The Study Of Inhibiting Of Mouse Oligodendrocyte OMgp Gene Expression By Short Interfering RNA

Background:As part of central nerve, optic nerve is different from spinal cord, it is composed of axon of optic ganglion and glial cells of optic nerve, doesn't contain neuron, that means optic nerve is composed of white matter, which is less advantage for axon growth compared with gray matter. As a result, it is more difficult for the injured optic nerve to regenerate than other central nerves. It is considered the reason that mature central nerve system lacks regeneration ability is mainly because of the protein inhibition caused by glial scar and Myelin. The latter includes Nogo-A, OMgp and MAG, they function all or in part by NgR. In 1987, Vidal et al cut optic nerve of adult rat, stitched a strand of sciatic nerve in between the cutting ends, and observed that the optic nerve axon grew along the grafted sciatic nerve and extended to superior colliculus. While similarly cut the sciatic nerve, and stitched by optic nerve, no similar regeneration was observed.Nogo-A and MAG are widely existed in central nerve system and peripheral nerve syste, while ligodendrocyte-myelin glycoprotein (OMgp ) only exists on the surface of neuroglial cells. It has been recently confirmed that OMgp has strong inhibition effects on axon regeneration[4]. These phenomena shows that, (1) optic nerve has weak ability to regenerate; (2) Nogo-A, Omgp and MAG existed in the micro-environment of central nerve are main reasons for central nerve regeneration difficulties, among them OMgp is exclusive to central nerve; (3) the optic nerve composed of white matter provides unique environment for Nogo-A, OMgp and MAG study. If optic nerve could be regenerated, then it will not be a big challenge for other central nerve regeneration.As a central nerve growth inhibitory factor, more study were focused on Nogo, while much rarely on OMgp. NgR seems to be the center for multiple inhibitor functions in central nerve system[5], so blocking NgR will bring many problems, makes it difficult for actual application. Therefore, we decided to study the relationship between OMgp and optic nerve regeneration.OMgp is one of the axon growth inhibitary protein, Kevin et al[4] confirmed that OMgp may function by Nogo receptor (NgR), Domeniconi also proved that after OMgp interatcts with NgR and develops complex, then it binds with PKC dependant P75 receptor, to inhibit the regeneration effect of injured axon. OMgp is only expressed in CNS, existed in much higher quantity in white matter than in gray matter. OMgp is a glycoprotein, with the relative molecular weight of 105,000, includes 440 animo acides, ancored at out layer of myelin sheath by glycosyl-phosphatidyl-inositol (GPI). OMgp was firstly recognized as a GPI coupling protein which is expressed on oligodendrocyte and neuron to combine Phytoagglutin, but until recently, it is confirmed as an important axon inhibitor in myelin sheath.RNAi nterference (RNAi) is a phenomenon that in vitro synthesize or in vivo short double stranded RNA (dsRNA) could induce the degradation of homologous RNA, and silence the corresponding gene. Because the RNAi acts on RNA level, it is also called posttranslational gene silence. RNAi can inhibit the corresponding gene expression, present the cell a phenotype with specific gene function loss, and abolish its biological effects. Gene knockdown technique is time-consuming and expensive, also it can inactivate the gene completely, makes it hard to use in treatment, and also difficult to study the dynamic changes of gene in different stages of the whole life cycle. While RNAi is highly efficient and specific, could recover gene activity after it's inhibition, shows enormous advantage in treatment.Because optic nerve and other partial central nerve system could not express OMgp when injured, and in vitro cultured optic nerve oligodendrocyte could not express endogenous OMgp, the present study expressed exogenous OMgp in optic nerve oligodendrocyte and cotransfected with chemically synthysized OMgp specific hairpin structure, to inhibit the exogenous oligodendrocyte ligodendrocyte-myelin glycoprotein. The results shows that the system designed in present study is effective, exogenous OMgp expression level decreased 51% by target gene and hairpin structure cotransfection. The next step is to introduce the identified PmU6-shRNA-Omgp into optic nerve injury model, and study OMgp function by inhibiting endogenous OMgp expression in optic nerve oligodendrocyte, and the role it plays in injured optic nerve axon, to resolve the difficulty involved in injured optic nerve regeneration. Objective:OMgp can inhibit axon regeneration of injured mammal central nerve system, so we study the function of OMgp in promoteing the regeneration of mammal central nervous system inhibition of OMgp expression.Methods:The present study was designed to answer this question. We constructed shRNA expression vector pmU6 driven by RNA polymerase III promoter. Firstly, we chose a interference target site within EGFP, and constructed EGFP specific shRNA expression vector mU6p-siEGFP; then PCR amplification hairpin structure containing U6 promoter, transfected into oligodendrocyte, and finally analyzed the protein expression level by restriction enzyme digestion and western blot. The results confirmed that the constructed pmU6 had the expected sequences, and chemically synthesized EGFP hairpin structure could effectively silence target gene. Secondly, we transcribed vector pMU6 to construct OMgp and GFP fusion protein, and detected gene expression by fluorescence microscope and Western Blot, to study the inhibition effects of siRNA on OMgp in mammal oligodendrocyte, and to lay a basis for the study of mice optic nerve axon injury regeneration. Finally, we chose two interference targets in OMgp gene, constructed OMgp specific shRNA expression vector mU6p-siOMgp; PCR amplification hairpin structure with U6 promoter, transfect into oligodendrocyte and analyze protein expression level by Western Blot.Results:The results showed that, we constructed OMgp gene targeting RNAi hairpin structure recombinant with U6 promoter, after being transfected into oligodendrocyte, protein expression level of Omgp gene was decreased dramatically, the relative inhibition rate at 48h post transfection was about 51%. The present study inhibited OMgp expression by RNAi technique, and confirmed that if Omgp expression was inhibited, it could provide an effective system for the study of the relationship between OMgp and mammal optic nerve axon regeneration.。Certainly, as the representative of central nerve, injured optic nerve regeneration is affected by many factors, such as internal environment changes, various factors effects. Some are promotion factors, some are inhibition factors. That means the inhibition or promotion of only one or several factors cannot resolve the whole regeneration process, but only if all relevant factors involved in optic nerve injury are fully illuminated, a more profound understanding of optic nerve regeneration could be realized. OMgp is well known as an important optic nerve growth inhibitory factor, and its expression level changes dramatically before and after injury, but still very little is known on it function. The present system is designed to study the role of OMgp expression inhibition in oligodendrocyte played in optic nerve regeneration. Optic nerve regeneration is a world-wide challenge, many reports were done in this area, but further study are still needed to illuminate the mechanisms involved in optic nerve neurotrophic factor and neuron growth inhibitor. Need to mention that, same as other central nerve regeneration, optic nerve requires a complete in vivo atmosphere, so various factors should be included in experiment. All in-depth study on relevant factors will provide strong theoretical support for optic nerve regeneration, but central nerve system exclusive OMgp means more for the improvement of optic nerve regeneration theory.