Role And Mechanism Of APC-Cdh1 In The Proliferation And Activation Of Oligodendrocyte Precursor Cells Following Stretch-induced Mechanical Injury

Research Background:With the rapid development of modern traffic and building industry,the incidence of spinal cord injury(SCI)is on the rise year by year,and this disease has become one of the major causes of disability among young people.Yet,the mechanism of spinal injury and repair remains unclear,and its diagnosis and treatment cannot meet patients’ need.Oligodendrocyte is the only cell that forms myelin in the central nervous system.It is very sensitive to injury stimulation like neurons.Researches in recent years believe that the apoptosis of oligodendrocytes following spinal cord injury causes the survived axons to demyelinate,which is the pathophysiological basis that affects neuron axonal regeneration and repair.Therefore,to study how to effectively inhibit apoptosis and promote the proliferation and activation of oligodendrocyte precursor cells(OPCs)is an important part to reduce SCI.Researches have shown that anaphase promoting complex(APC)and its co-activator protein Cdh1 play important roles in the growth,development and injury repair of the central nervous system.Recent studies have confirmed that Cdh1 participates in the pathophysiological process of hippocampal neuronal apoptosis after global cerebral ischemia and reactive astrocyte proliferation after oxygen–glucose deprivation and reperfusion.Yet,the role of APC-Cdh1 in the proliferation and activation of OPCs following SCI remains unclear.In this research,we proposed to culture primary OPCs and establish a model of stretch-induced mechanical injury in vitro,and to explore whether the proliferation and activation of OPCs after mechanical injury is associated with APC-Cdh1 activity.Second,the effect of down-regulating Cdh1 on the proliferation of OPCs after injury was investigated by using adenovirus-mediated RNA interference.Finally,we tried to identify the potential mechanism by which Cdh1 regulates the proliferation and activation of OPCs after mechanical injury by tracking the changes of Cdh1’s downstream substrate Id2 and skp2 after intervention.By following the regulation of cell cycle,this paper was to explore the role of APC-Cdh1 in the proliferation and activation of spinal OPCs after mechanical injury,thus providing experimental basis for selecting APC-Cdh1 as a new target for spinal cord protection.Part ⅠEstablishment and evaluation of a model of stretch-induced mechanical injury on cultured oligodendrocyte precursor cells from neonatal rat spinal cord in vitro Objective:To establish a model of stretch-induced mechanical injury on cultured oligodendrocyte precursor cells(OPCs)in vitro with the FX-4000T? system.Methods:The spinal cords of SD neonatal rats within 48 h were collected.Purified OPCs were obtained based on primary mixed glial cell culture by using the shaking method,and its specific marker A2B5 was used for immunofluorescence assay.FX-4000T? system was employed to establish a model of stretch-induced mechanical injury of OPCs from neonatal rat spinal cord.The purified OPCs were randomly divided into group A(control group),group B(5% tensile strain),group C(10% tensile strain)and group D(15% tensile strain).The morphology of each group was observed under inverted microscope;survival rate was measured by MTT assay;apoptosis was detected by double-staining flow cytometry.Results:OPCs were cultured successfully in vitro,and the percentage of purified OPCs was more than 90%.There were no significant differences between group A and group B in the survival rate and cell morphology.Compared with group A,the survival rates of group C and D were obviously decreased(P<0.05),and the apoptosis rates were remarkably increased(P<0.05),showing obvious pathological morphological change.However,there was a large loss of cell attachment and the survival rate of OPCs was found to be less than 50% in group D,which was unfavorable for further experiment.Conclusion:OPCs can be separated and purified by shaking and differential adhesion from SD rats spinal cord.The model of stretch-induced mechanical injury on cultured OPCs can be established by applying 10% tensile strain with the FX-4000T? system.Part Ⅱ Role and Mechanism of APC-Cdh1 in the Proliferation and Activation of Oligodendrocyte Precursor Cells Following Stretch-induced Mechanical Injury Objective:To explore the role of APC-Cdh1 in the proliferation and activation of OPCs following mechanical injury and the potential mechanism.Methods:The purified OPCs were randomly divided into four groups in the preliminary experiment: normal control group(no stretching),Stretch-2h group(stretched for 2h),Stretch-6h group(stretched for 6h)and Stretch-12 h group(stretched for 12h).After RNA interference in the further experiment,we set up four groups: normal control group(without adenovirus infection or stretching),Stretch group(stretched for 12 h without adenovirus infection),Ad-Control-Stretch group(stretched for 12 h after empty adenovirus vector infection)and Ad-Cdh1-Stretch group(stretched for 12 h after Cdh1-adenovirus vector infection).Next,each group was cultured for 48 h without stretching before cells were harvested.Cell proliferation was measured by MTT assay and cell cycle analysis by flow cytometry;the mRNA and protein expressions of Cdh1 and its downstream substrate Skp2 and Id2 were detected by real-time quantitative PCR and Western blot.Results:1.After 2h of mechanical stretching,proliferation activation began to decline continuously with time of stretching,and the most remarkable decline was observed in Stretch-12 h group.Western blot indicated that Cdh1 expression in Stretch-2h group,Stretch-6h group and Stretch-12 h group was much higher than that in the control group and increased with time,and the most significant up-regulation of Cdh1 protein observed in Stretch-12 h group(P<0.06).2.After RNA interference,Cdh1 expression in Ad-Cdh1-Stretch group was obviously lower than Ad-Control-Stretch(P<0.05),cell proliferation activity in Ad-Cdh1-Stretch group was obviously higher than Ad-Control-Stretch(P<0.05).3.The expressions of Cdh1’s downstream substrate Skp2 and Id2 varied with intervention of Cdh1,which was contrary to Cdh1: Skp2 and Id2 were down-regulated after mechanical injury,while adenovirus-mediated Cdh1 RNA interference could up-regulate the expressions of Skp2 and Id2.Conclusion:1.Stretch-induced mechanical injury causes the decrease of the proliferation of OPCs and leads to high Cdh1 expression in OPCs,and down-regulated Cdh1 expression in OPCs can significantly promote the proliferation after mechanical injury,suggesting Cdh1 protein expression levels associated with the proliferation of OPCs following mechanical injury.2.APC-Cdh1 involves in the regulation of the proliferation of OPCs,and its mechanism may be the degradation of downstream substrate Skp2 and Id2 by ubiquitination.