Investigation The Effection Of SUMO2/3 Gene On Neuron Growth And The Mechanism Of Its Alteration In Ischemia Neuron

PartⅠConstruction of the SUMO2/3 Gene specific MicroRNA expression vectorObjectives To Construct the SUMO2/3(small uniquitin-like modifier 2/3) gene specific MicroRNA expression vector, and prepare well for investigating the effect of silencing the SUMO2/3 gene in the B35 cell line.Methods Genomic sequences of SUMO2 and SUMO3 gene were retrieved from Genbank. Firstly the functional sequence of SUMO2 and SUMO3 gene were cloned and then they were inserted to pcDNA3.1 vector. Secondly miRNA specific silencing the SUMO2 and SUMO3 gene were designed which were called miR2 and miR3, at the same time we dedigned a negative control called negative-miR. All these sequences were inserted to pcDNA6.2-GW/EmGFP-miR. Recombinant vectors were then transformed to E-coli, The positive colony were selected and recombinant plasmids were extracted. After restricted enzyme digestion and sequencing, the plasmids were identified correctly. Then miR2 and miR3 were colonied into the same pcDNA6.2-GW/EmGFP-miR vector called pcDNA6.2-GW/EmGFP-miR2/3.The pcDNA6.2-GW/EmGFP-miR2/3 and counterpart pcDNA3.1-SUMO2 or SUMO3 vector were co-transfected into HT22 cells. After co-transfection, the cells were treated with 50μM H2O2, for 10 min. The cells were extracted with lysis buffer and runned Western-Blot to test expression of the SUMO2and SUMO3 proteins Results Recombinant plasmids were successfully transformated to E-coli cells, using the restricted enzyme and electrophoresis identified the right position of the sequence, the DNA sequencing was futher to prove the right position. pcDNA6.2-GW/EmGFP-miR2,pcDNA6.2-GW/EmGFP-miR3 and pcDNA6.2-GW/EmGFP-miR2/3 can successfully down-regulate the proteins respectively which were verified by Western-blot.Conclusions The correctly plasmids were constructed, after transfected into HT22 cells, it can obviously silence the SUMO2/3 gene expression. These plasmids were obstained for the future study.Part II The effection on B35 cell growth after SUMO2/3 gene silencing and the SUMO family protein changes after OGD treatmentObjectives To establish the stably transfected B35 cell line using pcDNA6.2-GW/EmGFP-miR2/3 plasmid and investigation the effection of the SUMO2/3 on B35 cell growth. And the stable B35 cell was treated using OGD which is common used for the ischemia model in vitro, and then we observed the changes of SUMO protein family using Western-blot.Methods The pcDNA6.2-GW/EmGFP-miR2/3 plasmid which has CMV promotor and the control plasmid were used to transfect into B35 cell line, then the stale cell line was selected by blasticidin, the changes of SUMO2/3 protein family was tested by Westen-blot and the cell growth was evaluated using CellTiter-Blue reagent; B35 cell was treated using OGD and then the SUMO protein family were investigated by Western-blot.Results SUMO2/3 protein decreased significantly in B35 cell experssed pcDNA6.2-GW/EmGFP-miR2/3 plasmid comparing to miR-Neg B35 cell. After silencing of the SUMO2/3 expression, B35 growth and proliferation decreased to 64% comparing to miR-Neg B35 cell at 3 days. SUMO1 protein didn't change after OGD treatment, while the SUMO2/3 in miR-Neg B35 cell decreased at Omin, then increased gradually and reached highest-level at 30 min, after this it decreased little by little and became normal at 3 h. In B35 cell stably expressed the pcDNA6.2-GW/EmGFP-miR2/3 plasmid, SUMO2/3 protein decreased obviously comparing control cell with all the time points because of the silencing of the SUMO2/3 gene.Conclusions Silencing of the SUMO2/3 using miR significantly repressed B35 cell growth and differentiation. SUMO2/3 protein expression in B35 cell stably expressed pcDNA6.2-GW/EmGFP-miR2/3 plasmid was drastically reduced after OGD treatment at all time points comparing to the normal B35 cell and miR-Neg B35 cell.Part III The alteration of SUMO gene expression in spinal cord ischemia injury and investigtion of its mechnismObjectives To development of mice spinal cord ischemia animal model and investigation the SUMO2/3 changes in this model.Methods Male C57B1/6J mice were anesthetized with isoflurane and endotracheally intubated. The middle segment of the thoracic aorta was clamped for 0,8,10 or 12 min via left lateral thoracotomy. Rectal temperature was maintained at 37.0±0.5℃. A laser Doppler probe was used to measure lumbar spinal cord blood flow during thoracic aorta cross-clamping. Open field locomotor function (BBB score)and rotarod performance were evaluated at 1 h and 1,3,5, and 7 days post-injury. Surviving neurons in the lumbar ventral horn were counted at 7 days post-injury using HE staining.Results In the 8 min ischemia group, the BBB score declined to 15 at 1 hour post-injury and then recovered to almost normal by 24 hours. In the groups of 10 or 12 min of ischemia, mice had a more severe decline in BBB scores and a slower recovery, there was significantly differences comparing to 8 min or control group. Similar to the BBB score, Rotarod performance were significantly different in 10 min and 12 min group from control and 8 min group(p<0.01,10 min or 12 min vs.0 min or 8 min). Cross-clamping the middle segment of the thoracic aorta resulted in approximately 90% blood flow reduction in the lumbar spinal cord. Neurological deficit and neuronal cell death were associated with ischemia duration. Another set of mice were subjected to 10 min aortic clamping or sham surgery,4 of 5 mice (80%) in the injured group survived 28 days and had significantly different BBB score and Rotarod performance comparing to control group(p<0.01).Conclusions Cross-clamping of the aorta via left thoracotomy is a simple and reliable method to induce spinal cord ischemia in mice allowing definition of long-term outcome. The SUMO2/3 was significantly increased comparing to the control group, this was similar to the cerebral ischemia.