摘要
目的检测葡萄胎发病新基因F10在人多种细胞系中转录水平的表达,从中选取F10低表达的细胞株,构建F10稳定转染的表达系统,研究F10的功能。方法荧光定量PCR检测F10在人A549、16HBE、Bel7402、HIC、HepG2、293、PC、MGC细胞株中的表达差异。采用电穿孔介导基因转染技术将已构建的pRc-CMV2-F10、pRc-CMV2转染A549细胞系。经G418筛选获得阳性单克隆细胞。细胞扩大培养后,荧光定量PCR技术鉴定F10基因在阳性克隆细胞中的表达。结果新基因F10在人的8种细胞系中呈不同程度的表达,其中在Bel7402、PC、MGC中呈高表达,在HepG2、HIC中表达次之,293中表达量较低,16HBE、A549中表达量最低;稳定转染细胞株A549具有F10基因的整合和相应mRNA的高表达。结论成功构建了稳定表达外源新基因F10的肺癌细胞系,为进一步研究F10基因的生物学功能提供了实验材料。
Objective To detect the transcriptional level of a novel gene F10 associated with the pathogenesis ofhydatidiform mole in human cell lines and screen the cell lines with low F10 expression to construct a stable eukaryotic expression system for F 10 gene. Methods The expression level of F 10 mRNA was detected with fluorescent quantitative PCR in A549, 16HBE, Be17402, HIC, HepG2, 293, PC and MGC cell lines. A549 cell line was transfected with plasmid pRc-CMV2-F10 via electroporation to allow stable F10 expression, and the positive cell clones were selected by G418. The insertion and expression of F10 gene in the A549 cells was analyzed using fluorescent quantitative PCR. Results F10 mRNA was expressed differentially in these cells lines, and the Be17402 cells, PC and MGC cells showed the highest F10 mRNA expression, followed by HepG2 and HIC cells and further by 293 cells, and 16HBE and A594 cells had the lowest expression. After transfection, A594 cells showed genomic integration of F 10 gene and high expression level of F 10 mRNA. Conclusion The pulmonary carcinoma cell line A549 with stable expression ofF 10 gene has been established, which may facilitate further study of the biological functions ofF 10 gene.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2008年第1期57-59,共3页
Journal of Southern Medical University
基金
国家自然科学基金(30470658
30672234)~~
关键词
葡萄胎
F10基因
稳定转染
hydatidiform mole
F10 gene
stable transfection
