山东医药2018，Vol.58Issue(5)：17-20,4.DOI:10.3969/j.issn.1002-266X.2018.05.005
DKK1基因过表达慢病毒载体构建及其在大鼠肾上腺嗜铬细胞瘤细胞PC12中的表达
Construction of lentiviral vector with DKK1 gene over-expression and its stable expression in rat adrenal pheochromocytoma cell line PC12
摘要
Abstract
Objective To construct the lentiviral vector with DKK1 gene over-expression and to explore whether it can be efficiently and stably expressed in rat adrenal pheochromocytoma PC12 cells.Methods PCR was used to amplify DKK1 gene sequence.Plasmid was transferred and integrated into the vector and GV358-DKK1 lentiviral expression vector was constructed.The 293T cells were co-transfected with the constructed target plasmids and the helper packaging plasmids to package the lentivirus.Fluorescence microscopy was used to observe the expression of genes in 293T cells.The virus titer was determined by virus gradient dilution method.The cultured PC12 cells were randomly divided into three groups:the empty vector negative control group,the PC12-DKK1 group which was infected with the empty vector lentivirus and GV358-DKK1 particles,respectively,and the blank control group which was routinely cultured.The relative expression of DKK1 mRNA and protein in the PC12 cells were detected by qRT-PCR and Westernblotting,respectively.Results The results of restriction endonuclease digestion and sequencing indicated that the recombinant lentiviral vector of GV358-DKK1 was successfully constructed and transfected into 293T cells to obtain high-titer virus.The relative expression of DKK1 mRNA and protein in the PC12 cells of the PC12-DKK1 group was higher than that of the the empty vector negative control group and the blank control group (all P ＜ 0.05).Conclusion The lentiviral vector overexpressing DKK1 gene is constructed successfully,meanwhile,DKK1 gene can be stably expressed in the PC12 cells.关键词
肾上腺嗜铬细胞瘤/DKK1基因/PC12细胞Key words
adrenal pheochromocytoma cells/DKK1 gene/PC12 cell分类
医药卫生引用本文复制引用
李慧华,仝书严,陈锐,张才溢,耿德勤..DKK1基因过表达慢病毒载体构建及其在大鼠肾上腺嗜铬细胞瘤细胞PC12中的表达[J].山东医药,2018,58(5):17-20,4.基金项目
江苏省科技项目(BK20151159). （BK20151159）
