| Objective:To investigate the expression of Golgi a-mannosidase Ⅱ (MANⅡ)in the mouse N2a cells upon oxygen-glucose deprivation/reperfusion(OGD/R) insult.Methods:1. The mouse neuroblastoma (N2a) cells were treated with6hours,12hours and24hours of reperfusion after4hours oxygen-glucose deprivation.2. The N2a cells were divided into5groups:normal control group, OGD4group,OGD4/R6group, OGD4/R12group,OGD4/R24group.3. Cellular viability was measured using the MTT assay.4. Confocal Immuno—fluorographs were taken to observe the intracellular location of MAN Ⅱ.5. Expression of MAN Ⅱ protein was detected by western blotting.6. Expression of MAN Ⅱ gene was detected by quantitative Real—Time polymerasechain reaction in every group.Result:1. The MTT of N2a cells decreased upon OGD compared with the control group(P<0.05). And with the prolongation of reperfusion time, MTT decreased significantly(P<0.01), indicating that the reperfusion reduced the activity of cells.2. Confocal Immuno—fluorographs revealed that MAN Ⅱ located in the cytoplasm and around the nucleus.3. The expression of MAN Ⅱ in protein was down a bit in the OGD4h group(P>0.05).When followed by reperfusion,the expression level was still low in the OGD4h/R6h and OGD4h/R12h groups(P>0.05). But the expression in protein was decreased remarkably in OGD4h/R24h group (P<0.05).4. The expression of MAN Ⅱ in mRNA was down a bit in the OGD4h group(P>0.05).In the course of reperfusion,the expression of MAN Ⅱ had no significant change in the OGD4h/R6h and OGD4h/R12h groups (P>0.05).But the expression level of MAN Ⅱ mRNA decreased remarkably in OGD4h/R24h group (P<0.05).Conclusion:The expression of MAN Ⅱ decreased upon OGD/R treatment, indicating that OGD/R damaged the function of glycosylation of Golgi apparatus. |
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