基因微矩阵技术在骨肉瘤发病相关基因研究中的应用

Exploration on the pathogenic genes and novel findings of osteosarcoma with gene microarray

目的探索骨肉瘤发病相关基因,研究基因表达水平对于骨肉瘤发病的重要意义。方法采用3个公认的骨肉瘤细胞株及1个成骨细胞株,提取总RNA,合成生物素标记的cRNA与AffymetrixGeneChipU133A芯片杂交。随机挑选10个差异表达基因,分别设计合成引物,用嵌合荧光法(SYBRGreenI)实时RTPCR法定量测量9例术中收集的新鲜骨肉瘤标本中各基因的表达水平,应用ABIPrism7000分析其与成骨细胞株之间的表达差异。结果以表达差异≥2.0倍为限,在AffymetrixHGU133A芯片包含的所有基因(约22000个转录本)中,3个骨肉瘤细胞株与成骨细胞株相比较,共同上调58个基因;共同下调142个基因。随机挑选10个差异表达基因的实时RTPCR结果与芯片结果完全相符。结论骨肉瘤是一种多基因病变,应用微矩列有助于发现该肿瘤的基因变化规律以及研究肿瘤内基因与基因的相互作用关系。

Objective To explore the pathogenesis-related genes in osteosarcoma and investigate the importance of gene expression to the pathogenesis of osteosarcoma. Methods Three well-known ATCC osteosarcoma cell lines and an osteoblastic cell line were collected, After the total RNA extraction, the finally synthesized biotinylated cRNAs were hybridized to Affymetrix GeneChip U133A arrays, After the design and the synthethesis of the primers, 10 of the differentially expressed genes were then chosen at random to further confirm the array results using the SYBR Green real-time RT-PCR method in 9 freshly collected osteosarcoma specimens during operation. The data analysis depended on the ABI Prism 7000 system, Results Using an entrance limit of ≥2.0, there were 58 up-regulated and 142 dowm-regulated genes identified by microarray which were commonly differentially expressed among all the three osteosarcoma cell lines in comparison with the osteoblastic cell line hfob 1.19. Many of the genes were first reported to be related to the pathogenesis of osteosarcoma. The array results were further confirmed by the real-time RT-PCR. Conclusion Many genes were involved in the pathogenesis of osteosarcoma. It is helpful to apply gene array to find the laws of the gene expression and to study the gene-gene interactional relationships in this tumor.