| Purpose:Establish a rat spinal cord injury(SCI)model.To determine whether the level of5 hmC after SCI changes with the time of injury by detecting the overall 5hmC level in the spinal cord tissue of rats with different injury time.Detection of spinal cord tissue in the injured group and spinal cord tissue of the control group,and detection of changes in expression of the TET protein family by Western blot.Detection of changes in the extent of damage to spinal cord tissue by inhibiting the expression of TET2 protein.Detection of changes in the expression of genes associated with necrosis and protection-related genes in spinal cord tissue by inhibiting the expression of TET2 protein.Method:Establishment of a spinal cord injury model: 32 SD rats aged 5-6 months were selected and divided into 7 groups according to time points(3h,6h,12 h,24h,48 h,3d,7d).4 surgical mice and 4 sham-operated mice..The rats were sacrificed at the selected time points and the spinal cord tissue at the injury site was removed and stored at-80 °C.Tissue DNA was extracted for Dot blot experiments.Extract the RNA from the spinal cord tissue of the control group and the 24 h time point of injury for QPCR to detect the transcription level of TET1,2,and 3.Western blot analysis was performed on the proteins of the spinal cord tissue from the control group and the 24 h time point of injury.32 SD rats aged 5-6 months were randomly divided into four groups,SCI group,SCI+SC-1 group,SC-1 group and control group.1SCI+SC-1 group: Spinal cord tissue was exposed,and 10 ul of TET2 protein inhibitor SC-1(concentration: 10 mM)was intrathecally administered with a 28 G needle half an hour prior to spinal cord injury,and spinal cord injury was modeled.2SC-1 group: After the spinal cord was exposed,only the drug was injected and the spinal cord tissue was not damaged.3SCI group:After the spinal cord was exposed,only the spinal cord tissue was damaged and no drug was injected.4 Control group: only the spinal cord tissue was exposed,that is,the spinal cord tissue was not damaged,and no drug was injected.The above rat model was sacrificed at 24 h,the spinal cord tissue at the lesion was removed,and the-20 ° C refrigerator was quickly frozen and sectioned.Gross sections were observed using TTC staining.The same method was used to establish four groups of models,SCI group,SCI+SC-1 group,SC-1 group,and control group.Extract RNA for quantitative real-time PCR(Q-PCR)detection.The DNA fragment containing 5hmC was enriched using the hMeDIP kit for QPCR detection.Result:1.Successfully produced a rat model of spinal cord injury.2.Tissue DNA was extracted for Dot blot experiments.The results showed that the5 hmC level of whole genome DNA was related to the injury time,reached the highest value at 24 h after injury,and then began to decline.The RNA of spinal cord tissue from the control group and the 24 h injury time was extracted by QPCR.The results showed that the transcription level of TET2 gene was increased after spinal cord injury.The WB experiment was performed on the protein of the spinal cord tissue from the control group and the 24 h time point of injury.The results showed that protein expression of TET2 increased after spinal cord injury compared with the control group.Four groups of rats: SCI group,SCI+SC-1 group,SC-1 group and control group TTC staining results: the necrotic area after SC-1 treatment was larger than the necrotic area of spinal cord injury group.QPCR results showed that the transcriptional levels of Bcl-2,Bax,HO-1 and iNOS were markedly increased after spinal cord injury,and Bcl was compared with the spinal cord injury group after treatment with SC-1.HO-1transcription levels decreased,while iNOS and Bax expression levels increased.Conclusion:This study successfully established the SCI model of rats,and found that the level of 5hmC modification was significantly increased after spinal cord injury,peaked at 24 hours,and then returned to the control level in the next few days.At the same time,itwas found to catalyze the apparently modified TET enzyme(Ten-eleven translocation enzymes)significantly increased TET2.Inhibition of TET2 enzyme expression aggravates spinal cord injury and neuronal cell death.However,it is unclear how TET2 enzyme and DNA 5hmC modification affect cell death by regulating the expression of death-related genes. |
PDF Full Text Request