Effects Of ShRNA Against Lingo-1 Gene Expression On Repair Of Spinal Cord Injury

In recent years, the incidence of Spinal cord injury (SCI) has risen year by year. It has already become the key factor of functional disability. The nerves functional recovery is very difficult after SCI because of deficiency of neurotrophic factor, necrosis of nerve cell, growth of nerve axon slowly, and glial scar, et al. At same time, the impaired axon is surrounded by the axon growth inhibiting factors, which include Nogo-A, MAG, and OMgp. The GIFs educe effect through a same acceptor (NgR), which Lingo-1 is important part of. After RNA interference (RNAi) is found, it offers a new approach to restrain gene expression. RNAi is belong to post transcription gene silence, the isogenesis sequence transcribed by endogenic and exogenous double stranded trigger target gene can degrade and decompound the function of mRNA. In the world, there are seldom reports about cure SCI by RNAi inhibiting LINGO-1.The aim of this experiment is to promote the reactivate of spinal cord by adenovirus-mediated shRNA restrained LINGO-1 expression.PartⅠConstruction and recombination adenovirus vector of rAd LINGO-1According to principle of RNAi, we design the homologous target , synthesiz the primer of LINGO-1, synthesiz U6 contained shLingo-1 with PCR, connect the production to T trager, and introduct the last production to competent. Sequencing effects indicate that pTU6shLingo-1 is right. We use AdMax? Adenovirus Creation System to construct the rAd Lingo-1. rAd Lingo-1 (1),rAd Lingo-1 (2),rAd Lingo-1 (3) and rAd Lingo-1 (4) titer were 10 8.0 TCID50 /ml,10 8.2 TCID50 /ml,10 9.5 TCID50 /ml,10 8.14 TCID50 /ml respectively.PartⅡExperiment of rAd Lingo-1 transfection rat spinal cord Experiment in vitro: We culture the primary rat spinal cord oligodendrocyte as aim cell invitro and introduct the aim cell to rAd Lingo-1. The basic control group is not introduct viral vector ,the non-specificity inhibition control group is introduct incorrelated shRNA,the intra reference isβ-actin .The change of Lingo-1 mRNA level and protein level is semiquantitatively surveyed by RT-PCR and Western Blot.We conclude that compared with Lingo-1 (4), the inhibition ratio of rAd Lingo-1 (1),rAd Lingo-1 (2),rAd Lingo-1 (3) is 60%,80.5%,22.7% by RT- PCR。The effect of Lingo-1 inhibited by rAd Lingo-1 (2) is the best at the level of mRNA. The next result is compared with Lingo-1 (4), the inhibition ratio of rAd Lingo-1 (1),rAd Lingo-1 (2),rAd Lingo-1 (3) is 52.86%,71.15%,45.9% by Western blot, The effect of Lingo-1 inhibited by rAd Lingo-1 (2) is the best at the level of protein. According to above results, we find that rAd Lingo-1 (2) is the best one.Experiment in vivo:SCI rat animal model was made. Then rAd Lingo-1 (2) was injected into rat. Lingo-1 mRNA level was assayed by RT-PCR, and Lingo-1 protein level was assayed by Western Blot. The results show us that, Lingo-1 mRNA relative level of spinal cord was downtrend from 99% of 1st to 58% of 21st after SCI rat was treated, and the treated group level of 7th was lower than that of normal group; Lingo-1 protein relative level of spinal cord was downtrend from 98% of 1st to 56% of 21st after SCI rat was treated, and the treated group level of 7th was lower than that of normal group; GAP43 protein relative level of spinal cord went up from 36% of 1st to 94% of 14th after SCI rat being treated, the number of 3rd had statistical significance, and it raised obviously at 7th, which authenticated efficiently that spinal axon had regenerated significantly.So, we can conclude that Lingo-1 gene restrained by adenovirs-mediated shRNA can inhibit rat spinal cord Lingo-1 protein expression, and promote spinal cord regeneration successfully.