CaMK Ⅱ活性对H_2O_2所致SK-N-SH细胞氧化应激损伤的影响  

Effect of CaMK Ⅱ on oxidative stress-dependent impairment induced by H_2O_2 in SK-N-SH cells

作　　者：于剑锋[1,2] 耿霞 王钢 陈静 高文洁[1] 黄科昌[1] 

机构地区：[1]潍坊医学院麻醉学系,潍坊261053 [2]潍坊医学院认知神经科学实验室,潍坊261053 [3]东营市人民医院疼痛科,东营257091

出　　处：《解剖学杂志》2017年第5期535-538,共4页Chinese Journal of Anatomy

基　　金：国家自然科学基金(81241041);山东省自然科学基金(ZR2012HM077);潍坊医学院前沿探索重点研究基金(K11TS1002)

摘　　要：目的:评价钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)活性对过氧化氢所致人源性神经母细胞瘤细胞氧化应激损伤的影响。方法:SK-N-SH细胞生长至对数期时,接种于96孔培养板或6孔培养板,采用随机数字表法,将其分为4组:正常对照组,培养基中添加H_2O_2组(H_2O_2组),培养基中只添加CaMKⅡ活性抑制剂KN93组(KN93组),培养基中同时添加H_2O_2和KN93组(H_2O_2+KN93组),倒置显微镜观察细胞形态学,采用CCK-8法测定细胞活力,采用Fura-2/AM荧光法检测细胞内Ca^(2+)浓度([Ca^(2+)]_i),采用免疫印迹测定CaMKⅡ和磷酸化CaMKⅡ的表达,采用RT-PCR法检测CaMK mRNA表达。结果:与正常对照组比较,H202组细胞体呈圆形的细胞和脱落细胞增加,细胞突起长度缩短,细胞存活率降低,[Ca^(2+)]_i升高,磷酸化CaMKⅡ表达上调,CaMKⅡmRNA表达下调。与H_2O_2组比较,H_2O_2+KN93组脱落细胞明显减少,突起变长,细胞存活率增加,[Ca^(2+)]_i降低,磷酸化CaMKⅡ表达下调,CaMKⅡmRNA表达水平上调。结论:CaMKⅡ增强了H_2O_2诱导的人源性神经母细胞瘤细胞氧化应激损伤。Objective：To evaluate the effect of Ca2＋-calmodulin-dependent protein kinase Ⅱ （CaMKⅡ）on oxidative stress-dependent impairment induced by H2 O2 in SK-N-SH cells.Methods：SK-N-SH cells in logarithmic phase of generation were cultured on 96-well culture plate or 6-well culture plate.Using the random number table method,SK-N-SH cells were randomly divided into 4 groups：normal control group （control group）,H2 O2-pretreated group （H2 O2 group）,CaMKⅡinhibitor KN93 group （KN93 group）,and H2 O2 plus KN93 group （H2 O2＋KN93 group）.Cell morphological analysis was assayed by inverted phase contrast microscope.Cell viability was determinated by CCK-8 method.Intracellular calcium concentration [Ca2＋]i was tested by the Ca2＋-sensitive fluorometric dye Fura-2.Protein expression of CaMKⅡ and CaMKⅡmRNA were analysed by Western blotting and RT-PCR respectively.Results：Compared with control group,round cell body and exfoliated cell increased,cell processes were shortened,cell survival rate decreased,[Ca2＋]i and p-CaMKⅡ expression increased,CaMKⅡ mRNA expression decreased in H2 O2 group.Compared with H2 O2 group,H2 O2＋KN93 group showed small exfoliated cells,increased cell survival rate,decreased [Ca2＋]i ,high expression level of p-CaMKⅡand CaMKⅡmRNA.Conclusion：CaMKⅡmay enhance oxidative stress-dependent impairment induced by H2 O2 in SK-N-SH cells.

关 键 词：过氧化氢 氧化应激 钙钙调蛋白依赖性蛋白激酶2型 神经母细胞瘤 

分 类 号：R363[医药卫生—病理学]