摘要
目的用SK-N-SH细胞构建PARK2基因过表达/敲除稳转株,用于阐明PARK2和锰毒性的关系。方法慢病毒转染SK-N-SH细胞构建PARK2基因过表达/敲低细胞稳转株,分为3组:空载质粒组、PARK2过表达组和PARK2敲低组。以900μM Mn^(2+)对各组细胞染毒24 h后,用荧光探针检测细胞内ROS水平,电镜下观察细胞内自噬小体数量,WB法检测OPTN、LC3Ⅱ/Ⅰ、P62蛋白的表达,ELISA法检测细胞培养基中DA水平。结果与空载质粒组比,PARK2过表达组细胞内Parkin蛋白表达增多、ROS水平降低、自噬小体数量增多、P62和OPTN蛋白表达下调、LC3Ⅱ/Ⅰ值增加及DA水平升高(P<0.05)。PARK2敲低组细胞内Parkin蛋白表达降低、P62和OPTN蛋白表达上调(P<0.05),而ROS水平、自噬小体数量、LC3Ⅱ/Ⅰ值以及DA含量与空载质粒组无差异。结论过表达PARK2基因能提高Parkin蛋白的表达,促进细胞内线粒体自噬来清除受损的线粒体,从而保护细胞。OPTN和P62蛋白可能作为PARK2的下游蛋白参与线粒体自噬过程。
Objective To construct PARK2 gene overexpression and knockdown cells would help elucidate the relationship between PAK2 and Manganese toxicity.Methods Lentivirus-transfected SK-N-SH cells were used to construct PARK2 gene overexpression/knockdown cell models,which were divided into empty-load plasmid group,PARK2 overexpression group,and PARK2 knockdown group.After treating cells of each group with 900μM Mn^(2+)for 24 hours,the ROS level was detected by fluorescence probe,the number of autophagosomes in the cells was observed by electron microscopy,the related target proteins(Optineurin,LC3Ⅱ/Ⅰ and P62)were detected by WB,and the content of dopamine in the cell culture medium was detected by ELISA.Results Compared with the empty-load plasmid group,the PARK2 overexpression group showed Parkin pretein expression increased,Ros level decresed,autophagosome number increased.P62 and OPTN protein expression decreased.LC3Ⅱ/Ⅰincreased and DA level increased(P<0.05).In the PARK2 knockdown group,the intracellular ROS level,the number of autophagosomes,LC3Ⅱ/Ⅰand the content of DA remain unchanged,while Parkin pretein expression decreased both P62 and OPTN protein expression increased(P<0.05).Conclusion Overexpression of PARK2 gene can increase the level of Parkin-mediated mitophagy to eliminate damaged mitochondria,and OPTN and P62 proteins may be involved in mitophagy as a downstream protein of PARK2 gene modulation.
作者
张玥
胡宏涛
赵颖
蒋智钢
周远忠
范奇元
Zhang Yue;Hu Hongtao;Zhao Ying;Jiang Zhigang;Zhou Yuanzhong;Fan Qiyuan(School of Public Health,Zunyi Medical University,Zunyi Guizhou 563099,China;Department of Health Management,Zunyi Medical and Pharmaceutical College,Zunyi Guizhou 563006,China)
出处
《遵义医科大学学报》
2023年第1期44-51,共8页
Journal of Zunyi Medical University
基金
国家自然科学基金资助项目(NO:81860568)。