血红蛋白诱导的HO-1对离体星形胶质细胞损伤作用的研究  

The role of heme oxygenase-1 on hemoglobin-induced astrocytes injury in vitro

作　　者：陈浩[1] 王兴启[2] 崔桂云[1] 赵辉[3] 宋远见[4] 高丽[5] 荆佳[1] 沈霞[1] 

机构地区：[1]徐州医学院附属医院神经内科,江苏徐州221004 [2]南京大学生命科学学院,江苏南京210093 [3]徐州市中心医院神经内科,江苏徐州221009 [4]徐州医学院神经生物学教研室,江苏徐州221004 [5]南京医科大学附属脑科医院神经内科,江苏南京210029

出　　处：《中国现代医学杂志》2011年第31期3847-3850,3855,共5页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30800446);江苏省教育厅课题(No:09KJB310016);江苏省脑病生物信息重点实验室开放课题(No:Jsbl0901)

摘　　要：目的探讨血红蛋白(hemoglobin,Hb)诱导的血红素氧合酶-1(heme oxygenase-1,HO-1)对离体星形胶质细胞的损伤作用。方法体外培养纯化获得星形胶质细胞,采用荧光标记胶质纤维酸性蛋白(glialfibrillary acidic protein,GFAP)鉴定细胞;实验分为正常组、Hb组及HO-1活性抑制剂锌原卟啉(ZnPPIX)+Hb组。Hoechst 33342及CCK-8法检测细胞损伤情况;Western blot检测HO-1、p-JNK、JNK、Bcl-2及Bax等蛋白的表达情况;RT-PCR检测HO-1的mRNA表达情况。结果 GFAP鉴定显示97%以上的细胞为星形胶质细胞;HO-1检测结果显示:40μmol/L的Hb可显著诱导HO-1 mRNA及蛋白的表达(P<0.001);Hoechst33342及CCK-8检测结果显示:40μmol/L的Hb对离体星形胶质细胞具有损伤作用,而ZnPPIX可明显减轻上述损伤;Western blot检测结果显示:HO-1被诱导高表达时p-JNK及Bax蛋白的表达水平也显著升高而Bcl-2蛋白的表达水平显著下降(P<0.01),ZnPPIX处理后与Hb组相比p-JNK及Bax蛋白表达水平有所降低而Bcl-2蛋白表达水平则有所升高(P<0.05)。结论 40μmol/L Hb诱导的HO-1对离体星形胶质细胞具有损伤作用。【Objective】 To investigate the effect of heme oxygenase-1（HO-1） on hemoglobin-induced astrocytes injury in vitro.【Methods】 Astrocytes were cultured in vitro and identified by immunofluorescent staining with GFAP antibody.They were divided into control group,hemoglobin（Hb） 40 μM group and Hb 40 μM ＋ ZnPPIX group.The morphology changes of astrocytes were observed by using Hoechst 33342 staining.Besides,the survival quality of astrocytes was measured by WST-8 assay.The proteins expression of HO-1,p-JNK,Bax and Bcl-2 were quantitated by western blotting.In addition,the mRNA of HO-1 was measured by RT-PCR.【Results】 Immunofluorescence staining showed that about 97% of the purified cells were astrocytes.Western blotting and RT-PCR assay showed that the protein and mRNA expression of HO-1 were significantly up-regulated by Hb（40μmol/L）（P 0.001）.Compared with the control group,the survival quality was dramatically reduced when astrocytes were incubated in the medium containing Hb.Compared with Hb group,the quality was significantly enhanced in Hb＋ZnPPIX group.Furthermore,the levels of Bax and p-JNK were significantly up-regulated while Bcl-2 was significantly down-regulated in Hb group（P 0.01）,and the effect were attenuated when cells were pretreated by ZnPPIX（P 0.05）.【Conclusions】 Astrocytes could be injured by 40μmol/L Hb-induced HO-1 in vitro.

关 键 词：血红蛋白 血红素氧合酶-1 星形胶质细胞 Bcl-2 JNK 

分 类 号：R743[医药卫生—神经病学与精神病学]