CAJ | 학술논문

目的探讨星形胶质细胞缺氧复氧损伤后细胞周期蛋白D1( Cyclin D1)的表达以及亚低温干预对其的影响.方法原代培养取材于新生SD大鼠皮层脑组织的星形胶质细胞(astrocytes,AC),传至第3代自然纯化后采用神经胶质纤维酸性蛋白(GFAP)化学染色法对其进行鉴定,并将其分为正常(37℃)对照组(C)、常温(37℃)缺氧复氧组(H/R)、亚低温(32℃)干预缺氧复氧组(M+H/R).以95％ N2和5％ CO2的培养条件致细胞缺氧8h,其后恢复正常供氧,分别复氧4,12,20h等,建立缺氧复氧模型.观察各时点AC的形态学变化、台盼蓝染色法测定AC的存活率、细胞免疫荧光及Western-blot检测Cyclin D1的表达变化.结果 1.自然纯化后的第3代AC 95％以上呈GFAP阳性；2.缺氧8h后,常温组及亚低温组AC的形态和存活力变化均不明显,但随着复氧时间延长,两组细胞死亡数均显著增高(按复氧时间常温组分别为:12.87 ±2.76、31.55 ±3.00、46.40±8.50；低温组分别为:6.77 ±1.53、15.97 ±4.00、28.33±5.69),但亚低温组AC较常温组AC损伤明显减轻(P＜0.05),细胞存活力增强.3.免疫荧光和Western-blot检测示AC正常氧供时Cyclin D1表达无明显变化,缺氧8h后两组AC的Cyclin D1表达均增加,且随复氧时间延长呈明显增高趋势,亚低温组Cyclin D1的免疫表达较常温组显著减少.结论 Cyclin D1是星形胶质细胞缺氧复氧损伤的敏感指标,通过抑制Cyclin D1表达调控细胞周期可能是亚低温脑保护的作用机制之一.
목적 탐토성형효질세포결양복양손상후세포주기단백D1( Cyclin D1)적표체이급아저온간예대기적영향.방법 원대배양취재우신생SD대서피층뇌조직적성형효질세포(astrocytes,AC),전지제3대자연순화후채용신경효질섬유산성단백(GFAP)화학염색법대기진행감정,병장기분위정상(37℃)대조조(C)、상온(37℃)결양복양조(H/R)、아저온(32℃)간예결양복양조(M+H/R).이95％ N2화5％ CO2적배양조건치세포결양8h,기후회복정상공양,분별복양4,12,20h등,건립결양복양모형.관찰각시점AC적형태학변화、태반람염색법측정AC적존활솔、세포면역형광급Western-blot검측Cyclin D1적표체변화.결과 1.자연순화후적제3대AC 95％이상정GFAP양성；2.결양8h후,상온조급아저온조AC적형태화존활력변화균불명현,단수착복양시간연장,량조세포사망수균현저증고(안복양시간상온조분별위:12.87 ±2.76、31.55 ±3.00、46.40±8.50；저온조분별위:6.77 ±1.53、15.97 ±4.00、28.33±5.69),단아저온조AC교상온조AC손상명현감경(P＜0.05),세포존활력증강.3.면역형광화Western-blot검측시AC정상양공시Cyclin D1표체무명현변화,결양8h후량조AC적Cyclin D1표체균증가,차수복양시간연장정명현증고추세,아저온조Cyclin D1적면역표체교상온조현저감소.결론 Cyclin D1시성형효질세포결양복양손상적민감지표,통과억제Cyclin D1표체조공세포주기가능시아저온뇌보호적작용궤제지일.
Objective To explore the changes of astrocytes in vitro on the expression of Cyclin D1 under normal and hypoxia/reoxygenation conditions.Methods The primary astrocytes were isolated from the cortex of SD fetal rats(less than 24 hours) and identified by immunosytochemical method with anti-GFAP antibody after 3 passages.Astrocytes of passage 3 were divided into normal group ( control group),hypoxia/reoxygenation group ( H/R 37℃ ),and mild hypothermiaintervention group( H/R 32℃ )separately.Astrocytes from the later tow groups were reoxygenated with 4,12,and 20 hours separately after exposed to hypoxia conditions for 8 hours.Trypan blue staining was employed to detect the survival rates and immunofluorescence,western-blot were used to analyse the expressin of Cyclin D1 of of astrocytes of different groups and time points.Results 1.The GFAP positive astrocytes from passage 3 exceeded 95 ％.2.With regard to morphology and survival rates,there is no difference between astrocytes of normal and hypothermia groups after 8 hours exposure to hypoxia conditions.Reoxygenation could obviously rise astrocytes mortality with time went by ( H/R 37 ℃ group:12.87 ± 2.76 ( R4 ),31.55 ± 3.00 ( R12 ),46.40 ±8.50(R20) ;H/R 32℃ group:6.77 ± 1.53( R4),15.97 ±4.00(R12),28.33 ±5.69(R20) ;all P＜0.05).3.Immunofluorescence and western-blot revealed that reoxygenation increased Cyclin D1 expression markedly,which was proportional to the duration of reoxygenation.Mild hypothermia could reduce Cyclin D1 expression of astrocytes severely under reoxygenation condition.Conclusion Cyclin D1 expression can be regarded as a sensitive index of damage to astrocytes caused by hypoxia/reoxygenation conditions.