Effect of mild hypothermia on the expression of Cyclin D1 in astrocytes under hypoxia/ reoxygenation / 中华行为医学与脑科学杂志

ABSTRACT

ObjectiveTo explore the changes of astrocytes in vitro on the expression of Cyclin D1 under normal and hypoxia/reoxygenation conditions.MethodsThe 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 ℃ group12.87 ± 2.76 ( R4 ),31.55 ± 3.00 ( R12 ),46.40 ±8.50(R20) ;H/R 32℃ group6.77 ± 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 D1expression markedly,which was proportional to the duration of reoxygenation.Mild hypothermia could reduce Cyclin D1 expression of astrocytes severely under reoxygenation condition.ConclusionCyclin D1 expression can be regarded as a sensitive index of damage to astrocytes caused by hypoxia/reoxygenation conditions.