稳定表达EGFP-LC3蛋白SH-SY5Y细胞系的构建

Construction of SH-SY5Y cell line stably expressing EGFP-LC3 protein

目的:建立稳定表达微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)与绿色荧光蛋白(EGFP)形成的融合蛋白(EGFP-LC3)的人神经母细胞瘤株SH-SY5Y细胞系,验证EGFP-LC3点状聚集物能够实时直观地反映自噬小体和自噬流。方法:以人神经母细胞瘤细胞(SH-SY5Y细胞)的cDNA为模板,克隆LC3,并连接EGFP绿色荧光蛋白。将EGFP-LC3连接入慢病毒载体GV348中,构建慢病毒表达载体EGFP-LC3-GV348。慢病毒转染人神经母细胞瘤细胞(SH-SY5Y细胞),使用3μg/mL嘌呤霉素筛选并处理细胞1月,筛选得到EGFP-LC3稳转系。诱导自噬后,用共聚焦显微镜观察LC3荧光斑点并定量分析。Western blot检测目的蛋白表达情况。结果:经酶切证实,EGFP-LC3-GV348慢病毒表达载体构建正确。在激光共聚焦显微镜下,可以观察到细胞内的绿色荧光EGFP代表的自噬小体,活体染料LysoTracker Red DND-99将溶酶体染成红色荧光,红色荧光与绿色荧光重叠代表自噬溶酶体。无血清诱导自噬后,自噬小体(绿色荧光斑点)明显增加,差异有统计学意义(P<0.001)。Western Blot免疫印迹可见EGFP-LC3融合蛋白表达条带。结论:成功构建了稳定表达EGFP-LC3的SH-SY5Y细胞系,EGFP-LC3荧光斑点可以直观实时反映自噬囊泡,为进一步探讨神经退行性疾病的自噬机制提供了实验基础。

Objective:To establish a human neuroblastoma SH-SY5Y cell line stably expressing the fusion protein(EGFP-LC3)formed by microtubule associated protein 1 light chain 3(LC3)and green fluorescent protein(EGFP),and to verify that the EGFP-LC3 dot-like aggregates can directly reflect the autophagy bodies and autophagy flow in real time.Methods:The cDNA of human neuroblastoma cells(SH-SY5Y cells)was used as a template to clone LC3 and connect EGFP green fluorescent protein.EGFP-LC3 was ligated into lentiviral vector GV348 to construct lentiviral expression vector EGFP-LC3-GV348.Human neuroblastoma cells(SH-SY5Y cells)were transfected with lentivirus,and the cells were screened and treated with 3 ug/mL puromycin for 1 month to obtain a stable EGFP-LC3 cell line.After autophagy induction,LC3 fluorescence spots were observed by confocal microscope and quantitatively analyzed.The expression of target protein was detected by Western blot.Results:Enzymatic digestion confirmed that EGFP-LC3-GV348 lentiviral expression vector was constructed correctly.Under the laser confocal microscope,the autophagosome represented by green fluorescence EGFP in the cells can be observed.LysoTracker Red DND-99 in vivo dye dyed the lysosome into red fluorescence,and the overlapping of red fluorescence and green fluorescence represented the autophagosome.After serum-free induction of autophagy,autophagosomes(green fluorescent spots)increased significantly(P<0.001).Western Blot showed EGFP-LC3 fusion protein expression band.Conclusion:SH-SY5Y cell line stably expressing EGFP-LC3 is successfully constructed.EGFP-LC3 fluorescent spots can intuitively and real-time reflect the autophagy vesicles,which provides an experimental basis for further exploring the autophagy mechanism of neurodegenerative diseases.