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

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。

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

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.