H_2O_2诱导PC12细胞凋亡前后miRNA的变化及探讨

A Study on miRNA Alternation after H_2O_2-induced PC12 Cell Apoptosis Using Microarray Technique

目的用微小RNA(miRNA)基因芯片技术检测H2O2诱导凋亡的PC12细胞和正常PC12细胞miR-NA的表达谱差异。方法分别以不同浓度的H2O2处理PC12细胞12h,用MTT和流式细胞仪检测处理后细胞的生长活力和凋亡情况;分别提取A(0浓度H2O2处理组)和B(400nmol/LH2O2处理组)PC12细胞的miRNA做miRNA基因芯片检测。结果以A组作为对照,30、50、100、200、400nmol/L的H2O2处理的PC12细胞生存率分别为(92±9.80)%、(90±14.70)%、(80±13.85)%、(54±12.33)%、(22±7.35)%(P<0.01);0、30、50、100、200和400nmol/L的H2O2处理的PC12细胞早期凋亡率分别为2.6%、5.2%、7.2%、10.4%、16.6%、72.2%;在样品A中共筛选出68个有效表达的miRNA分子数据,在样品B中筛选出46个有效表达的miRNA分子数据,两者样品中均检测到有效表达的miRNA分子有39个,其中与样品A相比,在样品B中显著性下调表达的有6个。结论为脑缺血再灌注损伤中神经细胞凋亡的机制和治疗的研究提供了理论依据和新的研究思路。

Objective To screen the differentially expressed microRNAs（miRNA） between H2O2-induced PC12 cell apoptosis and normal PC12 cells with microarray chips. Methods PC12 cells were treated with different concentrations of H202 for 12 hours, and then the cell viabilities were evaluated by MTT assay and cell apoptosis rates were assessed by flow cytometry （FCM）. miRNAs were isolated from control （0 nmol/L H2O2） and H2O2 treated （at concentrations of 50, 100, 200, and 400 nmol/L H2O2, respectively） groups of the PC12 cells, respectively, and were then detected and analyzed by microarray chips. Results The P12 cell viabilities in control and H202 treated group were （92±9.8）%, （90±14.70）%, （80±13.85）%, （54±12.23）%, and （22±7.35）%, respectively. The apoptosis rates for them were 2.6%, 5.2%, 7.2%, 10.4%, 16.6%, and 72.2% accordingly. Expression of 68 miRNAs and 46 miRNAs were detected in both control group and the H2O2 treated groups. Of the 46 miRNAs in the H2O2 treated groups, 39 were similar to that of the control group and 6 were significantly down-regulated. Conclusion Our preliminary data may provide a new potential foundation for further study of pathogenesis and its treatment of nerve cell apoptosis caused by cerebral ischemia reperfusion.