稳定表达RFP-GFP-LC3的大鼠胰岛β细胞株的构建

Establishment of RIN-m5f Cell Strain Stably Expressing RFP-GFP-LC3

为了简便自噬相关机理药物在胰岛β细胞上的高通量筛选,实验通过慢病毒转染技术,将RFP-GFP-LC3质粒转入大鼠胰岛β细胞株(RIN-m5f),用G418对感染细胞进行筛选,激光共聚焦进行活细胞拍摄验证,得到100%表达RFP-GFP-LC3质粒的细胞株。无血清饥饿处理细胞,结果显示:饥饿组GFP/RFP荧光点比值较对照组下降, P62蛋白表达量以及S6K磷酸化水平降低,加入10μmol/L氯喹部分抑制自噬后, GFP/RFP荧光点比值回升。以上结果表明稳定表达RFP-GFP-LC3的RIN-m5f构建成功,并能够以简单的检测方式准确表达自噬全过程,为自噬在糖尿病药物机理方面的研究奠定了基础。

In order to facilitate the high throughput screening of autophagy related drugs on islet β cells, the RFP-GFP-LC3 plasmid was transfected into RIN-m5f cells by lentivirus transfection, and the infected cells were screened with G418. Laser confocal imaging verified that a cell line expressing RFP-GFP-LC3 was obtained. After different treatments of RIN-m5f, the results showed that the fluorescence point ratio of GFP/RFP in the starved group was lower than that in the normal group, and that expression of P62 protein and the level of S6K phosphorylation were decreased. After 10 μmol/L chloroquine was added to partially inhibit autophagy, the fluorescence point ratio of GFP/RFP increased. These results indicated that the rat islet β cell strain stably expressing RFP-GFP-LC3 was successfully constructed. The cells could exhibit the whole process of autophagy in a simple and accurate manner, which lays a foundation for the study of mechanisms of autophagy related to diabetic medicines.