亚低温预处理对谷氨酸诱导原代大鼠皮质神经细胞损伤的保护作用  

The protective effect of mild hypothermia pretreatment against injury to primary cultured cortical neurons induced of rat by glutamate

作　　者：薄丰山[1] 王迪芬[1] 刘文悦[2] 付江泉[1] 

机构地区：[1]贵阳医学院附属医院重症医学科,贵州550004 [2]河北沧州市人民医院急诊ICU

出　　处：《中华危重病急救医学》2014年第4期264-268,共5页Chinese Critical Care Medicine

基　　金：贵州省科技计划基金项目（20103079）;贵州省贵阳市科学技术计划基金项目（2010116）

摘　　要：目的 观察亚低温预处理对体外环境下培养大鼠大脑皮质细胞发生类缺血/再灌注（I/R）损伤的保护作用;比较亚低温单用或联合药物预处理的保护作用.方法 选择出生24 h内SD大鼠的大脑皮质细胞,体外培养至第3天,用阿糖胞苷（2.5 mg/L）培养基换液1次;培养6d后随机分为空白对照组、谷氨酸损伤组（加入含200 μmol/L谷氨酸的无血清培养基作用0.5 h）、亚低温预处理组（谷氨酸损伤前1d于33.5℃低温下培养24 h）、亚低温联合依达拉奉预处理组和亚低温联合丙泊酚预处理组（谷氨酸损伤前1d分别更换为100 μmol/L依达拉奉培养基和3 mg/L丙泊酚培养基后,再低温培养24 h）.各组更换正常培养基24 h后检测细胞存活率[四甲基偶氮唑盐（MTT）比色法]、细胞凋亡率、c-fos蛋白表达,苏木素-伊红（HE）染色后显微镜下观察细胞形态改变,铀-铅染色后透射电镜下观察细胞超微结构改变.结果 谷氨酸损伤组细胞存活率明显低于空白对照组[（0.20±0.02）％比（0.97±0.03）％,P＜0.01],细胞凋亡率和c-fos蛋白表达均明显高于空白对照组[细胞凋亡率：（9.85±0.76）％比（0.94±0.20）％,c-fos（ng/L）：6.96±0.75比1.65±0.59,均P＜0.01].亚低温预处理可明显逆转谷氨酸损伤细胞的存活率、凋亡率及c-fos水平,且以亚低温联合丙泊酚预处理作用最为明显[存活率：（0.80±0.04）％比（0.20±0.02）％,凋亡率：（2.26±0.54）％比（9.85±0.76）％,c-fos（ng/L）：2.98±0.46比6.96±0.75,均P＜0.01].显微镜下观察细胞形态发现,空白对照组大部分细胞形态大致正常;谷氨酸损伤组较多细胞质呈深红色,核呈蓝黑色,同缩状态,轴突萎缩明显,个别细胞突起断裂,细胞数量锐减;各预处理组细胞数量减少不明显,可见少量凋亡细胞.透射电镜下观察细胞超微结构发现,空白对照组神经细胞超微结构无明显改变;谷氨酸损伤组细胞质内细胞器Objective To investigate the effect of mild hypothermia preconditioning against ischemia/ reperfusion （I/R） injury of cultured primary cortical rats neurons,and to compare the protective effect of mild hypothermia only and with its combination with drugs.Methods Cortical neurons of neonatal Sprague-Dawley （SD） rat within 24 hours after birth were harvested and cultured in vitro.On the 3rd day,the cells were cultured in a medium containing 2.5 mg/L cytosine arabinoside to inhibit the growth of glial cells and fibroblast.Having cultured for 6 days they were randomly divided into blank control group,glutamate damaged group （cultured with 200 μmol/L glutamate for 0.5 hour after washing）,mild hypothermia preconditioning group （cultured under 33.5 ℃ for 24 hours before injury induced by glutamate）,mild hypothermia combining with edaravone preconditioning group,and the hypothermia combining with propofol preconditioning group （medium containing 100 μmol/L edaravone and 3 mg/L propofol）.They were cultured under 33.5 ℃ for 24 hours before injury induced by glutamate.After 24 hours of culturing in various medium,apoptosis ratio was observed by flow cytometry.Cell surviving rate was determined with methylthiazolete trazolium （MTT）,c-fos protein expression was assayed,and morphologic change of cells with hematoxylin-eosin （HE） staining under the microscope,and uhrastructure changes were observed after uranyl acetate and lead citrate staining under transmission electron microscope.Results The apoptosis ratio and c-fos protein in glutamate damaged group were significantly higher than those in blank control group [apoptosis ratio：（9.85 ± 0.76）％ vs.（0.94 ± 0.20）％,c-fos （ng/L）：6.96 ± 0.75 vs.1.65 ± 0.59,both P＜0.01],the cell surviving rate was significantly lower than that in blank control group [（0.20 ± 0.02）％ vs.（0.97 ± 0.03）％,P＜0.01].Mild hypothermia preconditioning reversed surviving rate,apoptosis ratio and c-fos protein,and the effect of hypothermia

关 键 词：亚低温 预处理 谷氨酸 脑皮质细胞 依达拉奉 丙泊酚 脑保护 

分 类 号：R454.5[医药卫生—治疗学]