雷帕霉素对光诱导下视网膜色素上皮细胞自噬的影响

Effect of rapamycin on autophagy of retinal pigment epithelial cells under light induction

目的:探讨雷帕霉素对光诱导下视网膜色素上皮细胞(ARPE-19)自噬的影响。方法:将自噬双标mRFP-eGFP-LC3质粒转染进ARPE-19细胞中获得稳定表达的转染细胞株,随后随机分为6 h对照组、6h模型组、6 h雷帕霉素组;12 h对照组、12 h模型组、12 h雷帕霉素组和24 h对照组、24 h模型组、24 h雷帕霉素组。对照组避光培养,模型组受光照刺激;雷帕霉素组加入10μmol/L雷帕霉素后受光照刺激,光源采用LED冷光灯,在(16500±500)lx光照强度下照射细胞6、12、24 h。检测转染效率,MTT检测ARPE-19细胞存活率;激光共聚焦显微镜对自噬流变化进行定性分析,透射电镜观察细胞自噬形态;蛋白免疫印迹法对Beclin1、LC3、P62自噬相关蛋白表达量进行检测。结果:与对照组相比,模型组光照6、12、24 h后,细胞存活率显著下降(P<0.001);雷帕霉素组经光照后细胞存活率高于模型组(P<0.05)。光照6、12、24 h后模型组红色荧光斑点较对照组逐渐增多,绿色荧光斑点也随之增加,Merge图中从光照12 h开始黄色斑点数量增多明显。雷帕霉素组在光照6、12、24 h时红色荧光斑点较模型组加强,绿色荧光较模型组弱。透射电镜观察发现模型组胞内见较多的自噬泡,雷帕霉素组细胞见大量聚集分布自噬囊泡。蛋白免疫印迹结果发现光照6、12、24 h后,模型组中自噬相关蛋白Beclin1、LC3Ⅱ/LC3Ⅰ的相对表达水平均增加,P62蛋白表达较低。而采用雷帕霉素干预后,与模型组相比,雷帕霉素组在光照6 h时Beclin 1蛋白表达量差异不明显、在12、24h时Beclin 1蛋白表达量逐渐升高;LC3Ⅱ/LC3Ⅰ的比值均高于模型组;P62蛋白表达量差异不显著。结论:光照可诱导ARPE-19细胞自噬现象的发生,且雷帕霉素能上调其自噬活性。

Objective:To investigate the effect of rapamycin on autophagy in retinal pigment epithelial cells under light induction.Methods:The autophagy double labeled mRFP-eGFP-LC3 plasmid was transfected into ARPE-19 cells to obtain a stable expression of transfected cell line,and then randomly divided into the 6 h control group,the 6 h model group and the 6 h rapamycin group;the 12 h control group,the 12 h model group,the 12 h rapamycin group and the 24 h control group,the 24 h model group,the 24 h rapamycin group.The control groups were incubated in tinfoil and protected from light;the model groups received light stimulation;the rapamycin groups received light stimulation after the addition of 10μmol/L rapamycin,and the cells were irradiated at(16500±500)lx light intensity for 6 h,12 h and 24 h using a LED elektrolumineszenz as the light source.The changes in autophagic flow were analyzed qualitatively and the expression of Beclin 1,LC3 and P62 autophagy-related proteins was quantified by Western blot assay.Results:Compared with the control group,the cell survival rate of the model group decreased significantly after 6 h,12 h and 24 h of light exposure(P<0.001).The rapamycin group had a significantly higher cell survival rate after light exposure than the model group(P<0.05).After 6 h,12 h,and 24 h of light exposure,the number of red fluorescent spots in the model group gradually increased compared with the control group,and the number of green fluorescent spots also increased.The number of yellow spots in the merge map started to increased significantly at 12 h of light exposure.The red fluorescent spots in the rapamycin group were stronger than those in the model group at 6 h,12 h and 24 h of light exposure,and the green fluorescence was weaker than that in the model group.Transmission electron microscopy showed that more autophagic vesicles were observed in the model group,and a large number of autophagic vesicles were observed in the rapamycin group.Western blot results showed that the relative expression levels of autophagy-related proteins Beclin1 and LC3Ⅱ/LC3Ⅰ in the model group increased after 6 h,12 h and 24 h of light exposure,and the expression of P62 protein was low.After rapamycin intervention,compared with the model group,the expression of Beclin 1 protein in the rapamycin group was not significantly different at 6 h of light exposure,and gradually increased at 12 h and 24 h of light exposure.The ratio of LC3Ⅱ/LC3Ⅰ was higher than that of the model group;The difference in P62 protein expression was not significant.Conclusion:Light irradiation can induce autophagy in ARPE-19 cells,and rapamycin can up-regulate the activity of autophagy.