二氮嗪对软骨细胞氧化损伤的影响  

Diazoxide influence on cartilage cells from oxidative damage

作　　者：彭磊[1] 程少文[1] 沈跃[2] 应晓洲[2] 赵志蓉[2] 安涛[2] 周英勇[2] 程晓杰[2] 陈庆玉[2] 吕传柱[3] 

机构地区：[1]海南医学院附属医院,海南海口570100 [2]温州医科大学,浙江温州325000 [3]海南医学院,海南海口570100

出　　处：《中医正骨》2014年第5期3-8,共6页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：海南省医学科研重点课题(琼卫2012ZD-08);海南省医学科研课题(琼卫2013资助-039号)

摘　　要：目的:探讨二氮嗪对双氧水诱导的软骨细胞氧化损伤的影响。方法:取SD乳鼠膝关节软骨细胞进行原代培养,将对数生长期的软骨细胞分成5组,分别为阴性对照组(A组),双氧水组(B组),H2O2+0.1μmol·L-1二氮嗪组(C组),H2O2+1.0μmol·L-1二氮嗪组(D组),H2O2+10μmol·L-1二氮嗪组(E组);A组细胞不做特殊处理,B组用0.3 mmol·L-1双氧水在37℃恒温箱内孵育4 h,C、D、E、F组预先分别用0.1μmol·L-1DZ,1.0μmol·L-1DZ,10μmol·L-1DZ,100μmol·L-1在37℃的恒温箱孵育30分钟,PBS洗涤细胞,再用0.3 mmol·L-1双氧水在37℃恒温箱内孵育4小时。分别检测ABCDE各组细胞的细胞活性,ROS的量,细胞凋亡情况。结果:①MTT法检测各组细胞活性,细胞活性率从大至小依次为D组>C组>E组>F组>B组;②用活性氧探针DCFH-DA检测各组细胞中的ROS的量,ROS产量从多至少依次为B组>E组>D组>C组>A组;③流式细胞仪检测各组细胞凋亡情况,各组细胞凋亡率由大至小依次为B组>E组>D组>C组。④Western bolt检测各组软骨细胞中HIF-1α蛋白的表达,各组软骨细胞中HIF-1α蛋白表达量依次为C组>D组>E组>A组>B组。结论:二氮嗪可降低双氧水诱导的软骨细胞的凋亡率,同时也降低双氧水诱导的软骨细胞ROS的产生,并诱导软骨细胞产生HIF-1α蛋白,可能存在二氮嗪诱导HIF-1α的表达,引起下游相关基因的转录,总体提高软骨细胞对氧化损伤的耐受力,阻碍自由基诱导的软骨细胞的凋亡。Objective：To investigate diazoxide infection in hydrogen peroxide induced cartilage cells.Method：We obtain the carilage cells from the SD neonatal rat articular cartilages.The cartilage cells were divided into five groups,namely the control group （A）,the hydro-gen peroxide group （B）,H2 O2 ＋0.1 μmol·L^-1 DZ group （C）,H2 O2 ＋1 .0 μmol·L^-1 DZ group （D）,H2 O2 ＋1 0 μmol·L^-1 DZ group （E）;group A had no special treatment,group B incubated with 0.3 mmol/L hydrogen peroxide for 4 hours in 37 ℃incubator,the C,D,E group were preincubated with 0.1 μmol·L^-1 DZ 1 .0μmol·L^-1 DZ,1 0μmol·L^-1 DZ in a 37 ℃incubator incubated 30 minutes,then in-cubated with 0.3 mmol·L^-1 hydrogen peroxide solution in 37 ℃ incubator.The amount of the ROS and the apoptosis rate of cells in each group were detected.Results：①.Using the MTT assay,the order of the rate of cells activity：group A〉group D 〉group C 〉group E 〉group B.②.The ROS productions were detected by the DCFH-DA ,the order of ROS production as follow：group B〉group E〉group D〉group C〉group A.③.The apoptosis rates of cells were detected by Flow cytometer.The order of apoptosis rates of cartilage cells as fol-low：group B〉group E〉group D〉group C group.④.the expression of HIF-1αin each chondrocytes group detected by western blot,as follow：group C〉group D 〉group E 〉group A 〉group B.Conclusion：Diazoxide can reduce the apoptosis rates of hydrogen peroxide-induced chondrocytes,while also reducing the ROS production of the hydrogen peroxide-induced chondrocyte.It can be inferred that,diazox-ide may reduce the apoptosis rates and mortality of chondrocyte by reducing the ROS production of chondrocyte and increasing the expression of HIF-1αprotein.

关 键 词：二氮嗪 软骨细胞 活性氧 凋亡 

分 类 号：R965[医药卫生—药理学]