摘要
目的探讨三氧化二砷(As2O3)诱导人神经母细胞瘤细胞(SH-SY5Y细胞)凋亡及叶酸(FA)和维生素B12(VB12)的保护作用。方法体外培养SH-SY5Y细胞,采用成组设计分为6组:对照组(正常培养)、砷暴露组(10.00μmol/L As2O3)、FA干预组(0.30 mmol/L FA+10.00μmol/L As2O3)、VB12干预组(0.06 mmol/L VB12+10.00μmol/L As2O3)、联合干预组(0.30 mmol/L FA+0.06 mmol/L VB12+10.00μmol/L As2O3)及试剂对照组(0.30 mmol/L FA+0.06 mmol/L VB12),各组细胞培养24 h(n=3)。通过流式细胞仪测定各组细胞凋亡率;透射电镜观察细胞超微结构变化;实时荧光定量PCR和蛋白质免疫印迹分别检测细胞凋亡相关指标B淋巴细胞瘤-2基因(Bcl-2)及Bcl-2相关X蛋白(Bax)的mRNA和蛋白表达情况;发光法检测细胞含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)3活性,并对以上指标进行统计分析。结果各组间细胞凋亡率比较,差异有统计学意义(F=213.036,P<0.05)。砷暴露组细胞凋亡率[(44.43±3.54)%]高于对照、FA干预、VB12干预、联合干预组[(1.80±0.06)%、(14.37±0.13)%、(19.10±1.56)%、(17.11±2.34)%,P均<0.05]。透射电镜下可见砷暴露组SH-SY5Y细胞凋亡小体增多、线粒体肿胀变性、染色质凝集等,FA和VB12单独及联合干预后细胞形态及细胞器改变明显改善。各组间细胞Bcl-2、Bax mRNA和蛋白表达水平比较,差异均有统计学意义(F=5.178、7.169,6.142、9.194,P均<0.05)。砷暴露组Bcl-2蛋白表达水平低于对照组(P<0.05),Bax mRNA和蛋白表达水平均高于对照组(P均<0.05);FA干预和联合干预组Bcl-2 mRNA和蛋白表达水平均高于砷暴露组(P均<0.05),VB12干预组Bcl-2 mRNA表达水平高于砷暴露组(P<0.05);FA干预、VB12干预和联合干预组Bax mRNA和蛋白表达水平均低于砷暴露组(P均<0.05)。各组间细胞Caspase 3活性比较,差异有统计学意义(F=84.604,P<0.05)。砷暴露组细胞Caspase 3活性高于对照、FA干预、VB12干预、联合干预组(P均<0.05)。结论砷暴露可导�
Objective To explore the arsenic trioxide(As2O3)-induced apoptosis of human neuroblastoma cells(SH-SY5Y cells)and the protection mechanisms of folic acid(FA)and vitamin B12(VB12).Methods SH-SY5Y cells were cultured in vitro and divided into six groups by group design:control group(normal cultured),arsenic exposed group(10.00μmol/L As2O3),FA intervention group(0.30 mmol/L FA+10.00μmol/L As2O3),VB12 intervention group(0.06 mmol/L VB12+10.00μmol/L As2O3),combined intervention group(0.30 mmol/L FA+0.06 mmol/L VB12+10.00μmol/L As2O3)and reagent control group(0.30 mmol/L FA+0.06 mmol/L VB12).Cells in each group were cultured for 24 h(n=3).Flow cytometry was used to determine the apoptosis rate of cells in each group.Transmission electron microscopy was used to observe the ultrastructural changes of the cells.The expression levels of mRNA and protein of apoptosis-related indicator B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X(Bax)were detected by fluorescence quantitative PCR and Western blotting.The activity of cysteinyl aspartate specific proteinase(Caspase)3 was detected by luminescent assay.The above indicators were statistically analyzed.Results There was statistically significant difference in the apoptosis rate among different groups(F=213.036,P<0.05).The apoptosis rate in arsenic exposed group[(44.43±3.54)%]was higher than that in control,FA intervention,VB12 intervention,and combined intervention groups[(1.80±0.06)%,(14.37±0.13)%,(19.10±1.56)%,(17.11±2.34)%,P<0.05].Under transmission electron microscope,the apoptotic bodies,mitochondria swelling and degeneration,chromatin agglutination were observed in SH-SY5Y cells exposed to arsenic.The morphological and organelle changes of SH-SY5Y cells were significantly improved after respective and combined intervention of FA and VB12.The expression levels of Bcl-2,Bax mRNA and protein were significantly different among different groups(F=5.178,7.169,6.142,9.194,P<0.05).The expression level of Bcl-2 protein in arsenic exposed group was lower than that in contro
作者
王乔雨
孙洪娜
周齐
王腾
高彦辉
孙殿军
Wang Qiaoyu;Sun Hongna;Zhou Qi;Wang Teng;Gao Yanhui;Sun Dianjun(Institute for Endemic Fluorosis Control,Center for Endemic Disease Control,Chinese Center for Disease Control and Prevention,Harbin Medical University,Harbin 150081,China;Center for Endemic Disease Control,Chinese Center for Disease Control and Prevention,Harbin Medical University,Harbin 150081,China)
出处
《中华地方病学杂志》
CAS
北大核心
2021年第1期5-11,共7页
Chinese Journal of Endemiology
基金
国家自然科学基金(81573097、81903265)。
关键词
砷
叶酸
维生素B12
凋亡
Arsenic
Folic acid
Vitamin B12
Apoptosis
