六价铬诱导L-02肝细胞应激损伤的microRNA研究

Study on Changes of Micro RNA in Hexavalent Chromium Induced L-02 Hepatocyte Stress Injury

目的通过体外实验,在细胞水平探讨六价铬[Cr(Ⅵ)]诱导的L-02肝细胞氧化应激状态及microRNA谱系的改变。方法实验中设计不同的Cr(Ⅵ)浓度梯度,选择不同的时间点进行观察,用四甲基偶氮唑蓝(MTT)比色法检测L-02肝细胞活力,用荧光探针DCFH-DA观察L-02肝细胞氧化应激状态,用实时定量PCR技术检测L-02肝细胞中多个毒物特异性microRNA表达。结果MTT实验显示5μmol/L浓度的Cr(Ⅵ)是L-02肝细胞70%存活的临界值,在各时间点细胞的存活率分别为84.15%、81.44%、74.25%和68.04%。5μmol/L浓度的Cr(Ⅵ)处理L-02肝细胞8 h可诱发最强的氧化应激状态,ROS相对荧光值达169.2%。在氧化应激最高点检测到的microRNA改变是:miR-370、miR-let-7和miR-125b表达上调,miR-122和miR-137表达下调,miR-298和miR-153表达差异无统计学意义。结论金属毒物Cr(Ⅵ)可诱导L-02肝细胞氧化应激状态,并出现特征性的microRNA表达谱。

Objective To investigate the oxidative stress and micro RNA expression changes of hexavalent ehromium（Cr^＋6） induced L-02 hepatocyte in vitro. Methods Experimental groups were divided according to different concentrations of Cr^＋6 and incubation time. The cell viability was measured by MTT assay. Fluorescent probe DCFH-DA was used to observe the oxidative stress of L-02 hepatocyte. Analysis of specific micro RNA expression of toxicants in L-02 hepatocyte was carried out by using real time（L1）PCR quantitative kit. Results MTT assay showed 5 μmol/L of Cr^＋6 was the critical value for 70% L-02 hepatocyte survival, and the viabilities at different time points were 84.15%, 81.44%, 74.25% and 68.04% respectively. The strongest oxidative stress was induced after treating the hepatocyte with 5 μmol/L Cr^＋6 for eight hours, with ROS relative fluorescence value being 169.2%. At the highest point of oxidative stress, changes of micro RNA detected were that miR-370, miR-let-7, and miR-125b were regulated upward, miR-122 and miR-137 were regulated downward, and miR 298 and miR-153 did not show remarkable difference. Conclusions Metallic toxicant Cr^＋6 could induce oxidative stress of L-02 hepatoeyte showing speeific micro RNA expression profile.