HBV转染肝癌细胞系基因表达谱改变的研究

摘要

目的 利用寡核苷酸芯片筛选HBV感染应答基因,探讨HBV感染分子致病机制.方法 选用Agilent Human 1A Oligo基因芯片进行乙型肝炎基因表达改变的研究,用Cy3标记实验细胞(HepG2.2.15细胞),Cy5标记对照组细胞(HepG2细胞),比较HepG2.2.15细胞与HepG2细胞之间的基因表达谱差异;用适时定量PCR(RQ-PCR)对部分差异基因进行验证.结果 通过杂交、扫描、统计学分析,根据P Value LogRatio值及gProcessed signal/rProcessed signal值从近20 000个基因中共筛选出差异表达基因744个,其中423个基因表达上调,321个基因表达下调.用RQ-PCR技术对其中4个相关基因胰岛素样生长因子2(IGF2)、甲胎蛋白(AFP)、干扰素同源序列结合蛋白(ICSBP)、核糖核苷酸还原酶缩多氨酸M1(RRM1)的表达差异进行了验证,与表达谱的结果基本保持一致.结论 通过验证,表达谱芯片的杂交结果真实可靠,为下一步继续进行差异表达基因的研究、了解HBV的致病机制打下了基础.%Objective To disclose detailed gene expression with HBV infection and how HBV proteins regulate host cellular gene expression. Methods Gene expression profiles of cells with HBV infection was analyzed and compared by Agilent Human 1A oligo microarray method. We selected HepG2.2.15 cells as the cell line of research and the HepG2 as cell line of control. HepG2.2.15 cells were labeled with Cy3 and HepG2 cells were labeled with Cy5 ,respectively. Results After hybridization,microarray scan ning and statistics analysis(P Value LogRatio and gProcessed signal/r Processed signal), 744 genes were differentially expressed in HepG2.2.15 cells contrast to HepG2 cells from almost 20000 genes. Expression levels of 423 genes of these 744 genes were in creased and 321 genes of these 744 genes were decreased. To validate the authenticity of the microarray, we adopted the technique of real time PCR to verify the genes of different expression. The results showed that the mRNA level of IGF2,AFP,ICSBP and RRM1 in HepG2.2.15 cells were up regulated 9.68,4.80 times and down regulated 3.03,4.02 times,respectively,comparing with HepG2 cells. The results of Oligo microarray were up regulated 9.89,4.56 times and down regulated 2.88,3.85 times, respectively. Conclusion From the results,it was concluded that the results of genes expression profile by oligo microarray method were true and reliable. The possible responsive genes to HBV infection derived from this microarray offers new information to the exploitation of pathogenesis of HBV and shows abundant potential targets for the treatment of HBV infection.