小鼠视锥细胞系661W细胞凋亡模型的构建及自噬对其的保护性作用

Establishment of apoptosis model in mouse cone cell line 661W cells and the primary research on protective effects of autophagy

目的构建小鼠视锥细胞系661W细胞凋亡模型,探讨不同自噬水平下细胞生存状态的变化。方法采用不同质量浓度抗Fas抗体处理细胞,采用Western blot法检测caspase-3蛋白表达量,并确定凋亡细胞模型适宜抗Fas抗体刺激浓度;分别采用不同浓度自噬抑制剂3-甲基腺嘌呤(3-MA)和自噬诱导剂雷帕霉素处理细胞,采用Western blot法检测细胞中微管相关蛋白质1轻链3(LC-3)Ⅱ/LC-3Ⅰ蛋白表达,筛选合适的作用浓度。将细胞分为空白对照组、单纯3-MA组、单纯雷帕霉素组、模型对照组、模型+3-MA组、模型+雷帕霉素组,采用Western blot法检测各组细胞诱导后0、3、6、12、24和48 h caspase-3、caspase-8、自噬相关基因5(Atg-5)和LC-3Ⅱ/LC-3Ⅰ蛋白表达,采用流式细胞术检测细胞凋亡率。结果成功构建661W细胞凋亡模型,抗Fas抗体的适宜刺激质量浓度为2.0μg/ml;在Fas抗体刺激下,661W细胞caspase-3、caspase-8相对表达量从6 h开始增加,12 h达高峰,并持续至48 h,而Atg-5和LC-3Ⅱ/LC-3Ⅰ蛋白相对表达量从3 h开始增加,24 h达高峰,并于48 h时降至基线水平。3-MA和雷帕霉素的适宜作用浓度分别为20 nmol/L和2.0 nmol/L。空白对照组、单纯3-MA组和单纯雷帕霉素组诱导后各时间点caspase-3、caspase-8的相对表达量及细胞凋亡率总体比较,差异均无统计学意义(均P<0.05)。单纯雷帕霉素组各时间点细胞Atg-5和LC-3Ⅱ/LC-3Ⅰ蛋白相对表达量显著高于相应时间点空白对照组,差异均有统计学意义(均P<0.05)。模型+3-MA组caspase-3、caspase-8相对表达量以及细胞凋亡率在诱导后3、6、12、24和48 h均显著高于模型对照组,Atg-5和LC-3Ⅱ/LC-3Ⅰ的相对表达量在诱导后3、6、12和24 h均明显低于模型对照组,差异均有统计学意义(均P<0.05);模型+雷帕霉素组caspase-3、caspase-8相对表达量以及细胞凋亡率在诱导后6、12、24和48 h均显著低于模型对照组,Atg-5和LC-3Ⅱ/LC-3Ⅰ的相对表达量在诱导后3、6、12和24 h均明显高于模型对照组,差异均有统计学意义(均P<0.05)。结论在抗Fas抗体诱导661W细胞凋亡条件下,增强自噬可降低细胞凋亡率,而抑制自噬则会增加细胞凋亡率,自噬可能对661W细胞起到保护性作用。

Objective To establish the apoptosis model in mouse cone cell line 661W cells and to investigate the viability of 661W cells in the conditions of different levels of autophagy.Methods Different concentrations of anti-Fas antibody were added to establish the apoptosis model of 661W cells,the expression of caspase-3 was detected by Western blot and the appropriate concentration of anti-Fas antibody was screened.Different concentrations of the autophagy inhibitor 3-methyladenine(3-MA)or autophagy inducer rapamycin were added to inhibit or induce autophagy,the expression of microtubule-associated protein 1 light chain 3(LC-3)Ⅱ/LC-3Ⅰwere detected by Western blot and the appropriate concentrations were also screened.The cultured cells were divided into 6 groups:control group,simple 3-MA group,simple rapamycin group,model group,model+3-MA group and model+rapamycin group.Western blot was adopted to detect the expression of caspase-3,caspase-8,autophagy related genes 5(Atg-5)and LC-3Ⅱ/LC-3Ⅰat 0 hour,3,6,12,24 and 48 hours after induction.Flowcytometer was adopted to detect the apoptosis rate of 661W cells.Results The apoptosis model of 661W cells was successfully established,and the appropriate concentration of anti-Fas antibody was 2.0μg/ml.After stimulated by the anti-Fas antibody,the expression of caspase-3 and caspase-8 of 661W cells increased from the time point of 6 hours,peaked at 12 hours,and sustained to 48 hours.However,the expression of Atg-5 and LC-3Ⅱ/LC-3Ⅰraised from the time point of 3 hours,peaked at 24 hours,and decreased to the basic level at 48 hours.In addition,the appropriate concentrations of 3-MA and rapamycin were 20 nmol/L and 2.0 nmol/L,respectively.There was no statistical difference among the control group,simple 3-MA group and simple rapamycin group on the expression of caspase-3 and caspase-8 and the apoptosis rate of 661W cells at different time points(all at P>0.05).The expressions of Atg-5 and LC-3Ⅱ/LC-3Ⅰin the simple rapamycin group were significantly higher than those in the control group at different time points(all at P<0.05).The expressions of caspase-3 and caspase-8 and the apoptosis rate in the model+3-MA group were significantlly higher than those in the model group at 3,6,12,24 and 48 hours after induction,while the expressions of Atg-5 and LC-3Ⅱ/LC-3Ⅰwere obviously lower than those in the model group at 3,6,12 and 24 hours after induction(all at P<0.05).The expressions of caspase-3 and caspase-8 and the apoptosis rate at 6,12,24 and 48 hours after induction in the model+rapamycin group were significantly lower than those in the model group,while the expressions of Atg-5 and LC-3Ⅱ/LC-3Ⅰat the time points of 3,6,12 and 24 hours after induction were obviously higher than those in the model group(all at P<0.05).Conclusions Under the condition of anti Fas antibody inducing apoptosis,enhancing autophagy can reduce the apoptosis rate of cells,inhibiting autophagy can increase the apoptosis rate.Autophagy may play a protective role in 661W cells.