脂多糖诱导巨噬细胞自噬相关LC3B-GFP荧光聚集体的形成

摘要

To construct the eukaryotic expression vector pEGFP-Cl/LC3B and transfect macrophages RAW264. 7, and observe the formation of autophagy-related LC3B-GFP fluorescent aggregates in macrophages induced by lipopolysaccharide ( LPS ) . Methods On the basis of the open reading frame ( ORF ) encoding murinemicrotubule-associated protein 1 light chain 3(3 ( LC3B ) to design primers, the LC3B mRNA was amplified by reverse transcription polymerase chain reaction ( RT-PCR ). The recombinant plasmid pEGFP-Cl/LC3B was constructed and transfected into RAW264. 7 cell lines with liposome. Fluorescence microscopy and Western blot were applied to identify the expression of LC3B-GFP fusion protein, and the stable expressing cell lines were gained by G418 selection. Confocal laser scanning microscope was used to observe the distribution of the green fluorescent aggregates in the stable transfected RAW264. 7 macrophages which stimulated by LPS at different time points. Results The eukaryotic expression plasmid pEGFP-Cl/LC3B was constructed successfully, and stably expressing fusion protein RAW264. 7 cell strain was obtained. The green fluorescent was detected in the stable transfected cells by fluorescence microscope. Western blot identified the expression of 43 ku LC3B-GFP fusion protein. The results which observed by confocal laser scanning microscope indicated that these LC3B-GFP fusion proteins were diffusely distributed in the cytoplasm before LPS stimulation, while they formed obviously green fluorescent aggregates and increased in time-dependent manner after LPS stimulation. Conclusion The plasmid pEGFP-Cl/LC3B is constructed and transfected into RAW264. 7 macrophages successfully, which can express LC3B-GFP fusion protein correctly and form the green fluorescent aggregates after LPS stimulated.%目的 构建pEGFP-C1/LC3B重组质粒并转染小鼠巨噬细胞RAW264.7,观察脂多糖(LPS)诱导其自噬相关LC3B-GFP荧光聚集体形成的过程.方法 根据编码小鼠微管相关蛋白1轻链3β(LC3B)的开放读码框(ORF)设计引物,RT-PCR扩增小鼠LC3B的ORF,构建pEGFP-C1 /LC3B重组质粒;脂质体法转染并筛选稳定表达RAW264.7细胞株,荧光显微镜观察及Western blot鉴定LC3B-GFP融合蛋白表达;LPS刺激稳定转染的RAW264.7细胞株,激光共聚焦显微镜检测不同时间绿色荧光聚集体的分布.结果 成功构建真核表达质粒pEGFP-C1 /LC3B,并获得稳定表达LC3B-GFP融合蛋白的RAW264.7细胞株,荧光显微镜下可见细胞内有绿色荧光蛋白分布,Western blot鉴定表达LC3B-GFP融合蛋白,激光共聚焦结果 显示LPS刺激前融合蛋白呈弥散性分布在胞浆中,而LPS刺激后细胞内有明显绿色荧光聚集体形成并呈时间依赖性逐渐增加.结论 成功构建pEGFP-C1/LC3B,转染RAW264.7细胞可正确表达LC3B-GFP融合蛋白,LPS刺激后显示绿色荧光聚集体形成.