The Study Of Inhibiting Of RhoA Gene Expression By Short Interfering RNA In Repair Of Spinal Cord Injury

Spinal cord injury (SCI) can be divided into the primary lesion and the secondary lesion, as the former is irreversible, so the focal point of the study and treatment of SCI is to prevent the secondary lesion, remain the surplus nervous function and promote nerve regeneration. It has been thought that after the injury of the central nervous system (CNS), its recovery is impossible. However in recent years, with the development of molecular biology and cell biology, it has been certificated that the regeneration of CNS is possible. It is believed that one of causes to defect axon regeneration is there are inhibitors in the local surrounding. Now it has been confirmed that Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgP) are the most inhibitors. They have the same receptor NgR. Myelin-derived inhibitory proteins, as well as other classes of known growth inhibitory proteins, block regeneration by signaling activation of Rho with the P75NTR involving.Rho GTPases are a family of highly related proteins that are present in all cells as important signaling switches.Rho is one member of small molecule GTPases supperfamily associated to ras. Rho GTPases include three members,Rho,Rac and Cdc42.They are related to nerve growth. When rho is active, it binds to and activates several protein kinases.One downstream target of Rho, Rho kinase(ROCK) is a key regulator of neurite growth. When ROCK is activated, it induces neurite retraction.RNA interference (RNAi) is the technology of gene silence of post-transcription, small or short inference (siRNA) can target some kind of monitoring progress of post-transcription, identify mRNA which has the homology sequence, the specifically cut the mRNA and obstruct its interpretation function. In our experiment, we silence RhoA by the way of RNA interference, effect the expression of RhoA, promote the axon regeneration and recover the nervous function. So we provide a new strategy aiming and approach for SCI.I Methods:In vitro experiment, we design two short hairpin sequences associated with RhoA gene of rat and make them link with plasmid vector pSilencer3.1 H1 Hygro to synthesis recombination plasmid. Then make them accept sequencing. At the same time we cultivate the oligodendrocyte of rat. Make recombination plasmid transfect into oligodendrocyte. Using Real time RT-PCR and Western Blot processes check the change of RhoA at mRNA and protein level. Then we evaluate the interference's result of the shRNA.In vivo experiment, at the first, we recombine retrovirus with the shRNA whose interference result has been confirmed the better. According to improved Allen's way, we manufacture the spinal cord injury model of rat, and divide them into two group random.Treat group are 15 and untreat group are 15.And choice 15 rats which have not been injured as normal group. At the 1,4,7,10,13d,three rats every group are executed random.Get the injured spinal cord and then make Real time RT-PCR, Western Blot and immunohistochemistry processes. Meanwhile, using Western Blot, we detect GAP-43 which is a special protein associated with axon regeneration.II Results:In vitro, we cultivate the oligodendrocyte of rat successfully. Through sequencing, we constructe two interference plasmids associated with rat's RhoA gene. After the recombinated plasmid transfecte into oligodendrocyte, the result of real time RT-PCR display that mRNA of RhoA gene has descent tendency, and at the seventh day it get to the lowest, then it has ascent tendency. The maximum inhibition ratio is 77 percent. Meanwhile,Western Blot proofes that the expression of RhoA protein has descent tendency, and at the seventh day it get to the lowest.In vivo, we manufacture the injury model of rat. We recombine retrovirus with the shRNA whose interference result has been confirmed the better, and get recombined retrovirus which can inhibit the expression of RhoA gene. We inject the recombinated retrovirus into the injured spinal cord.Western Blot and PCR progresses indicate that the expression of RhoA gene has descent tendency from the first day 96.74 percent to the thirteenth day 55 percent. the expression of RhoA gene of untreatment group has ascent tendency. Compared with untreatment group and normal group,the expression of treatment group has statistic difference,P<0.05. And the depression to RhoA may maintain at least 13 days which is our experimental limit. At the same time, compared with untreatment group, the expression of GAP-43 protein of treatment group increases. The data of two groups has statistic difference, P<0.05.III Conclusion:Targeting to RhoA, we construct the short hairpin RNA to RhoA gene successfully. And we demonstrate that RNA interference can effectively inhibit RhoA replication cultured rat oligodendroglial cell, the down-regulation in RhoA mRNA is sequence specifically, and the RNAi effect is the most effective on the seventh post transfection in cultured cells. In rat SCI model, the level of RhoA mRNA and protein is knocked down by the shRNA. The GAP-43 protein increasing expression could promote axon regeneration after SCI.To sum up, our future work is to investigate RhoA signal conduction pathway and other associated genes by multiple gene combination RNAi strategy. We hope to develop gene therapy medicine that inhibits RhoA proliferation specifically and effectively.