人α-突触核蛋白基因慢病毒稳定转染PC12细胞系的建立及其凋亡检测

Stably expression human α-synuclein gene in PC12 cells by lentivirus and apoptosis detection

目的构建及鉴定人野生型和突变型α-突触核蛋白（SNCA和SNCAmu）基因慢病毒表达载体，并观察在体外大鼠嗜铬细胞瘤细胞中的表达。方法在引物合成后采用PCR技术扩增目的基因，并将基因克隆到慢病毒载体表达质粒[pGC—FU，含绿色荧光蛋白（GFP）基因]中，构建SNCA基因慢病毒载体表达质粒（pGC—FU—SNCA—GFP）和SNCAmu基因慢病毒载体表达质粒（pGC—FU—SNCAmu—GFP），通过酶切、测序验证后，将pGC—FU—SNCA—GFP、pGC—FU—SNCAmu—GFP质粒和包装质粒pHelper1．0、pHelper2．0共同转染大鼠嗜铬细胞瘤细胞（PCI2细胞），获得稳定转染。结果通过检测标签蛋白GFP和目的蛋白，进一步验证pGC—FU—SNCA—GFP和pGC—FU—SNCAmu—GFP在靶细胞中的表达。通过噻唑蓝比色法检测稳定转染后细胞凋亡水平，稳定表达SNCA组（OE组）、SNCAmu组（OEm组）、不加慢病毒质粒的空白对照组（CON组）和加pGC—FU—GFP空载体对照组（NC组）共4组细胞，病毒转染后不同时间（1、2、3、4、5d）细胞增殖变缓（F=4．534、196．285、411．829、1282．049、3135．559，均P〈0．05）。碘化丙锭单染法检测细胞周期，CON组、NC组、OE组和OEm组G1期、G2期和M期细胞百分比差异有统计学意义（F=885．79、45．03、207．11，均P〈0．05），其中G1期细胞百分比（％）较高（CON组：59．10±0．35、NC组：68．24±0．60、OE组：71．73±0．11、OEm组：74．66±0．35）。结论人SNCA和SNCAmu基因转染到PC12细胞中，成功建立稳定表达的细胞系，并且对细胞凋亡水平和细胞周期有一定程度的改变。

Objective Construction and identification of wild-type and mutant human α-synuclein （SNCA） gene lentiviral expression vector, and its stable transfection into the rat pheochromocytoma cells. Methods The 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 plasmid and packaging plasmid pHelperl. 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 group ：59, 10 ± 0. 35, NC group：68.24 ±0.60, OE group：71.73 ±0.11, OE group：74.66±0.35）. Conclusion This study constructs a foundation for further investigation on the basic function of SNCA and apoptosis related diseases.