摘要
目的:利用基因芯片的高通量分析性能和丙型肝炎病毒(HCV)全基因组细胞培养系统,研究HCV对宿主细胞转录调节基因的影响和机制。方法:用人工构建的HCV感染Huh-7肝癌细胞株,待其发生细胞病变后收集培养细胞,以同步培养但不感染HCV的细胞作为阴性对照。提取感染细胞和对照细胞的总RNA、总蛋白和细胞培养上清。总蛋白用于Western blotting鉴定HCV蛋白在感染细胞内的表达情况。细胞培养上清用于检测HCV在感染细胞内合成后的释放情况。总RNA用10g/L琼脂糖凝胶电泳鉴定抽提情况,再纯化后进行逆转录cRNA合成、荧光标记和纯化后用于基因芯片分析,检测宿主细胞转录调节基因的差异表达。结果:在细胞培养上清液中可检测到HCV,感染细胞内可检测到HCV蛋白表达。基因芯片检测2倍差异表达转录调节基因发现,上调基因11个,下调基因11个。结论:HCV全基因组细胞培养系统为研究HCV对感染细胞转录调节基因表达的影响提供了良好工具。利用基因芯片技术,筛选出HCV全基因组细胞培养系统中差异表达的转录调节基因,为研究HCV感染对细胞基因转录的影响提供了重要资料,加深了对HCV和宿主细胞相互作用机制的认识。
Objective： To investigate the effects regulation genes of host cells by gene chip assays Methods： Huh-7 hepatoma cells were cultured and of hepatitis C virus （HCV） on transcription in cultured cells with intact HCV genome. infected with in vitro constructed HCV. The total RNAs,proteins and cell culture supernatants of HCV infected cells and control cells were isolated. Proteins and cell culture supernatants were used to detect the HCV replication and protein expression in cell culture system. The HCV protein expression was detected with Western blotting. Released HCV from infected cells was analyzed by real-time fluorescence quantitative PCR. Total RNA was qualified using 10 g/L agarose gel electrophoresis, cRNA was synthesized, fluorescence labeled and purified,then hybridized with Agilent oligo microarray （20173 probes）. Differential expression of genes related to transcription in cell culture system was analyzed. Results： HCV was positive in cell culture supernatants and HCV protein expression was also positive according to Western blotting results. Eleven up-regulated and 11 down-regulated genes related to transcription were found after Agilent gene chip screening. Conclusion： Intact hepatitis C virus cell culture system provides an useful tool for study on the affects of HCV infection on transcription regulation genes in host cells.
出处
《浙江大学学报（医学版）》
CAS
CSCD
2008年第2期164-169,共6页
Journal of Zhejiang University(Medical Sciences)
基金
国家自然科学基金资助项目(30470089)
关键词
肝炎
丙型
肝炎病毒
基因
调节
丙肝病毒
细胞培养
转录
基因
Hepatitis C
Hepacivirus
Genes, regulator
Hepatitis C virus
Cell culture
Transcription
Genes
