CAJ | 학술논문

目的评价线粒体分裂在大鼠海马神经元缺氧复氧损伤中的作用.方法选择出生24 h内的SD大鼠,原代培养海马神经元,以7×105/ml的细胞密度接种到25 mm×25 mm培养瓶中,采用随机数字表法,将其分为3组(n=18):对照组(C组)细胞不进行任何处理；细胞缺氧复氧组(I/R组)细胞在缺氧前加入赋形剂[二甲基亚砜(DMSO),终浓度＜0.1％]孵育40 min;线粒体分裂抑制剂组(M组)细胞在缺氧前加入50 μmol/L线粒体分裂抑制剂(溶于DMSO,DMSO浓度＜0.1％)孵育40 min.采用氧糖剥夺法建立大鼠海马神经元缺氧复氧模型,缺氧6h复氧20 h后用ELISA法检测活性氧(ROS)水平、用流式细胞仪检测细胞凋亡情况,Western blot法检测线粒体分裂蛋白、Bcl-2和Bax蛋白的表达水平,并计算Bcl-/Bax蛋白比值.结果与C组比较,I/R组海马神经元ROS含量、细胞凋亡率升高,线粒体分裂蛋白和Bax蛋白的表达上调,而Bcl-2蛋白表达下调,Bcl-2/Bax蛋白比值降低(P＜0.05).与I/R组比较,M组海马神经元ROS含量、细胞凋亡率降低、线粒体分裂蛋白和Bax蛋白的表达下调,Bcl-2蛋白表达上调,Bcl-2/Bax蛋白比值升高(P＜0.05).结论线粒体分裂可通过线粒体介导的凋亡通路参与大鼠海马神经元缺氧复氧损伤.
목적 평개선립체분렬재대서해마신경원결양복양손상중적작용.방법 선택출생24 h내적SD대서,원대배양해마신경원,이7×105/ml적세포밀도접충도25 mm×25 mm배양병중,채용수궤수자표법,장기분위3조(n=18):대조조(C조)세포불진행임하처리；세포결양복양조(I/R조)세포재결양전가입부형제[이갑기아풍(DMSO),종농도＜0.1％]부육40 min;선립체분렬억제제조(M조)세포재결양전가입50 μmol/L선립체분렬억제제(용우DMSO,DMSO농도＜0.1％)부육40 min.채용양당박탈법건립대서해마신경원결양복양모형,결양6h복양20 h후용ELISA법검측활성양(ROS)수평、용류식세포의검측세포조망정황,Western blot법검측선립체분렬단백、Bcl-2화Bax단백적표체수평,병계산Bcl-/Bax단백비치.결과 여C조비교,I/R조해마신경원ROS함량、세포조망솔승고,선립체분렬단백화Bax단백적표체상조,이Bcl-2단백표체하조,Bcl-2/Bax단백비치강저(P＜0.05).여I/R조비교,M조해마신경원ROS함량、세포조망솔강저、선립체분렬단백화Bax단백적표체하조,Bcl-2단백표체상조,Bcl-2/Bax단백비치승고(P＜0.05).결론 선립체분렬가통과선립체개도적조망통로삼여대서해마신경원결양복양손상.
Objective To evaluate the role of mitochondrial fission in anoxia-reoxygenation injury to rat hippocampal neurons.Methods Neurons were enzymatically isolated from hippocampi of newborn Sprague-Dawley rats (less than 24 h old).The primary hippocampal neurons were cultured and seeded in 25 mm × 25 mm culture flasks at a density of 7 × 105/ml.The cultured neurons were randomly assigned into 3 groups (n =18 each) using a random number table:control group (C group),anoxia-reoxygenation group (I/R group),and mitochondrial fission inhibitor mdivi-1 group (M group).In group I/R,the vehicle dimethyl sulfoxide (DMSO,final concentration ＜ 0.1％) was added prior to anoxia and the cells were then incubated for 40 min.In group M,mdivi-1 (dissolved in DMSO,final concentration of DMSO ＜ 0.1％) was added prior to anoxia and the cells were then incubated for 40 min.The hippocampal neurons were subjected to oxygen-glucose deprivation (OGD) for 6 h followed by restoration of O2 supply for 20 h.After 20 h of reoxygenation,the level of reactive oxygen species (ROS) (by ELISA),cell apoptosis (using flow cytometry),and expression of mitochondrial fission protein Drp1,Bcl-2 and Bax (by Western blot) were measured.The apoptosis rate and the ratio of Bcl-2 to Bax were calculated.Results Compared with C group,ROS content and apoptosis rate were significantly increased,the expression of Drp1 and Bax was up-regulated,the expression of Bcl-2 was down-regulated,and the ratio of Bcl-2 to Bax was decreased in I/R group (P ＜ 0.05).Compared with I/R group,ROS content and apoptosis rate were significantly decreased,the expression of Drp1 and Bax was down-regulated,the expression of Bcl-2 was up-regulated,and the ratio of Bcl-2 to Bax was increased in M group (P ＜ 0.05).Conclusion Mitochondrial fission is involved in anoxia-reoxygenation injury to rat hippocampal neurons via mitochondria-mediated apoptotic pathway.