5-脂氧酶/绿荧光蛋白转染评价PC12细胞损伤后5-脂氧酶核膜移位

Transfection with 5-lipoxygenase/green fluorescence protein for evaluating injury-induced 5-lipoxygenase translocation to the nuclear membrane in PC12 cells

目的:通过绿荧光蛋白(green fluorescence protein,GFP)/5-脂氧酶(5-lipoxygenase,5-LOX)稳定转染PC12细胞,以可视化方法观察5-LOX在不同损伤后的变化。方法:将带有5-LOX基因的pEGFP-C2质粒及pEGFP-C2质粒(对照),稳定转染至PC12细胞;在缺糖缺氧(oxygen-glucose deprivation,OGD)、H2O2和NMDA处理后,荧光显微镜下观察GFP/5-LOX在细胞内的定位;同时,以免疫细胞化学方法观察野生型5-LOX分布及其变化。结果:转染GFP/5-LOX的细胞内,GFP/5-LOX主要均匀表达于细胞核内;仅转染GFP的细胞GFP表达于胞核和胞浆。OGD和H2O2处理后,分别有50.6%和57.7%细胞的GFP/5-LOX移位到核膜;NMDA处理或仅转染GFP的细胞,未见核膜移位现象。野生型5-LOX分布在细胞核和细胞浆内,3种损伤处理均诱导核膜移位。结论:稳定转染GFP/5-LOX的PC12细胞,GFP/5-LOX主要分布在细胞核;不同损伤处理诱导的5-LOX核膜移位,有不同的特点。

Objective： To evaluate the translocation of 5-lipoxygenase （5-LOX） after injuries by transfection with green fluorescence protein （GFP）/5-LOX in PC12 cells. Methods： PC12 cells were stably transfected with pEGFP-C2/5-LOX （GFP/5-LOX） or pEGFP-C2 vectors （control）. After treatment with oxygen-glucose deprivation （OGD）,H2O2 or NMDA,GFP/5-LOX localization in the cells was observed under a fluorescence microscope. Wild-type 5-LOX was determined by immunostaining after the treatment. Results. In the GFP/5-LOX-transfected cells, GFP/5-LOX was primarily localized in the nucleus; while in the GFP-transfected cells,GFP was localized in both the cytoplasm and nucleus. After OGD and H2O2 treatments, GFP/5-LOX was translocated to the nuclear membrane in 50.6% and 57.7% cells respectively. However,after NMDA treatment or in GFP-transfected cells, no translocation was observed. Wild-type 5-LOX was distributed in the nuclei and cytoplasm,and all the 3 treatments induced 5-LOX translocation to the nuclear membrane. Conclusion： In the PC12 cells stably transfected with GFP/5-LOX, GFP/5-LOX is primarily distributed in the nuclei; the OGD-,H2O2- and NMDA-induced 5-LOX translocation exhibits different properties.