| BackgroundSpinal cord injury is one of the common injuries, often caused by falls, traffic accidents, sports injuries and violence. In recent years, the incidence of spinal cord injury gradually increased. Spinal cord injury can lead to sever sensorimotor dysfunction with a poor prognosis. Cervical spinal cord injury is the most common type of spinal cord injury, which involving the damage of upper extremity function, ruined the self-care ability and quality of life of patients. To Clarify the internal pathological mechanisms, to find effective targets for intervention, and based on this, to seek to improvement in motor function are extremely important.microRNAs are a kind of noncoding single-stranded RNAs which can regulate protein expression by regulating their target mRNAs. Recent studies proved a disregulation of miRNAs after spinal cord injury. And these disregulated miRNAs has important roles in inflammation response, cell apoptosis and inhibition of axon regeneration after spinal cord injury. A few of studies suggested that miRNA-133b can promote the growth of neuron axons. However, to our knowledge, there is no report for its role in mammalian spinal cord injury and regeneration in the pathological process.Part 1ObjectiveTo establish a stable mice model of cervical spinal cord contusion injury and analyze the trend of its motor disfunction. To clarify the expression change of miR-133b after spinal cord injury at differen time point.MethodThe mice were traind to accomplish the griping strength measurement before injuryed. After that, they were devied into sham group and injury group. Mice in sham group were underwent laminectomy at C5 level. Mice in injury group were underwent contusion at C5 level by Infinite Horizon spinal cord impactor. The grip strength measurment (GSM) and forelimb locomotor scale (FLS) were measured pre-op and at Id,3 d,1 to 8 weeks after spinal cord injury. The expression of mature miR-133b in the lesion site were tested at 1d, 3d,7d,14d post-injury by qRT-PCR.ResultsFor the mice of sham group, the grip strength were decreased temporaily at 1d and 3d post-injury because the wound and were gradully recovered to a normal level since 1 week post-injury. In the first week after injury, mice of injury group can no used their forepaws to make any action because of the sever damage of motor function. From 7d to 14d post-injury, the mice tryed to grip the rod, but can not fulfill a successful griping. From 21d to 56d post-injury, the grip strength of forepaws recovered slightly but was still signicantly lower than that of pre-injury. At each time point, the grip strength of injured mice was significantly lower than that of sham mice (P<0.05)At each post-injury time point, expression of mature miR-133b in spinal cord of sham-operated mice is maintained at a stable level. There is no significant difference (p> 0.05) between at time points for the relative expression values. For injured mice, the expression of mature miR-133b at first day after injury was slightly increased (p> 0.05). And it decreased sharply at 3d and reached the bottom at 7d(p<0.05). The expression rebounded slightly at 14d, but still lower than that of sham group.ConclusionForelimb grip strength measurement is a sensitive and reliable method to assess motor function in spinal cord injury in mice. And after spinal cord injury, miR-133b was significantly down-regulate over time.Part 2ObjectiveTo explore the key downstream target genes of miR-133b and investigate the regulation effect of miR-133b to these target genes.MethodExogenous miR-133b mimic, miR-133b inhibitor and miR negative control were used repectively for the overexpression, lowexpression and control. Tissues were collected at Id, 3d,7d post-injury. qRT-PCR were used to detect the expression change of mature miR-133b in the leision sites. Bioinformatics Database were used to search the relative target mRNAs for miR-133b. And finally, qRT-PCR and western blot were used to measure the expression changes of these target genes.ResultAfter spinal cord injury, mature miR-133b expression levels of each groups experience a significant change over time. Compared with the injury group and negative control group, treatment group had a higher mature miR-133b expression in leision site (p <0.05), which reached the highest value in the third days after surgery. Inhibitor group had a lower mature miR-133b expression than that of the surgery group and negative control group (p<0.05).By searching the bioinformatics database, the relative key downstream target genes were RhoA, Stathmin4. While Stathminl, a family member with stathmin 4 with a highly similar coding and structure, also included in this study. Results of qRT-PCR and Western blot revealed that injury group exprienced a high regulation in RhoA and a low regulation of Stathminl over time. And for the treatment group the expression of RhoA decreased and the expression of Stathminl protein levels increased compared with those in injury group.ConclusionExogenous miR-133b mimic can be absorbed by injured spinal cord via a local injection and generated into mature miR-133b. Overexpression of miR-133b lead to a change in expression of RhoA and Stathminl which are important nerve growth regulatory proteins. Changes in miR-133b expression after spinal cord injury may prompted axon regeneration by regulating RhoA/ROCK pathway and Stathminl.Part 3ObjectiveTo clarify the effect of miR-133b on the axon regeneration and cell apoptosis after spinal cord injury.MethodExogenous miR-133b mimic, miR-133b inhibitor and miR negative control were used repectively for the overexpression, lowexpression and control. Griping strength measurement was used evaluate the recovery of motor function. Corticospinal tract biotinylated dextran amine tracing and axon number count were used to evaluate the regeneration of of CST axon. Western blot were used to determine the expression of apoptotic proteins. GFAP and DAPI co-staining were used to evaluate the cell morphology, growth trends and apoptosis.ResultGrip force of mice in treatment group started to recove from the 7th day after injury and gradually improved over time. From 28d-56d after injury treated mice exprience a higher grip strength than those in injury group. In contrast, forelimb motor function of mice in inhibitor group recovered slower. At the 14th day after injury, the mice were still unable to have successfully grip. Grip strength of these mice improved gradually until the 21th day post-injury. But it was still significantly lower than that of injury group (P<0.05).By comparing the relative axon count of each section. We found that there was no statistically significant difference between the injury group and negative control group(p> 0.05). The relative axon count section in miR-133b group was higher than the injury group (P<0.05).The expression of Caspase 3 after injury 3rd day,7th days in miR-133b group were significantly lower than that of injury only group.Compared with the that in injury only group, the distribution of GFAP-labeled astrocytes silk protein was more regularity and showed a growth out of the leision site. And DAPI-labeled apoptosis cells were less in miR group than that of injury group.ConclusionOverexpression of miR-133b can lead to a out growth of axons into the leision site and lower neuronal apoptosis, which finally lead partial improvement of motor function. In contrast, inhibiting miR-133b delayed the recovery process of spinal cord injured mice. These results implyed that miR-133b plays an important role in regulating axonal regeneration after spinal cord injury, and may become an important target for the treatment of spinal cord injury. |
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