波动性高糖对氧糖剥夺/再复供神经元存活的影响  

Effect of intermittent high glucose on oxygen-glucose deprivation/refurnish neuronal survival

作　　者：陈秋月 张丹红 王莉 张叶青 陈慧红 陈富强 何泽宝 Chen Qiuyue;Zhang Danhong;Wang Li;Zhang Yeqing;Chen Huihong;Chen Fuqiang;He Zebao(Department of Neurology,Taizhou Central Hospital (Taizhou University Affiliated Hospital),Taizhou 318000,Zhejiang, China;Department of Science and Education,Taizhou Central Hospital (Taizhou University Affiliated Hospital),Taizhou 318000,Zhejiang,China;Department of Clinical Laboratory,Taizhou Central Hospital (Taizhou University Affiliated Hospital),Taizhou 318000,Zhejiang,China;Department of Public Health Unit,Taizhou Enze Medical Center Enze Hospital,Taizhou 318050,Zhejiang,China)

机构地区：[1]台州市中心医院(台州学院附属医院)神经内科,浙江台州318000 [2]台州市中心医院(台州学院附属医院)科教处,浙江台州318000 [3]台州市中心医院(台州学院附属医院)检验科,浙江台州318000 [4]台州恩泽医疗中心恩泽医院公共卫生中心,浙江台州318050

出　　处：《中华危重病急救医学》2019年第1期61-66,共6页Chinese Critical Care Medicine

基　　金：浙江省台州市科技项目(15yw02-3);浙江省台州市医学科研项目(TZSYXH15-19);浙江省台州恩泽医疗中心(集团)科研基金项目(15EZB8).

摘　　要：目的探讨波动性高糖对氧糖剥夺/再复供(OGD/R)后神经元存活的影响。方法原代培养小鼠海马神经元,待细胞融合达到80%左右时传代培养。取对数生长期细胞置于缺氧状态(37℃、5%CO2、95%N2)的培养箱中模拟细胞缺氧,将培养液更换为无糖Hank平衡盐溶液(HBSS)模拟细胞缺糖;设常糖常氧对照组。细胞缺氧缺糖处理6h后,倒置相差显微镜下观察细胞形态,用CCK-8细胞增殖检测试剂盒检测细胞活性,再进行分组实验。将细胞随机分为4组,分别采用含不同浓度葡萄糖培养基,置于常氧、5%CO2、37℃培养条件下继续培养72h,制备OGD/R模型模拟细胞缺血/再灌注。低糖对照组用含5.5mmol/L葡萄糖的培养基培养;恒定高糖组用含33.0mmol/L葡萄糖的培养基培养;波动高糖组日间用含33.0mmol/L葡萄糖的培养基培养3h,再用含5.5mmol/L葡萄糖的培养基培养2h,交替3次,夜间用含33.0mmol/L葡萄糖的培养基过夜;高渗对照组用含5.5mmol/L葡萄糖的培养基与甘露醇配制,渗透压与恒定高糖组相同,有效糖浓度与常糖常氧对照组相同,以排除高糖培养基引起的渗透压变化对结果的影响。各组细胞培养72h后,倒置相差显微镜下观察细胞形态学改变;用CCK-8试剂盒测定细胞活性;用流式细胞术测定细胞凋亡率。结果倒置相差显微镜下显示,常糖常氧对照组细胞饱满,折光性强,胞核明显,突起清晰,细胞活性较高。缺氧缺糖处理6h后,细胞皱缩,折光性差,核质不清,突起不清晰,细胞活性较常糖常氧对照组明显降低(A值:0.34±0.06比1.09±0.06,P<0.01),说明细胞氧糖剥夺(OGD)模型制备成功。更换不同浓度葡萄糖继续培养72h后,低糖对照组细胞皱缩,胞膜不完整,核质不清,坏死细胞较多;恒定高糖组细胞折光性差,大量细胞漂浮,胞核不明显;而波动高糖组细胞形态正常,细胞折光性稍降低,坏死细胞较少;高渗对照组与恒定高糖组细胞状态接近。与�Objective To investigate the effect of intermittent high glucose on oxygen-glucose deprivation/refurnish (OGD/R) neuronal survival. Methods The primary cultured hippocampal neurons of mice were sub-cultured when the cell fusion reached about 80%. Cells in logarithmic growth phase were placed in a hypoxic incubator (37 ℃, 5% CO2, 95% N2) to simulate cell hypoxia. The culture medium was replaced by glucose-free Hank equilibrium salt solution (HBSS) to simulate cell hypoglycemia. The normal glucose and oxygen control group was set up. Cell morphology was observed under inverted phase contrast microscope after 6 hours of hypoxia and hypoglycemia treatment, and cell viability was detected by CCK-8 cell proliferation assay kit, and then grouping experiment was carried out. The cells were randomly divided into four groups. The cells were cultured in different concentration glucose medium under normal oxygen, 5% CO2 and 37 ℃ for 72 hours to prepare OGD/R model of cell ischemia/reperfusion. The low-glucose control group was cultured in medium containing 5.5 mmol/L glucose. The constant high-glucose group was cultured in medium containing 33.0 mmol/L glucose. The intermittent high-glucose group was cultured in medium containing 33.0 mmol/L glucose for 3 hours then in medium containing 5.5 mmol/L glucose for 2 hours alternately for 3 times during the day, and overnight in medium containing 33.0 mmol/L glucose at night. The hyperosmotic control group was made up of 5.5 mmol/L glucose medium and mannitol. The osmotic pressure was the same as that of the constant high-glucose group, and the effective glucose concentration was the same as that of the normal glucose and oxygen group, so as to eliminate the effect of osmotic pressure changes caused by the high-glucose medium on the results. Cell morphology was observed under inverted phase contrast microscope after 72 hours of cell culture in each group. Cell viability was measured by CCK-8 kit, and apoptotic rate was measured by flow cytometry. Results The inverted phase con

关 键 词：波动性高糖 氧糖剥夺/再复供 海马神经元 存活 

分 类 号：R743[医药卫生—神经病学与精神病学]