【作者】 袁才佳；

【导师】 聂新民；

【作者基本信息】 中南大学 ， 临床检验诊断学， 2007， 硕士

【摘要】 目的:1建立稳定的NOR₁基因转染肝癌细胞系pcDNA3.1（+）/NOR₁-HepG2。2应用大通量的基因芯片技术和荧光定量Real-time PCR技术,分析转染NOR₁基因对肝癌细胞系HepG2基因表达谱的影响。3探讨NOR₁基因与差异表达基因在肝癌发生、发展中的可能机制。方法:1将NOR₁基因重组质粒pcDNA3.1（+）/NOR₁（实验组）和空白载体pcDNA3.1（+）（对照组）通过脂质体分别转染至HepG2细胞中,用G418筛选阳性克隆,建立稳定的转染细胞系pcDNA3.1（+）/NOR₁-HepG2（HepG2-NOR₁）和pcDNA3.1（+）-HepG2（HepG2-pc）扩大培养后提取细胞总RNA,用逆转录PCR（RT-PCR）对NOR₁基因的表达进行鉴定。2选择生长良好的实验组与对照组培养细胞,分别提取mRNA,逆转录成cDNA,用人17K基因表达谱芯片对实验组和对照组细胞基因组进行表达分析,统计表达差异在2倍以上的基因。3用Real-time PCR对芯片检测的部分差异表达基因进行进一步验证。结果1 RT-PCR结果显示实验组NOR₁基因表达明显高于对照组,表明pcDNA3.1（+）/NOR₁能在HepG2细胞中表达。2人17K基因表达谱芯片检测显示实验组与对照组比较有162个基因（或EST）表达差异在2倍以上,其中59条表达上调、103条表达下调,涉及细胞凋亡或肿瘤相关、细胞周期、转录调控、信号传导、翻译调控等基因。3 Real-time PCR对p15、MAGE-A6检测,实验组的表达量分别为对照组的2.692/2.989和2.535/2.118（Real-timePCR结果/芯片结果）,与芯片检测结果相符。cDNA基因芯片和Real-timePCR都是研究差异表达基因的有力方法,基因芯片筛选基因表达谱具有高通量大规模的特点,而荧光定量Real-time PCR则适合单个基因表达变化的研究,两者互为补充和印证。结论:1重组质粒pcDNA3.1（+）/NOR₁在肝癌细胞HepG2中稳定表达,转染pcDNA3.1（+）/NOR₁后肝癌细胞HepG2的基因表达谱发生改变。NOR₁基因可能是与肝癌相关的抑瘤/易感基因侯选者之一。基因芯片技术为大规模筛选疾病相关基因及各基因间相互作用提供了可能。2经Real-time PCR验证,P15和MAGE-A6基因在实验组与对照组的表达差异与芯片结果基本一致,分别为2.692/2.989和2.535/2.118（Real-time PCR结果/芯片结果）。NOR₁基因通过对P15 mRNA表达水平的影响是其对肿瘤细胞的生长抑制可能机制之一,这将为进一步系统研究NOR₁基因的功能及其在肝癌发生中的可能机制提供了探索的方向。更多还原

【Abstract】 Objective:1 To establish the stable transfection of hepatoma cell line pcDNA3.1（+）/NOR₁-HepG2.2 To study the effects of the gene expression profiles of human hepatocarcinoma HepG2 cells after transfection of NOR₁ gene with microarray assay and Real-time PCR.3 To discuss the possible mechanism between NOR₁ gene and up-regulated/down-regulated in the occurrence and progression of human hepatocarcinoma.Methods:1 The mammal expression vector of NOR₁（pcDNA3.1（+） /NOR₁）and eukaryotic expressing plasmid pcDNA3.1（+）were introduced into human hepatocarcinoma cell line HepG2 mediated by lipofectamin transfection.The stable G418-resistant clones were isolated. After extracting total RNA,the overexpression of NOR₁ gene were detected by RT-PCR.2 The differential expression profiles genes from NOR₁-transfected cells（HepG2-NOR₁）and empty vector cells（HepG2-PC）were determined using the human 17K cDNA expression chip with 17101 cDNA probes.3 The expression of p15 and MAGE-A6 genes were verified by Real-time PCR. Results:1 Recombinant plasmid pcDNA3.1（+）/NOR₁ can express in HepG2 cells.Stable expression of NOR₁ gene in HepG2 cells was confirmed by RT-PCR.The lever of NOR₁ mRNA increased apparently in HepG2-NOR₁ cells compared with HepG2-PC cells.2 From the scanning results of gene chips,162 genes were differentialiy expressed in the two cells lines,among which 59 genes（or EST）expression were up-regulated and 103 genes（or EST）expression were down-regulated in the HepG2-NOR₁ cells.The genes were correlated with cell apoptosis or tumor,cell cycle,cell transcription regulator activity,cell signal transducer activity,cell translation regulator activity,and so on.3 The expression of P15 gene in HepG2-NOR₁ cells increased to 2.692 compared with HepG2-PC cells,and MAGE -A6 increased to 2.533 by Real-time PCR.Conclusion:1 Over-expression of NOR₁ gene by transfecting NOR₁ can change the gene expression profiles in HepG2 cells,leading to change of cell growth,differentiation and apoptosis in HepG2-NOR₁ cells.cDNA chip is a useful method in understanding the mechanism of cancer with its high throughput characteristics.2 The expression of P15 gene in HepG2-NOR₁ cells increased to 2.692 compared with HepG2-PC cells,and MAGE-A6 increased to 2.533 by Real-time PCR.P15 is a very important factor in the occurrence and progression of human cancer,one of the mechanisms of NOR₁ gene inhibited the growth of HepG2-NOR₁ cells was NOR₁ gene induced p15 gene up-regulatation.The study laid a solid foundation for the further research of mechanism of NOR₁ gene during the occurrence and progression of hepatocarcinoma.更多还原