Microarray analysis for detecting biological characteristics in neuroblastoma

应用来自人1、2、3、11、13、17、19、21、22号染色体的细菌人工染色体，对神经母细胞瘤组织DNA进行CGH芯片检测。信号的改变，特别是染色体1，11和17，检测到不利的神经母细胞瘤，但没有在有利的肿瘤。使用来自胚胎脑的约6500个克隆的cDNA微阵列分析显示，在不利的肿瘤中有43个上调基因，在退化和成熟肿瘤中分别有20和32个上调基因。另一方面，微分类分析显示63和39个基因分别在不利和有利的肿瘤中上调。通过上述技术鉴定的上调基因中的真实的时间定量RT-PCR鉴定了21个已知基因，包括MYCN、hTERT、VGEF、Cyclin G1、CD 44、NGF和Caspase 8。在同一肿瘤内肿瘤表现不均一的“复合型”肿瘤组织中，我们采用显微切割技术收集高度异型性的部分或端粒酶阳性的部分的肿瘤细胞。并对化疗后肿瘤切片中残存的存活肿瘤细胞进行显微解剖。然后，扩增从这些收集的细胞中分离的总RNA，然后用于微阵列。这些结果鉴定了在高度恶性细胞中上调的23个基因。几乎所有的基因都包含在不利肿瘤中鉴定的基因中。退化的神经母细胞瘤表现出凋亡相关基因如caspase 8和9的上调，成熟的肿瘤表现出神经元分化因子如NTRK 1和NGF的上调。端粒酶上调肿瘤与其它肿瘤的比较显示，hsp 90、Rad 50等102个差异表达基因可能直接调控端粒酶。这些结果表明，神经母细胞瘤的分类有用的标志物是21个基因的表达和1，11，17号染色体的基因剂量。

DNA samples of neuroblastoma tissues were examined CGH array using bacterial artificial chromosomes derived from human chromosome 1, 2, 3, 11, 13, 17, 19, 21, 22. Alterations of signals, especially chromosomes 1, 11 and 17, were detected in unfavorable neuroblastomas but not in favorable tumors. Analysis of cDNA microarray using approximately 6500 clones derived from embryonal brain revealed 43 upregulated genes in unfavorable tumors, 20 and 32 upregulated genes in regressing and maturing tumors, respectively. On the other hand, microsort analysis revealed 63 and 39 genes upregulated in unfavorable and favorable tumors, respectively. Real time quantitative RT-PCR in the upregulated genes identified by above technologies identified 21 known genes including MYCN, hTERT, VGEF, Cyclin G1, CD44, NGF, and Caspase 8. In the "composite type" tumor tissues which have heterogeneity of tumor findings in the same tumors, we collected tumor cells in the part with highly dismorphologic findings or in the part with telomerase positive cells by microdissection technique. And we also microdissected the viable tumor cells remained in the postchemotherapeutic tumor sections. Then, total RNA isolated from these collected cells was amplified and then used for microarray. These results identified 23 genes upreglated in the highly malignant cells. Almost all genes were included in the genes identified in the unfavorable tumors. Regressing neuroblastomas showed the up-regulation of apoptosis-relating genes such as caspase 8 and 9 and maturing tumors showed the up-regulation of the neuronal differentiating factors such as NTRK1 and NGF. The comparison between telomerase upregulating tumor and others revealed 102 different expressing genes including hsp 90 and Rad 50, which may directly regulate telomerase. These results suggested that the useful markers for classification of neuroblastoma are the expression of 21 genes and gene dosage of chromosome 1, 11, and 17.