In Vitro Experimental Study On The Effect Of Circulatory Arrest At Different Temperatures On The Expression Of Small Ubiquitin-like Proteins In Nerve Cells

ObjectiveTo establish an in vitro model of multi-temperature hypothermia circulatory arrest(HCA)in human neuroblastoma cell line SHSY5Y,and to examine the hypothermia protection of neural system in multiple temperature under hypoxic and ischemic condition.MethodsOxygen-glucose deprivation(OGD)is established using Anaero pack and sealed cell culture box.Cell incubator is used to control different temperature.SHSY5Y cells are cultured in normal condition or with 4h OGD in different temperature.Morphology of the cells are recorded immediately after OGD,in order to reveal the damage caused by OGD,and to observe the hypothermia protection.Rewarming and reperfusion are processed subsequently.The secretion level of LDH and the expression level of Caspase-3 are measured 24h after reperfusion,in order to evaluate the apoptosis of the cells,and reveal the difference of hypothermia protection in multiple temperature.ResultsMorphology showed the cells under OGD condition were gathered with pyknosis and dark bubbles.The higher temperature during OGD resulted in more obvious morphological changes.Further results revealed that the secretion of LDH and expression of Caspase-3 both significantly increased after OGD,while hypothermia reduced them.ConclusionThe in vitro model of multi-temperature HCA was established,which threw light on the damage and protection mechanism of HCA.Hypothermia could effectively protect neural cells under hypoxic and ischemic condition.Both medium and shallow hypothermia contributed protection to the neural cells as well as the deep hypothermia.ObjectiveTo investigate the influence of hypothermia and hypoxia in different temperature on SUMO modification,and to evaluate the induction of brain ischemia tolerance in different temperature hypothermia based on detection of SUMO-associated proteins.MethodsBased on the multi-temperature HCA model,SHSY5Y cells OGD was processed in 37?,30?,26?,18?,respectively.The expression level of SUMO1,SUMO2/3,and UBC9 was measured by Western blot.The ubc9 gene expression was measured by qPCR.The combined results showed the change of SUMO-associated proteins during HCA process.ResultsCompared with the OGD cells in 37?,hypothermia increased the level of binding SUMO1,and the lower temperature resulted in the higher level.Binding SUMO1 was significantly higher in the 18? OGD cells than in 30?(p<0.05,n=6),whereas no significant difference between 18? and 26?(p>0.05,n=6).Hypothermia also increased both binding and free SUMO2/3(p<0.05,n=6),however the ratio of binding/free SUMO2/3 did not increase compared with the 37? OGD cells(p>0.05,n=6).OGD did not significantly influence the expression of UBC9(p>0.05,n=6).qPCR results showed no significant change of ubc9 expression.ConclusionHypothermia increases the expression of SUMO2/3 by increasing binding of SUMO1,thus enhance the tolerance of neural cells under hypoxia and ischemia.Within a certain range,the lower temperature can induce the better tolerance of neural cells,while the deep and medium hypothermia have similar effects.ObjectiveTo investigate the regulate relationship between miR-200c and UBC9.MethodsThe online bioinformatics tool TargetScan was used to analyze the sequence of UBC9 mRNA,and predict potential binding sites.Furtherly,the relationship was verified by the dual luciferase reporter assay.ResultsTargetScan analysis showed that a seed sequence of human miR-200c was found on UBC9 mRNA,with a context score of 98%,indicating a potential regulation site.The dual luciferase reporter assay showed that the miRNA-transfected cells expressed significantly lower luciferase than the control cells(p<0.05,n=3).While the luciferase expression of the UTR-mutated/miRNA-transfected cells was consistent with the control cells(p<0.05,n=3),and was significantly lower than the miRNA-transfected cells(p<0.05,n=3).ConclusionHuman miR-200c directly regulates the expression of UBC9.