Inhibition Of Expression Of Nogo-66 By RNA Interferencing In Oligodendrocytes

Spinal cord injury(SCI),whether of traumatic or non—traumatic aetiology, often cause many people paraplegia as a severe result of the devastating consequences of CNS injuries.Until today,there are no available methods to repair the damage of SCI.RNA interference(RNAi)through small interfering RNA(siRNA)technology has become one of the most widely used gene silencing techniques.These nucleic acid-basedsequences have higher reliability,efficiency,and specificity than previous methods.Objective:To study the efficiency and specifity of nogo—66 gene silence in oligodendrocytes of Rattus norvegicus in vitro by small interference RNA.Methods:1.The nogo—66 gene was amplified by PCR,using computer designed NOGO-66 PCR primer.The amplified fragment was then connected with T vector to construct a plasmid for later DNA sequencing.2.The GAPHD siRNA was transfected into oligodendrocytes cells with liposomes,and the GAPDH protein was detected by using Western-blot method,in order to decide siRNA's effective dose and effective time.3.Nogo-66 specific siRNA was brought into target cells by lipo-transfection. The transcription of nogo-66 gene was quantified with FQ-PCR(fluorescence quantitative PCR)and the expression of Nogo—A was detected with Immunofluroescence technique 48h after transfection.Western-blot was used to detect the Nogo—A P-gp at different time spot after transfection.Result:1.Oligodendrocytes SKOV3/TR30 cells were confirmed of highly expression nogo-66 gene by PCR.The plasmid was confirmed by restriction enzyme digestion identification and DNA sequencing,nogo-66 PCR amplified fragment could be used in later experiments.2.The data of GAPDH siRNA's effective dose and effective time were applied to nogo-66 siRNA transfection,nogo-66 cDNA PCR result 48h after transfection showed that inhibition was positive.3.FQ-PCR result showed the amount of nogo-66 mRNA in silenced cells was about 0.24%of that in not treated ones(p＜0.001).While the GAPDH amount of both cells were almost the same(P＞0.05).No fluorescence could be seen on silenced cells' membrane.The most effective time of silencing was 48h after transfection seen from Western-blot of P-gp.Conlusion:nogo-66 mRNA sequence specific siRNA could significantly and specifically inhibit nogo-66 mRNA's expression.RNA interference technique is a potent assistant therapy of SCI.