelin sheath. These myelin inhibitors include Nogo-A,myelin-associated glycoprotein (MAG)and oligodendrocyte myelin glycoprotein(OMgp). These myelin proteins induce growth cone collapse and inhibit neurite outgrowth, as expected for inhibitory components of CNS myelin. Recent findings indicate that they inhibit axon growth by binding a common receptor, the Nogo-66 receptor (NgR). NgR is a common receptor mediating inhibition by Nogo-66, MAG and Omgp.NgR is a 473 amino acid protein containing a signal sequence, a leucine-rich repeat (LRR)-type N-terminal domain, eight LRR domains, a cysteine-rich LRR-type C-terminal flanking domain, a unique C-terminal region, and a glycosylphosphatidylinositol (GPI) anchorage site.' The LRR domains of the NgR share moderate amino acid sequence similarity to many other LRR-containing proteins. Because other LRR proteins serve a wide variety of functions, they offer little insight into the mechanism of NgR signaling. This is particularly surprising because Nogo, MAG and OMgp are structurally unrelated proteins. To understand the basis of this multi-ligand binding, the crystal structure of NgR has been solved.NgR is a banana-shaped molecule consisting of short strands that together form a long parallel sheet that spans the concave surface of the molecule. The convex side of the receptor is formed by the loops connecting the. strands and several small helices.RNA interference (RNAi) is the process of sequence-specific posttranscriptional gene silencing triggered by double-stranded RNAs(dsRNAs) homologous to the silenced gene .Small interfering RNA (siRNA) is an extremely effective tool for reducing gene expression of target genes in a variety of organisms and cell types (e.g., worms, fruit flies, plants, and mammalian cells). The ability of transfected siRNAs to suppress the expression of specific transcripts has proved a useful technique to probe gene function, gene knockout, antiviral research, and gene therapy in mammalian cells. The ability to simply, effectively and specifically down-regulate the expression of genes in mammalian cells holds enormous scientific, commercial, and therapeutic potential.We hypothesize that specific inhibition of Nogo-A gene expression via RNAi may promote axonal growth and possible functional recovery after adult CNS injury.Methods:In the first experiment, U6 promotor and the fragment including Nogo-A reverse complement target sequence were obtained by PCR. It was detected by agarose gel electrophoresis and sequencing.Small hair pin coding for interfering Nogo-A was cloned into pDC316, pDC316 vector and adenovirus vector was transfected into 293 Cells. Recombinated adenovirus vector was transfected into oligodend- roglial Cells. To investigate the effect of shRNA knocking down the Nogo-A gene mRNA and protein expression by RT-PCR and Western-Blot.In the second experiment, model of Spinal cord injury using the modified halfway-cuting method that resulted in rats with incomplete paraplegia . Adenovirus vectors incuding Small hair pin were injected zones of Spinal cord injury, detected expression of Nogo-A mRNA by using RT-PCR in vivo.In the three experiment , Adenovirus vectors incuding Small hair pin were injected zones of Spinal cord injury, detected expression of GAP-43 protien by using Western-Blot.Results:We obtained successfully U6 promotor and the fragment including Nogo-A reverse complement target sequence by using PCR.RT-PCR and Western blot analysis were used to detect the Nogo-A mRNA and protein level, we drew a conclusion that the recombinant Adenovirus vectors could significantly inhibited the expression of Nogo-A in oligodendroglial Cells.In rats' SCI modle, we observed that Nogo-A gene expression Were inhabited by using RT-PCR after injection, the GAP-43 mRNA positive expression cells were detected in site spinal cord injury(SCI). The expression of CAP-43 mRNA and protein cells increased rapidly after operation and reached their peak at day 7 after SCI .Conclusions:We demonstrate that RNA interfierence can effectively inhibit Nogo-A replication in cultured rat oligodendroglial cells, the down-regulation in NgR mRNA is sequencespecific, and the RNAi effect is most effective on day 3 post transfection in cultured cells. In rat SCI modle, the level of NgR mRNA and protein was knocked down by the two shRNA. The GAP-43 protein increasing expression could promote axon regeneration of rats after SCI.To sum up, our future work is to investigate Nogo-A signal conduction pathway and other associated genes by multiple gene combination RNAi strategy. We hope to develop gene therapy medicine that inhibits Nogo-A proliferation specifically and effectively.