活性氧在微囊藻毒素-LR诱导细胞凋亡效应中的作用探讨

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

[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,37℃、5%CO_2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^(-8)mol/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^(-6)mol/L两个染毒组的肝脏细胞几乎全部死亡(>95%),1×1^(-7)mol/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)诱导细胞凋亡,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。

[ 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.