CAJ | 학술논문

目的评价血红素加氧酶-1(HO-1)对氧糖剥夺神经元细胞周期蛋白依赖激酶5(CDK5)-共济失调-毛细血管扩张突变基因(ATM)-P53信号转导通路的影响.方法培养7d的海马神经元,采用随机数字表法,将其随机分为4组(n=10):正常培养组(C组)、氧糖剥夺组(D组)、氧糖剥夺+血晶素(HO-1诱导剂)组(D+H组)和氧糖剥夺+血晶素+锌原卟啉(HO-1抑制剂)组(D+H+T组).C组采用正常培养方法培养.D组神经元进行缺糖、缺氧后复糖、复氧处理.D+H组神经元用10μmol/L血晶素处理24h后进行缺糖、缺氧后复糖、复氧处理.D+H+T组神经元同时用10μmol/L血晶素和10 μmol/L锌原卟啉处理24h后进行缺糖、缺氧后复糖、复氧处理.培养24h后,MTT法检测神经元存活情况,TUNEL法检测神经元凋亡情况,RT- PCR法检测HO-1 mRNA表达,Western blot 法检测HO-1、CDK5、ATM和P53蛋白表达.结果与C组比较,D组神经元HO-1 mRNA、HO-1、CDK5、ATM和P53蛋白表达上调,神经元存活率降低,神经元凋亡率升高(P＜0.01)；与D组比较,D+H组神经元HO-1 mRNA和HO-1蛋白表达上调,CDK5、ATM和P53蛋白表达下调,神经元存活率升高,神经元凋亡率降低(P＜0.01)；与D+H组比较,D+H+T组神经元HO-1 mRNA和HO-1蛋白表达下调,CDK5、ATM和P53蛋白表达上调,神经元存活率降低,神经元凋亡率升高(P＜0.01).结论 HO-1可通过阻断氧糖剥夺海马神经元CDK5- ATM-P53信号转导通路抑制神经元凋亡.
목적 평개혈홍소가양매-1(HO-1)대양당박탈신경원세포주기단백의뢰격매5(CDK5)-공제실조-모세혈관확장돌변기인(ATM)-P53신호전도통로적영향.방법 배양7d적해마신경원,채용수궤수자표법,장기수궤분위4조(n=10):정상배양조(C조)、양당박탈조(D조)、양당박탈+혈정소(HO-1유도제)조(D+H조)화양당박탈+혈정소+자원계람(HO-1억제제)조(D+H+T조).C조채용정상배양방법배양.D조신경원진행결당、결양후복당、복양처리.D+H조신경원용10μmol/L혈정소처리24h후진행결당、결양후복당、복양처리.D+H+T조신경원동시용10μmol/L혈정소화10 μmol/L자원계람처리24h후진행결당、결양후복당、복양처리.배양24h후,MTT법검측신경원존활정황,TUNEL법검측신경원조망정황,RT- PCR법검측HO-1 mRNA표체,Western blot 법검측HO-1、CDK5、ATM화P53단백표체.결과 여C조비교,D조신경원HO-1 mRNA、HO-1、CDK5、ATM화P53단백표체상조,신경원존활솔강저,신경원조망솔승고(P＜0.01)；여D조비교,D+H조신경원HO-1 mRNA화HO-1단백표체상조,CDK5、ATM화P53단백표체하조,신경원존활솔승고,신경원조망솔강저(P＜0.01)；여D+H조비교,D+H+T조신경원HO-1 mRNA화HO-1단백표체하조,CDK5、ATM화P53단백표체상조,신경원존활솔강저,신경원조망솔승고(P＜0.01).결론 HO-1가통과조단양당박탈해마신경원CDK5- ATM-P53신호전도통로억제신경원조망.
Objective To investigate the effect of heme oxygenase-1 (HO-1) on cyclin-dependent kinase 5 (CDK5)-ataxia telangiectasia mutated (ATM)-P53 signal transduction pathway in rat hippocampal neurons subjected to oxygen-glucose deprivation (OGD) injury.Methods Hippocampal neurons of newborn Wistar rats ( ＜ 48 h) were cultured for 7 days in vitro.The primary cultured neurons were randomly divided into 4 groups with 10 wells in each group:control group (group C),OGD (group D),OGD + hemin (HO-1 inducer) group (group D + H ) and OGD + hemin + zinc protoporphyrin ( HO-1 inhibitor) group ( group D + H + T).For OGD experiments,cultures were washed three times in a glucose-free balanced salt solution (BSS).They were then placed in deoxygenated glucose-free medium and sealed under 95％ N2-5％ CO2 in an anaerobic chamber equilibrated to 37°C and 100％ humidity for 45 min.OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 95％ air-5％ CO2.The OGD model was established after the neurons were preconditioned with hemin 10 μmol/L for 24 h in group D + H.The OGD model was established after the neurons were preconditioned with hemin 10 μmol/L and zinc protoporphyrin 10 μmol/L for 24 h in group D + H + T.After 24 h of culture,the neuronal viability,apoptosis rate,and expression of HO-1 mRNA and protein,and CDK5,ATM and P53 protein were detected.Results Compared with group C,the expression of HO-1 mRNA,and HO-1,CDK5,ATM and P53 protein was up-regulated,the neuronal viability was significantly decreased,and the apoptosis rate was significantly increased in group D (P ＜ 0.01 ).Compared with group D,the expression of HO-1 mRNA and protein was up-regulated,the expression of CDK5,ATM and P53 protein was down-regulated,the neuronal viability was significantly increased,and the apoptosis rate was significanlly decreased in group D + H ( P ＜ 0.01 ).Compared with group D + H,the expression of HO-1 mRNA and protein was down-regulated,the expression of CDK5,ATM and P53 protein was up-regulated,the neuronal viability was significantly decreased,and the apoptosis rate was significantly increased in group D + H + T ( P ＜ 0.01 ).Conclusion HO-1 can inhibit neuronal apoptosis through blocking CDK5-ATM-P53 signal transduction pathway in rat hippocampal neurons subjected to OGD injury.