| Objective To investigate the effects of down-regulation of MSK1 on inflammatory response,apoptosis,astrocyte proliferation and motor function recovery after spinal cord injury in rats by lentivirus-mediated RNA interference.In order to explore the role and mechanism of MSK1 in the repair of spinal cord injury,and to provide new ideas and targets for the basic research and clinical treatment of spinal cord injury.Methods 1.The lentivirus was transfected into well-differentiated PC12 cells.They were divided into blank control group(NC),lentivirus negative control group(LV-GFP),interference group 1(MSK1siRNA1),interference group 2(MSK1siRNA2)and interference group 3(MSK1siRNA3).The transfection efficiency was observed by fluorescence microscope.The interference efficiency of MSK1 was verified by Western blot.2.The rats were randomly divided into operation group and sham operation group.the spinal cord injury model was established in the operation group.in the sham operation group,only the lamina was resected and the spinal cord tissue was not injured.The lentivirus vector was transfected into spinal cord tissue by local injection of lentivirus.the green fluorescence expression of spinal cord tissue at different time points after lentivirus injection was observed by fluorescence microscope to determine the best transfection time point of lentivirus.3.The rats were randomly divided into SCI group(simple spinal cord injury),LV-GFP group(injection of negative control lentivirus after spinal cord injury)and LV-MSK1 group(injection of MSK1 siRNA lentivirus after spinal cord injury).The expression of MSK1 protein was detected by Western blot on the 7th and 14 th day after lentivirus transfection.4.After down-regulating the expression of MSK1,the expression of TNF?,IL-1 ?,IL-6,IL-10 and PCNA was detected by Western bolt.The expression of PCNA,GFAP and Caspase3 was detected by immunofluorescence staining.The apoptosis was detected by TUNEL staining,and the ultrastructural changes of spinal cord neurons were observed by transmission electron microscope.The recovery of hindlimb motor function after spinal cord injury was evaluated according to BBB score.To determine the effects of down-regulation of MSK1 on inflammatory response,apoptosis,astrocyte proliferation and motor function recovery after spinal cord injury.Results 1.After 72 hours of lentivirus transfection,a large number of green fluorescence was observed in PC12 cells in each group,and about 80% of the cells were infected with lentivirus.After 96 hours of lentivirus transfection,the expression of MSK1 protein in MSK1-siRNA1 MSK1-siRNA2 and MSK1-siRNA3 groups was significantly lower than that in NC and LV-GFP groups.Compared with LV-GFP group,MSK1-siRNA2 lentivirus vector has the highest interference efficiency.2.The expression of green fluorescence was observed on the 1st day after lentivirus transfection,increased gradually on the 3rd and 5th day,reached the peak on the 7th day,and decreased gradually on the 14 th day.3.The expression of MSK1 protein in LV-MSK1 group was significantly lower than that in SCI and LV-GFP groups on the 7th and 14 th day after lentivirus injection.4.Western blot showed that the expression of TNF-?,IL-6,IL-1 ? and PCNA in LV-MSK1 group was significantly higher than that in SCI and LV-GFP groups,and the expression of IL-10 in LV-MSK1 group was significantly lower than that in SCI and LV-GFP groups.5.Immunofluorescence staining showed that the average immunofluorescence intensity of PCNA and GFAP positive cells and the number of NeuN + Caspase3 positive cells in LV-MSK1 group were significantly higher than those in LV-GFP group.TUNEL staining showed that the number of TUNEL positive cells in LV-MSK1 group was significantly higher than that in LV-GFP group.6.Transmission electron microscopy(TEM)showed that neurons in SCI and LV-GFP groups showed typical apoptotic characteristics,such as nuclear pyknosis,chromatin condensation,nuclear membrane wrinkling,irregular myelin morphology and thinning.In LV-MSK1 group,severe damage of cell membrane,swelling of mitochondria and demyelination were found.In LV-MSK1 group,the nerve cell membrane was severely damaged,mitochondria swelled,accompanied by demyelination.7.The BBB score of hindlimb motor function in LV-MSK1 group was lower than that in SCI and LV-GFP groups on the 5th,7th and 14 th day after injury.Conclusion 1.MSK1 siRNA lentivirus vector can effectively transfect well-differentiated PC12 cells and rat spinal cord tissue,and effectively inhibit the expression of MSK1 in vitro and in vivo.2.Down-regulation of MSK1 expression promoted inflammatory response,astrocyte proliferation and apoptosis and inhibited the recovery of motor function after SCI 3.MSK1 may play a role in the repair of spinal cord injury by regulating inflammatory response,astrocyte proliferation and apoptosis. |
PDF Full Text Request