活性氧在微囊藻毒素-LR诱导细胞凋亡效应中的作用探讨

[目的]研究微囊藻毒素．LR（MC—LR）对原代大鼠肝脏细胞的促凋亡效应以及探讨活性氧（ROS）在这一效应中的作用。[方法]采用胶原酶灌注法获取原代大鼠肝脏细胞，用William’s E培养液重悬后接种于铺被鼠尾胶原的孔板内，分为1个对照组和4个染毒组，370C、5％CO2条件下培养3h后换液并染毒各梯度浓度的MC—LR，使各染毒组毒物终浓度分别为1×10^-5、1×10^-6、1×10^-7、1×10^-8mol／L。染毒相应时间后，采用中性红比色实验检测细胞存活率，采用流式细胞术检测细胞凋亡和细胞内ROS含量。[结果]中性红比色实验发现，至染毒期末观察到1×10^-5、1×10^-6mol／L两个染毒组的肝脏细胞几乎全部死亡（〉95％），1×10^-7mol／L染毒组部分肝脏细胞死亡（约30％）；流式细胞术观察到MC—LR染毒后早期（5min）诱导细胞调亡，同时观察到肝脏细胞内短时间（15min）快速产生大量的ROS。[结论]MC—LR诱导原代大鼠肝脏细胞快速产生大量的ROS，这可能是MC-LR诱导细胞凋亡并最终导致细胞死亡的毒性作用机制之一。

Preliminary Study on the Role of ROS in the Apoptosis-inducing Effect of Microcystin-LR on Primary Cultured Rat Hepatocytes

[ Objective ] To study the apoptosis-inducing effeci of microcystin-LR （MC-LR） on primary cultured rat hepatocytes and the role of reactive oxygen species （ ROS ） in this effect. [ Methods ] Primary cultured rat hepatocytes were obtained through collagenase perfusion method, resuspended in William＇s E medium, and cultured in orifice plates coated with rattail collagen at 37℃, 5% CO2. They were divided into 1 control group and 4 dose groups, and treated with gradient concentrations of MC-LR. Neutral red colorimetric assay was used to detect the viability of hepatocytes, and flow cytometry to detect apoptosis as well as ROS ]evel in hepatocytes. [ Results ] It was observed that the rat hepatocytes in two groups（ MC-LR 1×10^-5, 1×10^-6 mol/L ） almost totally died out, and in one group （MC-LR 1×10^-7 mol/L ）died partly by neutral red colorimetric assay; and that MC-LR induced rapid apoptosis and ROS formation by flow cytometry. [ Conclusion ] MC-LR induces rapid massive ROS in primary cultured rat hepatocytes, which may be one of the mechanisms that MC-LR induces apoptosis and death of the cells finally.