Stably expression human α-synuclein gene in PC12 cells by lentivirus and apoptosis detection / 中华神经科杂志

ABSTRACT

ObjectiveConstruction and identification of wild-type and mutant human o-synuclein (SNCA) gene lentiviral expression vector, and its stable transfection into the rat pheochromocytoma cells.MethodsThe genes were synthesized with particular primer, amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU ( with green fluorescent protein (GFP) gene) to construct a lentiviral vector expression plasmid pGC-FU-SNCA-GFP and pGC-FU-SNCA-GFP.After digestion and sequencing,pGC-FU-SNCA-GFP,pGC-FU-SNCA-GFP plasmidandpackaging plasmidpHelper1. 0,pHelper2. 0 were co-transfected into rat pheochromocytoma cells (PC12 cells).A stable transfection was established in the PC12 cells. Results By detecting the level of tagged protein of GFP and the target protein, the pGC-FU-SNCA-GFP and pGC-FU-SNCA-GFP expression in target cells was verified. MTT assay was employed to detect cell apoptosis in lentiviral pGC-FU-SNCA-GFP transfected group, lentiviral pGC-FU-SNCAmu-GFP transfected group ( experimental groups), without virus group ( control group) and empty vector group( total four groups cells). After transfection, at different timepoints ( 1, 2, 3, 4 and 5 d) the proliferation of cells was slowed ( the F value was 4. 534, 196. 285, 411. 829, 1282. 049, 3135. 559, all P ＜0.05). PI single staining was used to examine the cell cycle. The percentages of G1 phase, G2 phase,M phase cells were all statistically significant ( the F value was 885.79, 45.03,207.11 ,all P ＜0. 05). The percentage of G1 phase cells in the four groups cells increased significantly (CON group59. 10 ±0. 35, NC group68.24 ±0.60, OE group71.73 ±0. 11, OE group74.66 ±0.35). Conclusion This study constructs a foundation for further investigation on the basic function of SNCA and apoptosis related diseases.