Investigation Effects Of NgR On Microglia And Axon Regeneartion In EAE Model By Ad-shRNA-NgR Interfering

Objective Multiple sclerosis (MS) is a inflammatory demyelinating disease in central nervous system (CNS). With the development of magnetic resonance (MRI), The incidence of MS is increasing. The pathogenesis of MS is unclear. Furthermore there is still no good way to treat this disease. So to search for a new method for nerve protection in this disease is very important. EAE is a classical model of MS. It can simulate MS no matter in clinic or in pathology. Nogo receptor is the same receptor for MAG, OMgp and NgR. The three proteins above are the inhibitors for axons regeneration in CNS. Right now some projects indicate that NgR may participate in inflammatory. And in the CNS tissue of MS, the expression of NgR is increased. It seems that NgR may play some role in MS. RNAi can reduce the expression of objective gene. Ad-shRNA-NgR can down regulation the expression of NgR. Microglias is the principal member of macrophage family in CNS. These cells might sustain and propagate inflammation within the CNS during autoimmune inflammation. So by transfecting the Ad-shRNA-NgR into the experimental autoimmune encephalomyelitis (EAE) model'lateral cerebral ventricle and observation the microglias and remyelination could help us to find the effects of NgR on microglias and remyelination.In this research, after transfection the Ad-shRNA-NgR into the experimental autoimmune encephalomyelitis(EAE) model'lateral cerebral ventricle, we investigated:the expression of nogo receptor(NgR)mRNA and protein, the aggregation of inflammation cells, the depletion of myelin sheath and the clinical invasion of the EAE model. By doing this we can find the contribution of NgR myelin regeneration and microglias. This project can provide a therapeutic approach for MS and other inflammation diseases of neurology.Method 1.Established the EAE model and MRI scanning, the dyeing of HE, Luxol fast blue, microglias were carried out for evaluation.2. The recombinated adenovious was amplified in 293 cells(human embroyonic kidney cells)and purified by Sartorius Vivapure AdenoPACK 20 kit.The virus titer was determined by TCID50. Three different titers of recombinated adenovious(high titer, middle titer, low titer) were used to transfect the brain tissue of EAE. Histopathological examination was performed to evaluate the local inflammatory reaction. And green fluorescent protein (GFP) expression was observed under fluorescence microscope. The optimal transfective virus titer was selected;3.48 female Wistar rats were randomed into normal control group,blank control group, Ad-shRNA-HK intervention group(negative control group), Ad-shRNA-NgR intervention group (specific RNAi group). Reverse transcription polymerases chain reaction (RT-PCR) was used to determine the changes of NgR mRNA expression at the 7th and 14th day after transfection.The stain of HE,Luxol Fast Blue,NgR and CDllb/C was also carried out at the 7th and 14th day after transfection.Result 1.The MRI scanning and pathological observations of the rats were consistent with those of classical EAE model.2. After 24 hours of transfection the fluorescence was observed in 293 cells. The titer of amplified recombined adenovirus Ad-shRNA-NgR and Ad-shRNA-HK were 2.93×1010pfu/ml and 2.84×1010pfu/ml respectively. Green fluorescence protein(GFP) could be found in the brain of the high, middle and low titer group at the 7th day of transfection. GFP expression in the high and middle titer group was obviously higher than that in the low titer group. While there was no obvious difference between the high and middle titer group. HE staining showed that acute inflammatory infiltration was only observed in the high titer group;3. NgR protein in the brain of EAE was significantly increased at the 14th and 21th day after induction (p<0.01). NgR mRNA and protein was reduced in the brain of EAE at the 7th and 14th day after RNAi intervention (p<0.01);4.Compared with the blank control group, the Ad-shRNA-NgR specific intervention group had longer latency (p<0.05),lower clinical score;at the d21,the more significant regeneration of myelin sheath, while the slower extinction of inflamation.Conclusion The EAE model was induced successfully. The middle titers of adenovirus can transfect the brain tissue of EAE model efficiently and safely. The expression of NgR was increased in the brain of EAE. Ad-shRNA-NgR interfering can degrade the expressing of NgR remarkably and enhance the regeneration of myelin sheath, but it's not good for extinction of activated microglias.