miRNA-99a对过氧化氢诱导neuro-2a细胞氧化损伤的影响  

Effects of microRNA-99a on Neuro-2a cells against oxidative injury induced by hydrogen peroxide

作　　者：陶真[1] 王荣亮[1] 赵海苹[1] 罗玉敏[1] 

机构地区：[1]首都医科大学宣武医院脑血管病研究室,北京100053

出　　处：《首都医科大学学报》2014年第3期305-309,共5页Journal of Capital Medical University

基　　金：国家自然科学基金项目(81201028;81271461)~~

摘　　要：目的研究microRNA-99a(miR-99a)对过氧化氢所诱导神经细胞neuro-2a氧化损伤的影响。方法常规培养neuro-2a细胞并分为3组:正常对照组,过氧化氢100μmol/L刺激组,miR-99a预处理组(转染miRNA-99a mimics+过氧化氢100μmol/L刺激),用CCK-8试剂盒检测细胞存活率,生化试剂盒检测还原型烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NADH)含量和总超氧化物歧化酶(total superoxide dismutase,T-SOD)、锰超氧化物歧化酶(manganese superoxide dismutase,MnSOD)活性,Western blotting检测突触小体相关蛋白(synaptosoma associated protein of molecular mass 25 000,SNAP25)及Mn-SOD、细胞外超氧化物歧化酶(extracellular SOD,EC-SOD)的蛋白表达水平。结果与正常对照组相比,过氧化氢刺激的2组细胞存活率明显下降,分别为85%和89%(P<0.05),但miR-99a预处理组的细胞存活率下降程度较小仅为11%(P<0.05)。进一步研究发现,过氧化氢刺激引起细胞T-SOD、Mn-SOD活性降低(P<0.05),转染miR-99a mimics不但能增强T-SOD和Mn-SOD的活性,还能促进Mn-SOD和EC-SOD的表达(P<0.05)。过氧化氢导致细胞中NADH含量降低(P<0.05),miR-99a能够增加NADH含量甚至高于正常细胞水平(P<0.05)。miR-99a还有增加neuro-2a细胞表达SNAP25的趋势。结论 miR-99a对过氧化氢诱导neuro-2a细胞引起的氧化损伤具有保护作用。Objective To investigate the effects of microRNA-99a（miR-99a） on neuro-2a cells against oxidative injury induced by hydrogen peroxide（H2 O2 ）. Methods Neuro-2a cells were divided into 3 groups by different treatments： control group, control＋H2 O2 （100 μmol/ L） group and miR-99a mimics＋H2 O2 group. Cell viability was measured by cell counting kit-8. Total superoxide dismutase （T-SOD） activity, manganese superoxide dismutase （ Mn-SOD） activity, and content of reduced nicotinamide adenine dinucleotide （NADH） were assayed by a variety of biochemical kits according to the instructions respectively. Protein expression levels of synaptosoma associated protein of molecular mass 25 000（SNAP25）, Mn-SOD and extracellular SOD（EC-SOD） were assessed by Western blotting. Results Compared with control group, cell viability of neuro-2a in the control ＋ H2 O2 group and miR-99a ＋ H2 O2 group, decreased significantly to 85% and 89% respectively upon hydrogen peroxide stimulation（P〈0. 05）. However, miR-99a＋H2 O2 group appeared to have less reduction of 11% than control＋H2 O2 group（P〈0. 05）. Further studies showed that enzyme activities of T-SOD and Mn-SOD were depressed in control＋H2 O2 group in comparison with control group（ P〈0. 05）. Whereas, with pretreatment of miR-99a mimics transfection, T-SOD and Mn-SOD activities were enhanced to a significantly higher level, relative to the normal level of control group（P〈0. 05）, as well as greatly increased protein expression of Mn-SOD and EC-SOD, in contrast to control group and control＋H2 O2 group（P〈0. 05）. Besides, NADH content of neuro-2a in control＋H2 O2 group was reduced compared with control group（P〈0. 05）, while in miR-99a＋H2 O2 group, NADH content exceeded the normal level of control group（ P〈0. 05）. It was also found that protein expression of SNAP25 increased in the two groups of hydrogen peroxide stimulation. And miR-99a＋H2 O2 group had a tendency of more increase in protein expres

关 键 词：miR-99a neuro-2a 过氧化氢 氧化损伤 

分 类 号：R743.9[医药卫生—神经病学与精神病学]