| Part ?:Modified Allen's method to establish moderate contusion model of spinal cord injury model in rats.Objective:To establish moderate contusion model of spinal cord injury model in rats by modified Allen's method,and to observe histological differences and hind limb function score(BBB score)after spinal cord injury in the SCI group and the sham operation group.Methods:A rat model of thoracic 9 moderate contusion SCI was established.Changes in motor function of the hind limbs after SCI were evaluated by BBB scoring method,and histological changes of the two groups after SCI were observed by beta III-tubulin labeled immunofluorescence staining.Results:After spinal cord moderate contusion in thoracic 9 rats,the neurons in the sham operation group were basically complete in structure,normal in shape,well-proportioned in distribution,well-proportioned in intercellular matrix,and well-aligned.However,in the SCI group,the neuron structure was incomplete,the neural protrusion basically disappeared,and the intercellular matrix was blurred and confused.The difference of BBB score between the SCI group and the sham operation group was statistically significant(p<0.05).Conclusion:1.The moderate contusion model of spinal cord injury in rats was successfully established;2.BBB score of the SCI group was significantly lower than that of the sham operation group.Part ? Expression of miR-21 and apoptosis factors PDCD4 and PTEN after spinal cord injury in ratsObjective:To elucidate the expression changes of miR-21,the expression time distribution and role of apoptosis factors PDCD4 and PTEN after spinal cord injury in rats.Methods:60 adult rats with SPF level and body weight of 200-250g were used to establish the T9 contusion spinal cord injury model,by using modified Allen's method in the SCI group,and laminectomy only was performed in the sham operation group.Samples were taken from each group at 4h,8h,1d,3d and 7d after opertation,and the changes of miR-21 expression before and after spinal cord injury were detected by real-time PCR.Meanwhile,the expressions of PDCD4 and PTEN of miR-21 related target genes were detected by Western-blot to observe whether miR-21 affected the repair of spinal cord injury by regulating the expressions of PDCD4 and PTEN.Results:compared with the sham operation group,the expression level of mir-21 was lower than that of normal tissue at 4 hours,8 hours and 1 day after SCI,and there were statistical differences between each group and the sham operation group(P<0.05).However,mir-21 level at 3 and 7 days after injury was significantly higher than that in the sham group(P<0.05),which first decreased and then increased.The expression level of PDCD4 increased at 1 day after injury,peaked at 3 days after injury,and decreased at 7 days after injury.Compared with the sham group,the expression of PDCD4 at 1 and 3 days after injury was statistically different from that before injury(P<0.01).The expression of p-pten was the highest 1 day after spinal cord injury,decreased 3 days after injury,and decreased to the pre-injury level 7 days after injury.The expression levels at 1 day(P<0.01)and 3 days(P<0.05)after spinal cord injur.decreased 3 days after injury,and decreased to the pre-injury level 7 days after injury.The expression levels at 1 day(P<0.01)and 3 days(P<0.05)after spinal cord injury were statistically different from those in the sham group.The expression level of p-pten decreased to the level before spinal cord injury 7 days after SCI.Conclusion:Within one week of SCI,the expression level of PDCD4 and PTEN decreased with the increase of miR-21 expression,indicating that miR-21 inhibited the expression of apoptosis factors PDCD4 and PTEN in the stage of secondary injury,and played an important role in promoting post-injury repair and inhibiting the formation of glial scar.The correlation between the expression of PDCD4 and PTEN and the expression of miR-21 after spinal cord injury indicates that miR-21 can inhibit glial scar and promote repair after injury by regulating apoptosis factors.Part ? recombinant adeno-associated virus vectors mediate overexpression of mir-21 and promote axonal regeneration by inhibiting apoptotic protein PDCD4Objective:To establish a rat spinal cord injury model of contusion type by using modified Allen's method,and to investigate the regulatory effect of recombinant adeno-associated virus vector raav-mirna-21 on target protein PDCD4 and its effect on axonal growth.Methods:Primary neurons of spinal cord of fetal rats were isolated and cultured.rAAV-pri-miRNA-21,rAAV-in-miRNA-21 and rAAV-nc-miRNA were infected with fetal rat spinal cord neurons by hU6-MCS-CMV-EGFP rAAV vector,respectively.Axonal growth of each group was detected by immunofluorescence labeled by ?-? tubulin,and PDCD4 expression was detected by Western blot.Results:In rAAV-in-miRNA-21 transduced neuron cultures,the neurons either extended very short neurite or not at all.In contrast,neurons overexpressing miRNA-21 showed significantly increased neurite growth compared to the control group or the nc-miRNA group.The results of quantifying the average length of axons of total neurons showed that miRNA-21 significantly increased the average length of axons of total neurons(415±16.7mm),while the control group(198±11.2mm)and the nc-miRNA group(185±10.6mm)(p<0.05).The results of quantifying the mean length of the axon of the longest neuron showed that miRNA-21 significantly increased the axon of the neuron with the longest mean length(234±7.1mm),while the control group(121±3.9 mm)and the nc-miRNA group(109±4.7 mm),respectively(p<0.04).Western blot analysis showed that compared with the control group,PDCD4 expression decreased by 35%(p<0.05),and PTEN protein level showed no significant change.Conclusion:Overexpression of miR-21 can increase neurite extension and promote neurite growth of spinal cord neurons in rats after birth by regulating the expression of target protein PDCD4. |
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