肿瘤相关基因表达检测寡核苷酸芯片的制备及其初步应用

Preparation of Oligonucleotide Microarray for Tumor-associated Gene Expression Detection and Its Primary Application

为研制肿瘤相关寡核苷酸芯片 ,并实现其在抗肿瘤反义核酸“癌泰得”作用机理研究方面的初步应用 ,制备了包含近 450种肿瘤相关基因特异寡核苷酸探针的寡核苷酸芯片 ,建立了相应的质控标准 .“癌泰得”用脂质体转染HepG2肿瘤细胞 ,提取细胞总RNA反转录并荧光标记cDNA ,用制备的寡核苷酸芯片检测肝癌细胞HepG2的肿瘤相关基因表达水平 ,用软件分析获得其差异基因表达谱 .0 4μmol/L的反义核酸“癌泰得”作用于HepG2细胞 15h后 ,MDNCF、DHS等基因mRNA表达下调 ,MUC2、MPP11、LAT、HRIF B、JNK3A1等mRNA基因表达上调 ,初步检测到了“癌泰得”的抗肿瘤作用可能的相关基因 ,为进一步的分子作用机理的探讨奠定基础 .结果表明 ,制备的肿瘤相关芯片敏感度高、特异性高、重复性均较好 ,可用于检测肿瘤相关基因的表达谱 。

Preparation of oligonucleotide microarray for gene expression detection of tumor-associated gene and its primary application in study on molecular mechanism of antisense oligonucleotide 'cantide' were carried out. An oligonucleotide microarray consisting of about 450 kinds of tumor-associated genes was constructed and the standards of quality controls of it were founded at the same time. Tumor cells were treated with 'cantide' mixed with lipofectin and total RNA was extracted. Through reverse-transcription, cDNA was synthesized and labeled with Cy5 or Cy3. After hybridized with the prepared microarray, signal intensities were detected and expression profiles were analyzed by software. The results indicate that the oligonucleotide microarray prepared has high specificity, good sensitivity and stablity etc. Compared to cells treated with lipofectin alone, the mRNA expression levels of seven genes appeared to show significant change. The mRNA expression of MDNCF, DHS genes were downregulated, while the mRNA expression of MUC2, MPP11, LAT, HRIF-B, JNK3A1 genes were upregulated after HepG2 cells treated with 'cantide' for 15 It. The data should provide some candidate genes that may be involved in antitumor effect of ' cantide and it is helpful to make a further investigation on the molecule mechanism. It can be concluded that the prepared oligonucleotide microarray can be used to investigate the gene expression profile of tumor-associated genes and provide techonical paltform for clinical diagnosis and basic research.