LC3真核表达载体构建及其在293T细胞中自噬诱导研究

Study on Construction of LC3 Eukaryotic Expression Vector and Its Autophagy Induction in 293T Cells

关键词:  LC3  细胞自噬  绿色荧光蛋白  人胚肾293T细胞

Key words:  LC3  autophagy  green fluorescent protein  human embryonic kidney 293T cells

摘要:

[目的]构建pEGFP-N1-LC3真核表达载体,并将其转染进人胚肾细胞293T,通过Earle's盐平衡液(Balanced salt solution,EBSS)饥饿诱导观察细胞自噬现象。[方法]RT-PCR扩增LC3基因,并将其插入pEGFP-N1真核表达载体构建重组质粒。将重组质粒转染至人胚肾细胞293T,显微镜观察、Western blot检测GFP-LC3融合蛋白表达情况。对转染细胞进行Earle's盐平衡液饥饿诱导,通过Western blot验证自噬指标,激光共聚焦显微镜观察自噬泡形成。[结果]成功构建pEGFP-N1-LC3真核表达载体,并在293T细胞中成功表达目的蛋白,在进行Earle's盐平衡液饥饿诱导后观察到自噬泡的形成,Western blot检测到LC3-Ⅰ向LC3-Ⅱ的转化。[结论]本实验为研究自噬在癌细胞病理进程中的机制提供了实验素材。

Abstract:

[Objective] The pEGFP-N1-LC3 eukaryotic expression vector was constructed and transfected into human embryonic kidney 293T cells and the expression of the fusion protein in the state of autophagy was observed by Earle's Balanced salt solution (EBSS) starvation induction.[Methods] The LC3 gene was amplified by RT-PCR and inserted into pEGFP-N1 eukaryotic expression vector to construct a recombinant plasmid.The recombinant plasmids were transfected into 293T cells and the expression of the GFP-LC3 fusion protein was observed under fluorescent microscope and detected by Western blot.The transfected cells were subjected to starvation with Earle's Balanced salt solution, then the autophagic development was confirmed by Western blot and the formation of autophagic vacuoles was observed by confocal laser scanning microscope.[Results] The eukaryotic expression vector pEGFP-N1-LC3 was constructed and the recombinant protein was efficiently expressed in 293T cells.Autophagic vacuoles were observed after starvation induced by Earle's Balanced salt solution, and then the conversion of LC3-Ⅰ to LC3-Ⅱ was detected by Western blot.[Conclusion] This experiment provided an experimental material for the later study on the mechanisms of autophagy in tumorigenesis.