The Experimental Study That Gene Silencing EphB3Effects On The Rat Spinal Cord Injury Repair And Regeneration

Objective1.By detecting the expression of erythropoietin prime hepatocytes b3receptor （Erythropoietin producing hepatocyte b3receptor,EphB3）at eachtime point of spinal cord tissue in the normal adult rats and spinal cord injurydault rats,the experiment grasps the difference of expression of EphB3in spinalcofd tissue between normal rats.2.Successfully constructed4group of the silence EphB3gene that can besilence and the vector of lentiviral vector (pGCSIL) of SiRNA carrying a redfluorescent protein (Red fluorescent protein,RFP) and a group of out-of-order ofSiRNA. Through the application of SiRNA injury in rat target genes that EphB3silence,blocking gene activity to observe the expression of EphB3and thegrowth associated protein Gap-43,and to discuss the role of gene silencingEphB3repair and regeneration of spinal cord axons.MethodsFirstly,choosing the healthy adult rats,finding the rats EphB3receptor genesequences,screening SiRNA sequence and analysis genome-wide scan andsequence homology.Then synthesis purification Eph B3SiRNA sequence andbuild4SiRNA expression bector lentiviral: EphB3-SiRNA1-pGCSIL-copRFP,EphB3-SiRNA2-pGCSIL-copRFP, EphB3-SiRNA3-pGCSIL-copRFPEphB3-SiRNA4-pGCSIL-copRFP and an out-of-order SiRNA:Negative-SiRNA-pGCSIL-copRFP.Then randomly divide the844-month-old male SD rats into seven groups (n=12),and number them from A to G.Settinggroup A for the normal group,group B for the damage control group.TheC-G,respectively,corresponds to the experimental group EphB3-SiRNA(1-4) andNegative-of-SiRNA.B-G rats were produced thoracic T9spinal cord injurymodel.Observe the1w、2w、4w at all time points in spinal cord Eph B3genereceptor mRNA transcription and protein exptession by RT-PCR and Westenblot.Immunofluorescence lentiviral transfection and immunohistochemicalmethod to observe the Gap-43expression in rat spinal cord injury organizationsaxon.Results1.Protein receptor in spinal cord tissue in the injury control group (groupB)Eph B3gene is highly expressed and the3d time points is lower than theother time points.While Eph B3gene receptor expression is no significantchange in1w、2w、4w（P>0.05）.Spinal cord tissue in the normal group (groupA),Eph B3gene receptor almost no expression.The results between A and B aresignificantly different（P<0.05）.2.In addition to Negative-SiRNA-pGCSIL-copRFP group(group G),theexperimental group(ie:group C,group D,group Eand group F)show that thetranscription and expression of Eph B3are lower than group B by PCR andWestern blot（P<0.05）The group E Eph B3expresses the lowest whichdemonstrates gene silencing is the best.Group G and Group B is basically thesame expression level（P>0.05）.3.The immunofluorescence results show that group C,D,E,F,G lentiviralvector successfully transfect rat spinal cord tissue.The immunohistochemicalresults show growth associated protein Gap-43expression in eachgroup.Normal group A expression is the lowest.Injury control group B andnegative control group G is slightly higher compared with group A(P<0.05).Eachgroup C,D,E,F expression is significantly higher (P<0.05) and group E highexpression of the most significant.Conclusion1.Spinal cord injury caused by Eph B3is long stable and high expression.2.Gene silencing Eph B3helps spinal cord injury repair and regeneration.