人源性ApoE4基因转染PC12细胞株建立及麦芽酚铝对其细胞活力影响  

Establishment of human ApoE4 gene transfected PC12 cell line and the effect of maltol aluminum on its cell viability

作　　者：张婷[1] 李立荣 王姗姗 赵宇卿 张淑惠 王翡 牛侨[1] 

机构地区：[1]山西医科大学公共卫生学院,山西太原030001

出　　处：《中国职业医学》2017年第6期677-682,共6页China Occupational Medicine

基　　金：国家自然科学基金(81430078)

摘　　要：目的建立人源性载脂蛋白E4(ApoE4)基因转染的大鼠肾上腺嗜铬细胞瘤细胞株PC12细胞(以下简称"PC12细胞"),探讨麦芽酚铝对该细胞株细胞活力的影响。方法将携带有人源性ApoE4基因重组慢病毒载体转染PC12细胞株,用嘌呤霉素进行筛选,获得ApoE4稳定过表达PC12细胞株(PC12-ApoE4组)和阴性空载体对照PC12细胞株(PC12-NC组)。采用实时荧光定量聚合酶链反应法分别检测PC12组、PC12-NC组、PC12-ApoE4组细胞的R-Apo E和(或)H-Apo E-FLAG的mRNA相对表达量,以鉴定构建效果。分别用浓度为0.00、100.00、200.00和400.00μmol/L的麦芽酚铝溶液对PC12-ApoE4组和PC12组细胞染毒24 h后,采用CCK-8法检测细胞存活率。结果 PC12-ApoE4组、PC12-NC组细胞荧光显微镜下均可见荧光表达,提示转染成功;PC12组细胞未见荧光表达。PC12组细胞R-Apo E基因、PC12-NC组细胞R-Apo E基因、PC12-ApoE4组细胞H-Apo E-FLAG基因的mRNA的相对表达量的中位数分别为1.00、1.01和148.74;其中,PC12组与PC12-NC组细胞R-Apo E基因的mRNA的相对表达量均低于PC12-ApoE4组细胞H-Apo E-FLAG基因(P<0.01)。麦芽酚铝染毒后,细胞存活率在染毒剂量及细胞类型的主效应以及交互效应上均有统计学意义(P<0.01);其中,麦芽酚铝在0.00~400.00μmol/L浓度下,随着染毒剂量的增加,2种细胞的细胞存活率均降低,均呈剂量-效应关系(P<0.01)。结论成功构建稳定表达人源性ApoE4基因的细胞株。麦芽酚铝和ApoE4基因对PC12细胞存活率的影响存在交互作用,ApoE4基因可增强麦芽酚铝对PC12细胞的细胞毒性。Objective To establish a PC12 cell line with stable expression of human apolipoprotein E （ApoE4） gene by transfection with a lentiviral vector carrying human ApoE4 gene and to investigate the effect of maltol aluminum on the viability of transfected PC12 cells. Methods The lentiviral vector carrying human ApoE4 gene was transfected into PC12 cells. PC12 cells with overexpression of ApoE4 gene and negative control vector were obtained after puromycin screening. The mRNA relative expression of R-ApoE and （or） H-ApoE-FLAG of cells in PC12, PC12-NC and PC12-ApoE4 groups were detected by real-time fluorescent quantitative polymerase chain reaction, and the effect of cell construction was identified. PC12-ApoE4 cells and PC12 cells were exposed to maltol aluminum solution at concentrations of 0. 00, 100. 00, 200. 00 and 400. 00 μmol/L respectively for 24 hours, and cell viability was detected by Cell Counting Kit-8 （ CCK-8 ） assay. Results PC12-ApoE4 and PC12-NC cells under the fluorescence microscope showed fluorescence expression, suggesting that transfection was successful. The expression of PC12 cells showed no fluorescence. The relative expression of H-ApoE-FLAG gene mRNA （the median amount） of PC12-ApoE4 cells was 148.74, which was higher than the R-ApoE gene in PC 12 cells （1.00） and PC12-NC cells （ 1.01 ） （ P 〈 0. 01 ）. After exposure to mahol aluminum, the cell survival rates in terms of the main effect and interaction effect of dose and cell type were statistically significant （P 〈0. 01 ）, among them, the cell viabilities were decreased in the concentration range of 0. 00-400. 00 μmol/L with the dose of mahol aluminum exposure increased, showing dose-effect relationship （P 〈 0. 01 ）. Conclusion The cell line stably expressed human ApoE4 gene was constructed successfully. There was interaction between the effects of maltol aluminum and ApoE4 gene on the survival rate of PC12 cells, and ApoE4 gene could enhance the cytotoxicity of maltol aluminum on PC12 cells.

关 键 词：载脂蛋白E基因 PC12细胞 慢病毒转染 基因重组 聚合酶链反应 CCK-8 麦芽酚铝 

分 类 号：R135.1[医药卫生—劳动卫生] R114[医药卫生—公共卫生与预防医学]