| Peripheral nerve injury,one of the most common neurosurgical disease with high rate of deformity,affects 13 to 23 per 100000 persons each year.Peripheral nerve injury is easy to cause nervous system partial or complete damage resulting in considerable long-term disability and neurological disorders.Neuroanastomosis,nerve transfer and other practical surgeries are used to repair nerve injury with small defects.Reinnervation,tubulization and other bridge operation are usually used to restore the long segment neurological defects.Due to the potential risk of transmission and immunologic rejection of allograft,autogenous nerve is preferred in clinical treatment for peripheral nerve injury.However,autologous nerve grafting is limited by implant sources,which often result in denervation of the donor site,and can not match to acceptor area,resulting in dysfunction in dominated area.How to reduce the neuron apoptosis and promote nerve regeneration after peripheral nerve injury is one of the most challenging surgical problems.Proliferation and migration of Schwann cells are the important processes in peripheral nerve regeneration.A series of cellular and molecular biology degeneration occurs after a nerve severed.The distal portion begins to degenerate and the axon and myelin begins to breakdown,followed by dissolution of the cell membrane.Wallerian degeneration of the distal stump involves invasion by mononuclear macrophage that removes tissue fragments and initiate dedifferentiation and mitosis in Schwann cells.Schwann cells generate Bungner' band surrounding the axon and guide the growth direction of growth cones after proliferation,redifferentiation and migration.Simultaneously,Schwann cells secrete a variety of cell adhesion molecules,neurotrophic factors and extracellular matrix,promoting the peripheral nerve axon regeneration and myelin formation,and finally repair the injured nerve.MiRNAs are important regulators at post-transcription in the growth and physiological process.Hundreds of miRNAs were identified with significant expression variance at different points after peripheral nerve injury,indicating that miRNAs are involved in the process of nerve repair and regeneration after nerve injury.miR-210 was identified as a key player of cells response to low oxygen tension.miR-210 inhibits cell apoptosis and promotes cell survival at hypoxic and oxidative stress condition.Furthermore,miR-210 promotes nerve regeneration after spinal cord injury.Animal and cell experiments were designed to explore the effects and molecular mechanism of miR-210 in the process of nerve regeneration following peripheral nerve injury.Sciatic nerve injury model in rats were constructed and the expression level of miR-210 in proximal stumps at different points after sciatic nerve injury was validated.Lentiviral vectors were employed to up-regulate or down-regulate the expression level of miR-210,and the effects on proliferation and migration of Schwann cells and the expression level of nerve regeneration associated proteins were determined.The results will provide theoretical basis for the molecular targeted therapy of sciatic nerve injury and give the target for developing new drugs.Part 1.The expression variance of miR-210 at different points after sciatic nerve injuryObjective: Constructing rat model of sciatic nerve injury and testifying the expression variance of miR-210 at different points after sciatic nerve injury,to provide theoretical basis for subsequent experiments.Method: The left hind limb sciatic nerves were exposed and 1 cm long segment was then resected at the site just proximal to the sciatic nerve piriformis' lower hole to construct rat sciatic nerve injury model.The experiment was divided into 0 d,1 d,4 d,7 d,14 d of control group and model group according the time after nerve injury.The expression level of miR-210 in proximal stumps at different points after sciatic nerve injury was validated by Real-time PCR.Result: Rat sciatic nerve injury model was constructed successfully.The results of Real-time PCR showed that there were no obvious changes of the expression of miR-210 in the control group at 0 d,1 d,4 d,7 d and 14 d.The expression of miR-210 in the model group gradually increased with the passage of time.The relative expression level of miR-210 was 0.91±0.02?1.24±0.04?1.94±0.08?2.43±0.05?3.45±0.19 separately.Compared with the 0 d group,the expression level of miR-210 significantly increased at 1 d,4 d,7 d and 14 d,and the difference was statistically significant(P<0.01).Conclusion: The expression of miR-210 increased after sciatic nerve injury,and miR-210 was involved in the nerve regeneration after sciatic nerve transaction.Part 2.Effect of miR-210 on cell proliferation and apoptosis in Schwann cellsObjective: To explore the effect of miR-210 on cell proliferation and apoptosis in Schwann cells.Method: The primary Schwann cells were separated from sciatic nerve of newborn SD rats.Lentiviral vectors were used to up-regulate or down-regulate the expression level of miR-210 in Schwann cells.The experiment was divided into four groups: mimic NC,miR-210 mimic,inhibitor NC and miR-210 inhibitor.The effect of miR-210 on Schwann cells proliferation was detected by MTT assay.The effect of miR-210 on Schwann cells migration was measured by transwell assay and wound healing test.Result: The primary Schwann cells were successfully separated from sciatic nerve of newborn SD rats,and cells were observed spindle or fusiform under optical microscope.The expression level of miR-210 was significantly increased after miR-210 mimic transfection(P<0.01).The results of MTT assay showed that overexpression of miR-210 promoted cell proliferation(P<0.01).Transwell and wound healing assay indicated that the cell migration ability was significantly increased in miR-210 mimic group compared with the mimic NC group(P<0.01).The expression level of miR-210 was significantly decreased after miR-210 inhibitor transfection(P<0.01).The decreased expression of miR-210 inhibit cell proliferation and migration ability compared with inhibitor NC group(P<0.01).Conclusion: Overexpression of miR-210 promotes cell proliferation and migration in Schwann cells.Schwann cells proliferation and migration ability decreased when the expression of miR-210 was silenced.Part 3.The effects of miR-210 on nerve regeneration related proteins in Schwann cellsObjective: To clarify the effect of miR-210 on growth-associated protein-43(GAP-43),myelin associated glycoprotein(MAG)and myelin basic protein(MBP)in Schwann cells.Method: The experiment was divided into four groups: mimic,miR-210 mimic,inhibitor NC,miR-210 inhibitor.The nerve regeneration related proteins expression of GAP-43,MAG and MBP were determined by Western blot analysis.Result: over-expression of miR-210 promotes the expression of GAP-43 and MBP.Compared with the mimic NC group,the expression level of GAP-43 and MBP increased 152.00 % and 72.00 % separately(P<0.01).Silencing miR-210 decreased the expression level of GAP-43 and MBP.Compared with the inhibitor NC group,the expression level of GAP-43 and MBP decreased 46.23 % and 74.55% separately(P<0.01).The expression trend of MAG differs from GAP-43 and MBP.Over-expression of miR-210 inhibit the expression of MAG.The expression level of MAG in miR-210 mimic group decreased 44 % compared with mimic NC group,and increased 210 % in miR-210 inhibitor group compared with the inhibitor NC group.The difference was statistically significant(P<0.01).Conclusion: miR-210 promotes the expression of GAP-43 and MBP,inhibit the expression of MAG in Schwann cells,which affects biological activity of Schwann cell,and promotes nerve regeneration after peripheral nerve injury. |
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