米托蒽醌通过活性氧诱导大鼠肝癌 RH35细胞凋亡的研究

摘要

试验旨在探讨米托蒽醌(mitoxantrone,MTN)对大鼠肝癌 RH35细胞毒性及其作用机制。以 MTT 法检测 MTN 的细胞毒性,显微镜观察细胞形态变化,流式细胞仪检测细胞凋亡率及细胞中活性氧(reactive oxygen spe-cies,ROS)的产生,Western blotting 检测相关蛋白的表达。结果显示,MTN 时间和剂量依赖性地抑制 RH35细胞的生长；15μmol/L MTN 作用于细胞24、48 h 后可使细胞皱缩、变圆,并出芽形成明显的凋亡小体,且其可时间依赖性地诱导凋亡细胞比率的增加及 ROS 的产生；ROS 清除剂 NAC 可以极显著降低 MTN 诱导的 RH35细胞的ROS 的产生和凋亡率(P <0.01)；15 mmol/L NAC 可以下调 MTN 诱导的 caspase-3、Bax 及 CytC 表达增加,上调MTN 诱导的 Bcl-2表达降低。结果提示,MTN 通过增加细胞内 ROS 而诱导 RH35细胞发生凋亡。%The assay was aimed to study the cytotoxicity and mechanisms of mitoxantrone (MTN) in rat hepatama cell line RH35.MTT assay was performed to assess the cytotoxicity of MTN, and microscope was used to observe cellular morphologic changes.Apoptotic ratio and intracellular ROS generation were measured by flow cytometric analysis.The protein expression was examined by Western blotting analysis.The results showed that MTN inhibited RH35 cell growth in a time-and concentration-dependent manner.The morphologic changes were observed in the cells, including cell shrinkage,membrane blebbing and apoptotic bodies when treated with 15 μmol/L MTN for 24 and 48 h.And MTN also induced the increase of apoptotic ratio and generation of ROS in a time dependent manner.In addition,the intracellular ROS generation ratio and the apoptotic ratio of MTN treated group were extremely decreased by the ROS scavenger NAC (P <0.01).The pretreatment of RH35 cells with 15 mmol/L NAC thoroughly reversed the MTN-in-duced enhancement of caspase-3,Bax and CytC level and attenuation of Bcl-2 level.In conclu-sion,MTN induced apoptosis in RH35 cells through increasing the generation of ROS.