p38MAPK在体外培养星形胶质细胞缺氧/复氧损伤中的表达

目的 建立SD大鼠星形胶质细胞缺氧复氧损伤模型,探讨p38MAPK活性变化与星形胶质细胞损伤的关系.方法 体外培养新生SD大鼠星形胶质细胞,实验设正常对照组（N）、SB203580组（SB组,10 μmol/L）、缺氧/复氧组（H/R组）和缺氧/复氧组＋SB203580阻断p38MAPK组（H/R＋SB组）.应用MTT法、WB法、ELISA法检测缺氧4 h、8 h、复氧6 h、12 h、24 h、48 h时细胞存活率,p38MAPK、p-p38（磷酸化p38MAPK）及TNF-α的变化.结果 培养星形胶质细胞GFAP阳性表达率大于97%.缺氧/复氧使星形胶质细胞活力降低,SB203580阻断p38MAPK细胞活力高于H/R组,各组星形胶质细胞总p38MAPK水平无显著变化,缺氧复氧干预后p-p38表达上调,TNF-α水平显著增高.用SB203580阻断p38MAPK通路后,SB＋H/R组较H/R组p-p38、TNF-α水平降低.SB组总p38MAPK、p-p38、TNF-α水平与N组比较无显著变化.结论 p38MAPK信号通路参与了星形胶质细胞缺氧复氧损伤过程.

Effect of P38MAPK signaling pathway on astrocytes impairment under hypoxic/reoxygenation

Objective To establish astrocytes hypoxia-reoxygenation induced injury model,observe the astrocytes impairment under hypoxic/reoxygenation and investigate the relationship between p38MAPK signaling and astrocytes induced by hypoxic/reoxygenation in vitro.Methods Reference to the methods of McCarthy,the primary cells from the cortex of SD fetal rat,the cultured astrocytes were divided into four groups： normal contrast group（N）,hypoxia/reoxygenation group（H/R）,SB203580 group（SB）,and hypoxia/reoxygenation＋the SB203580 interrupt p38MAPK group（H/R＋SB）.SB203580 with a final concentration of 10 μmol/L,to add in 1 hour before hypoxia.After 4,8 hours of hypoxia,it began to reoxygenation.Each group reoxygenation 6 hours,12 hours,24 hours,48 hours.MTT was used to detect the cell activity.Western-blot analysis of semi-quantitative detected the p38 and p-p38 expression.TNF-α secretion of astrocytes was measured by TNF-α ELISA.Results Homogeneity of the cell populations was evaluated by immunofluorescence examinations with ant-glia fibrillary acid protein（GFAP）,the number of GFAP positive cells exceeded 97%.Hypoxia/reoxygenation reduced the astrocyte activity,cell viability was higher than the H/R group with SB203580 block p38MAPK,no significant changes in total p38MAPK level among the four groups.p-p38 expression increased after the intervention of hypoxia and reoxygenation and TNF-α level was increased.When p38MAPK pathway was inhibited by SB203580 in the H/R＋SB group,the levels of p-p38 and TNF-α were reduced compared with H/R group.The levels of p38MAPK,p-p38 and TNF-α in the SB group had no changes compared with the normal group.Conclusion p38MAPK signaling pathway is involved in the process of astrocytes in hypoxia-reoxygenation injury