The Study On Treatment Of Spinal Cord Injury By Lentiviral Vector-mediated SiRNA Interfering Gene PTEN

ObjectiveTo observe the regeneration of axons of PTEN siRNA in vitro, and inject lentivirus carrying siRNA locally during the acute period of spinal cord injury (SCI) to explore the role of treatment to SCI.MethodsTwo siRNAs which had the greatest specific effect on PTEN were selected and constructed the lentiviral RNA interference (RNAi) vector of rat PTEN gene. The Lentivirs vector was transfected into the rat dorsal root ganglion cells. DRG were randomly divided into three groups:control group (A), PTEN-sham group (B) and PTEN-RNAi group (C), we evaluated PTEN gene suppressing by using Western blot and measured the length of neurons axon regeneration after1week. In vivo,60female Wistar rats were randomly divided into three groups as follows:control group (group A, n=20), PTEN-sham group (group B, n=20), and PTEN-RNAi group (Group C, n=20). Impactor Model-II device was used to induce SCI model at the level of Chest10. After SCI, group A was injected saline locally immediately, group B accepted local injection of lentivirus with nonspecific siRNA, group C accepted local injection of lentivirus carrying siRNA immediately. Then the hindlimb function behavior score (BBB scale) was analyzed at the time of24hour,3rd day,5th day,1st week,2nd week,3rd week,4th week,5th week,6th week,7thvweek,8th week after SCI. The spinal cord tissue was taken to observe the expression of EGFP under a fluorescence microscope at the time of the8w after SCI, and to observe the axonal regeneration through immunohistochemical (neurofilament protein200, NF-200) staining.ResultsLentivirs vector carrying the PTEN-siRNAs could infect the cultured rat DRG cells and knock down the expression of PTEN. PTEN knockdown in DRG cells resulted in improved neurite outgrowth and elongated neuritis. At the time of1d,3d,5d,1w,2w after SCI, there was no significant difference among BBB scores of the three groups (p>0.05), and the BBB score of group C was significantly higher than the group A and group B at each time point after the3rd week (p<0.05). The NF-200immunohistochemical staining shown that the number of survival neuron was significantly more in group C than in group A and group B (p<0.05), and a significant increase in neuronal size was observed in group C as compared with group A and group B (p<0.05), but there was no significant difference in the the density of astrocytes around the cavities of the three groups (p>0.05).ConclusionLentivirus carrying siRNA can infect the cultured rat DRG cells and disturb the expression of PTEN. PTEN suppression can promote the regeneration of axons in vitro. In vivo, PTEN suppression ablation can promote neuron surviva and increase neuronal size obviously, which demonstrate a neuroprotective role of PTEN ablation. These results give indication for research and clinical practice for the therapeutic interventions of SCI.