摘要
目的筛选干扰素-α真核表达质粒转染HepG2细胞后细胞内表达下调的基因,以进一步探讨干扰素-α作用的分子生物学机制。方法将pcDNA3.1(-)-IFN-α以及pcDNA3.1(-)空载体分别转染肝母细胞瘤HepG2细胞系,用抑制性消减杂交技术筛选pcDNA3.1(-)-IFN-α转染HepG2细胞内表达下调的基因。以pcDNA3.1(-)转染组细胞作为tester,pcDNA3.1(-)-IFN-α转染组作为driver,建立消减文库,随机挑选阳性克隆,测序并进行序列比对,得到下调表达的基因。应用RT-PCR技术,进一步证明IFN-α对Hsp90的下调表达作用关系。结果成功构建了IFN-α真核表达质粒转染HepG2细胞后表达下调的cDNA消减文库。从文库中随机挑选克隆,测序并进行生物信息学分析后,得到下调表达的基因有铁蛋白,热休克蛋白90,S-腺苷甲硫氨酸脱羧酶1,真核翻译延伸因子1β,信号序列受体β(SSR2),组织因子路径抑制子,RAD23同源物B。RT-PCR进一步表明,IFN-α真核表达质粒转染的HepG2细胞内Hsp90mRNA表达水平下调。结论应用SSH技术成功构建了pcDNA3.1(-)-IFN-α转染的HepG2细胞内表达下调的cDNA消减文库,并筛选出下调表达基因,半定量RT-PCR表明IFN-α对Hsp90mRNA具有下调表达作用。为进一步阐明IFN-α作用的分子生物学机制提供了重要的理论依据。
Objective To screen the down-regulated genes in HepG2 cells transfected by pcDNA3.1（-）-IFN-α to further investigate the biological mechanism of IFN-α Methods PeDNA3. 1 （-）-IFN-α was transfected into HepG2 cells as treatment group and the PeDNA3. 1（-） transfected cells as the control to perform suppression subtractive hybridization （SSH）. The subtractive library for down- regulated genes was obtained when peDNA3. 1 （-） transfccted HepG2 as tester arid peDNA3. 1 （-）-IFN-α transfected cells as driver. A housekeeping gene, G3PDH, was used to estimate the subtractive efficiency. Genes with lower expressions in IFN-a transfected HepG2 cells were obtained from the library after being randomly sequenced and analyzed. Among the obtained genes, regulation of IFN-α on heat shock 90kD （Hsp90） mRNA was further investigated by RT-PCR Results G3PDH was subtracted efficiently indicating that the subtraetive library was constructed successfully. 50 positive clones were randomly isolated for PCR identification. Results showed that most of the plasmids in the clones contained 200-1000bp inserts. The down-regulated genes obtained were as follows： eukaryotic translation elongation factor 1 beta, ferritin, RAD23 homolog B, signal sequence receptor 2 （SSR2）, tissue factor pathway inhibitor, heat shock 90kD protein 1, and adenosylmethionine decarboxylase 1. RT-PCR showed that hsp90 mRNA had a reduced expression in pcDNA3. 1 （-）-IFN-α transfected HepG2 cells Conclusion The down-regulated cDNA subtractive library in HepG2 cells after pcDNA3. 1 （-）-IFN-α transfection was constructed successfully. IFN-α could down-regulate the hsp90 mRNA expression.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第1期35-38,共4页
Medical Journal of Chinese People's Liberation Army
关键词
干扰素-Α
细胞系
肿瘤
抑制性消减杂交技术
interferon-alpha
cell line, tumor
suppression subtraetive hybridization
