The Effect Of Lentivirus Mediated SiNgR-199 Cortical Injection After Spinal Cord Transection In Rat

BackgroundThe axon is hard to regenerate after spinal cord injury. and it's the most important reason that the functional recovery is very limited. Now,most researchers belive that it is mainly for the inhibitors in the microenvironment that play an important role inhibiting the axonal regeneration ability.Recent studies shows that the myelin-associated inhibitors is one of the crucial factor to inhibit axonal regeneration of CNS(central nervous system).It was found through research that the inhibitors of growth present in CNS myelin sheath, these myelin inhibitors include NogoA (also known as reticulon 4), MAG (myelin-associated glycoprotein) and OMgp (oligodendrocyte myelin glycoprotein) inhibit neurite outgrowth. Further studies indicated that these myelin inhibitors bind a common receptor, the Nogo receptor (NgR). NgR is a neuronal specific receptor with high effect and affinity by binding Nogo-A, MAG and Omgp,mediating the inhibition of axonal regrowth and induction of growth cone collapse. Being the convergence of the three myelin inhibitors, suppressing Nogo receptor protein can decrease Nogo-A, MAG and Omgp's inhibition at the same time. In the research of the myelin-associated inhibitors in CNS,anti-NogoA antibody IN-1 , myelin or associated cDNA vaccine and a NogoA-derived peptide, NEP1-40, have been delivered to rats with spinal cord injury.Functional recovery and axonal regeneration were improved after treated with them. But there are some deficiencies in these studies, IN-1 only inhibits Nogo-A, myelin or associated cDNA vaccine can not pass the blood brain barrier efficiently. Meanwhile,other myelin inhibitors'function can not be decreased in this method. Although NEP1-40 can decrease Nogo-A, MAG and Omgp's inhibition at the same time, it is not stable enough in the environment,and effect as a transient way.If Nogo receptor gene expression can be suppressed, we can find a attractive strategy to avoid deficiencies in IN-1, vaccine and NEP1-40 treatment .A gene therapy ,RNA interference, known as the small RNA induced gene silencing is a possible method. RNA interference (RNAi) is a post transcriptional gene silencing process targeting homologous mRNA for degradation,and induced by Double-stranded RNA (dsRNA). In this process, targeted gene can be transcripted as normal,but mRNA can not be accumulated for it's degradation.It is reasonable to suppressed axonal regeneration inhibitors'gene or silencing NgR gene to suppress myelin inhibitors'function,especially the latter is a good choice. Recent studies shows that RNAi is effective at suppressing specific gene expression in a number of primary cells including neurons. In 2000, Krichevsky and Kosik found that the rat hippocampus and forebrain can be effectively suppressing endogenous and heterologous genes induced by small RNA interfering. In 2003,Hommel and Sears knocked down localized gene, encoding the dopamine synthesis enzyme tyrosine hydroxylase in the brain, by using viral vector-mediated RNA interfering.These experiments indicated that neurons can get gene silence by RNA interfering.The condition of axonal regeneration after SCI is that the neuron can express growth gene and be supplied with neurotrophy and axon growth substantia basilaris, and inhibiting factor of axon growth must be prevented. Therapeutic regimens about SCI might be foetus neural graft, peripheral nerve tissue or cell graft, and gene therapy. Foetus neural graft is restricted by poor source and ethics problem though foetus neural graft has a good therapeutic effect to SCI for low rejection and strong fissionability. Peripheral nerve tissue has a poor prostecdtive efficacy to SCI for high rejection. Gene therapy has a lower rejection than peripheral nerve tissue and can act a long-term effectiveness after single therapy. For this reason, the most of scholar consider that gene therapy is one of the best Therapeutic regimens about SCI .RNA interference (RNAi) is a post transcriptional gene silencing process targeting homologous mRNA induced by Double-stranded RNA (dsRNA), and it can specially silence internal source or extrinsic source target gene. Nowadays, RNA interference has been applied with many kinds of creature including plants, eumycete, virus, mammalia. Applying vitro synthesis siRNA is the latest development of RNAi and establish a foundation for RNA drug treatment. In mammalian cell, vitro synthesis siRNA can specially degradatiohn homologous mRNA and silence target protein expressing, and can prevent from non-specificity degradation and cell death.In our prophase study, we had successfully inhibited the expression of endogenous NgR genes effectively in cultured rat neuron by applying chemical synthetic siRNA, and sieved out a high-performance RNA interference chi sequence siNgR199. Then we designed siNgR199 to little hare cadette frame and cloned into plasmid or viral vector, they might long-term interfere with target gene in neuron after infection. The plasmid has low transducing rate. Lentiviral vector is more suitable to infect unseparated nerve cell than adeno-associated viral vector and other viral vectors because its high tropism to stem cell and other cells in vitro culture. Lentiviral vector has powerful tropism to neuron but no counter transport ability. As a vector, Lentiviral vector is perfect to mediate RNAi in CNS.In this study, we first constructed lentivirus siNgR recombinant to optmize the process of synthetizing and transmitting siNgR. We selected siNgR199 as homologous NgR RNA succession, because it had the greater specific effect on NgR gene silence. Then, we applied recombinant to infect rat cortical neuron in vitro for checking the effectivity of recombinant silencing target gene. At last, we verified the capability of recombinant about inducing nerve fiber regeneration and promoting functional recovery in rat SCI model.Objective:1.To construct a recombinant of RNA interference with lentivirus vector and siNgR199;2.To check the effectivity of the recombinant silencing NgR gene in the rat cortical Cells and optimize the dose and time dependent in vitro ;3.To investigate the effect of the recombinant in inducing nerve axon regeneration and promoting functional recovery of nerve in rat SCI model.Methods:First, we selected siNgR199 which was designed into a short hairpin RNA construction as homologous NgR RNA succession, and cloned it into lentivirus vector using the E1bashir's criteria and the RNAi vector rule; Then, we evaluated the recombinant by enzyme cutting and gene sequencing test.Second, we introducted recombinant into the cultured nerve cell from the rat cerebral cortex with different concentrations, studying the transfection of recombinant by observing the marker cGFP gene expressing, evaluating the effect of suppressing NgR gene with real-time PCR. Third, we created a transection model of SCI in rat, and saline, lentivirus vector and recombinant was injected into hindlimb motor area of cerebral cortex after 7 days, 8 weeks after administration, nerve tracer agent was also injected into hindlimb motor area of cerebral cortex .Fourth, every week postinjury we assessing the hindlimb motor functional recovery.9 weeks after administration, the rats were killed for histologic studies after the last assess of the functional recovery.Results:1. We had successfully constructed a recombinant targeting NgR-specific siNgR199 with Lentiviral vector. The recombinant molecular weight and gene sequence is correct by checking with enzyme cutting and gene sequencing test.2. we introducted the recombinant into the neuron cultures, finding that the marker cGFP gene began to express 96h after transfection, and expressed powerfully 146h after transfection. The best MOI value for effective transfection is 3. NgR mRNA had been suppressed 61% at 192h after transfection by checking with real-time PCR test.3. we finded that NgR mRNA expressed more little in rats injected with recombinant than in rats injected with saline or lentivirus vector 8d after administration, and NgR masc-grana was also more little in rats injected with recombinant than other groups. There is statistical difference between them(P<0.05).These results demonstrated that NgR protein was suppressed effectively by injecting recombinant in vivo.4. We assessed the functional recovery using BBB score, all rats was 0 in the first day postinjury, until 5 weeks after injury,it became 3. The functional recovery improved slowly in the last 2 weeks finally up to 8. All rats were observed to have the hindlimb functional recovery, and the BBB scores were no statistical difference between each group(P>0.05). Moreover, we finded that some nerve fiber passed the injured region and more medullary sheath and glial cell were present in the spinal cord injured region in these rats injected with recombinant.Conclusions:We had successfully constructed a recombinant targeting NgR-specific siNgR199 with Lentivirs vector. The recombinant can effectively suppress NgR mRNA expression in rats'cortical nerver cells in vitro or vivo, and can promote neurological functional recovery and axon regeneration in rat SCI model.which indicates that lentivirus mediating RNAi may be a potential therapeutic method for SCI. The possible mechanism is that the recombinant stably express siNgR199 in cortical nerver cells, and long-term induce silencing NgR gene, decreasing the combination of NgR with Nogo,MAG and OMgp, which result in promoting the regeneration of axon at the lesion site after SCI.