Lentiviral Vector-mediated RNA Interfere Gene NgR To Repair Spinal Cord Injury

BackgroundThe therapy of spinal cord injury is a lasting tough problem which puzzles medical workers and sociologists, and the regeneration and recovery of central nervous system after injury are always hot spots and nodus in neuroscience research. Now many scholars consider that the main reason of aborting neuaxon regeneration is a lack of central neuron regeneration capacity and the presence of growth inhibitors, and the latter might be the obvious physiological factors suppressing axon regrowth in CNS myelin sheath. These myelin inhibitors include NogoA, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), which induce growth cone collapse and inhibit neurite outgrowth, as expected for inhibitory components of CNS myelin. These myelin proteins are resided in exterior and interior rings of myeline sheath and producted by oligodendroglial cell and some neuron. Recent findings indicate that the Nogo-66 receptor (NgR) is a common receptor mediating inhibition with Nogo-66, MAG and Omgp, and NgR which functional domain has been definite is expressed in many kinds of neuron including cortical neuron, hippocampus neuron, cerebellum Purkinje's cell and tuberculum annulare neuron. On the whole, the distribution of NgR protein is conformity with NgR mRNA. Moreover, researcists find that NgR mRNA is not present in white matter which oligodendroglial cell express Nogo-A. So, they think that NgR in the cinereal mediates nhibition activity of Nogo-A in white matter by specific ligand receptor binding.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 establishs 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 two high-performance RNA interference chi sequence siNgR199 and siNgR964 which gene silencing efficiency is 93.5% and 92.14%. But their effect is transient and interfered with environmental factor easily. If siNgR199 and siNgR964 are designed to little hare cadette frame and cloned into plasmid or viral vector, they might long-term interfere with target gene in neuron after infection. It is trend to apply lentiviral or adeno-associated viral vector which can infect unseparated cell as nerve cell for mediating RNAi to silence target gene in CNS. 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 can take along 9kb gene token and 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. The last, we verified the capability of recombinant about inducing nerve fiber regeneration and promoting functional recovery in rat SCI model.Objective:To construct a recombinant of RNA interference with lentivirus vector and siNgR199; To check the effectivity of the recombinant silencing NgR gene in the rat cortical Cells and optimize the dose and time dependent in vitro ; 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.Secondly, 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.The third, we created a moderate contusion model of SCI in rat with the modified Allen's method, and saline, lentivirus vector and recombinant was injected into hindlimb motor area of cerebral cortex after 7 days, assessing the hindlimb motor functional recovery every week postinjury. 6 weeks after administration, nerve tracer agent was also injected into hindlimb motor area of cerebral cortex .8 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 Lentivirs 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. In morderate rats'SCI modle, 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. 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, but 8 on the time for administration one week after injury. The functional recovery improved significantly in 3 weeks after injury, and slowly 5 weeks after injury. Although all rats were observed to have the hindlimb functional recovery, these rats injected with recombinant had better hindlimb functional recovery than others showing by more BBB score (P<0.01). The BBB scores were no statistical difference between in rats injected with saline and in rats injected with lentivirus vector(P>0.05). Moreover, we finded that some nerve fiber passed the injured region and lesser cavity, 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.