氧糖剥夺、复氧后损伤星形胶质细胞中MMP-9的表达变化与意义

目的通过观察金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)在氧糖剥夺、复氧培养后损伤星形胶质细胞中基质的表达变化并抑制其活性,探讨其在氧糖剥夺、复氧后细胞损伤中的意义。方法将星形胶质细胞分为正常组、模型组和GM6001组。模型组细胞在无糖DMEM培养基,1%O2的培养条件下行3、5、7 h时长的氧糖剥夺后复氧至24 h。GM6001组细胞在5 h氧糖剥夺、复氧的过程中加入外源性基质金属蛋白酶抑制剂(GM6001,10μmol/ml),复氧至24 h。用RT-PCR测定不同时长氧糖剥夺后星形胶质细胞MMP-9 mRNA表达变化,ELISA法测定细胞培养上清液中MMP-9蛋白的浓度变化;用光学显微镜观察各组的细胞形态变化,LDH漏出率、CCK-8法检测细胞的损伤情况及存活率。结果①RT-PCR及ELISA法检测示:与正常组相比,氧糖剥夺、复氧后,模型组细胞MMP-9 mRNA表达量和细胞培养上清液中MMP-9蛋白的浓度均有所增加(P<0.05),随着氧糖剥夺时间的延长,均呈现不断上升趋势;②与正常组相比,氧糖剥夺、复氧后,模型组细胞明显损伤,随着氧糖剥夺时间的延长,细胞损伤加重,光镜下细胞出现明显肿胀,并有部分细胞呈凝固性坏死,LDH漏出率不断增高(P<0.01)、存活率则逐渐下降(P<0.05,P<0.01),且培养上清MMP-9蛋白浓度与细胞LDH漏出率的增高程度呈正相关(r=0.693,P<0.05);与模型组相比,5 h氧糖剥夺、复氧后,GM6001组细胞形态学损伤表现明显减轻,LDH漏出率水平也有明显下降(P<0.01),细胞存活率则显著上升(P<0.05)。结论 MMP-9过表达可能是氧糖剥夺、复氧后引起细胞损伤的重要因素之一。

Significance of MMP-9 expression in astrocytes after oxygen-glucose deprivation/reoxygenation in vitro

Objective To investigate the significance of matrix metalloproteinase-9(MMP-9) expression in astrocytes after oxygen-glucose deprivation/reoxygenation(OGD/R) in vitro.Methods Astrocytes were divided into control group,OGD/R group and GM6001 group.Astrocytes in OGD/R group were treated with OGD for 3,5,and 7 h,respectively,and then reoxygenated for 24 h.In GM6001 group,astroctyes were treated with the inhibitor of matrix metalloproteinase(GM6001,10 μmol/ml) during 5 hours’ OGD/R.The expression of MMP-9 at mRNA and protein levels in the astrocytes were detected after 24 hours’ reoxygenation by RT-PCR and ELISA.The morphological alterations of cells in each group were observed by light microscopy.The cell injury was inspected by LDH activity(LDH %) and Cell Counting Kit-8(CCK-8).Results Compared with normal group,as the OGD time prolonged,the expression of MMP-9 at mRNA and protein levels were increased gradually(P<0.05) after OGD/R,which were further increase with time elapse.OGD resulted in in significant swelling astrocytes and obviously injured with time prolonged,and some cells seemed to be coagulation necrosis,the LDH % was increased(P<0.01) and the survival rate was decreased severely(P<0.05,P<0.01).Protein level of MMP-9 in the supernatant was positively correlated with LDH %(r=0.693,P<0.05).Compared with OGD/R group,cell injury following 5 hours’ OGD/R was remarkably decreased,as survival rate was raised significantly.Conclusion Over-expression of MMP-9 might be a important factor in OGD/R-induced cell injury.