Role of developmental regulated genes in biological properties of medulloblastoma

最近，Wnt和Sonic Hedgehog信号被阐明与髓母细胞瘤的发生有关。除了一些其他信号，如Notch，已知在这一现象中也有作用，这些信号之间存在串扰，但肿瘤发生的分子机制尚不完全清楚。另一方面，随着人类基因组计划的完成，建立了人类基因组数据库，并发展了DNA微阵列和SAGE等多种方法，可以一次分析大部分转录本。此外，还提出了肿瘤干细胞的概念。近年来，一些研究人员报道了肿瘤干细胞理论及其在增殖、复发和/或转移中的作用的证据。我们分析和鉴定了许多在人和啮齿动物模型动物髓母细胞瘤发生中的发育调控基因。阐明这种现象背后的分子机制及其对b…的影响。对于髓母细胞瘤肿瘤干细胞的更多生物学特性，我们进行了进一步的分析。用抗CD133单抗作为肿瘤干细胞标记物，对髓母细胞瘤细胞系DAOY和D286MED进行流式细胞仪分析。少数细胞呈阳性反应。在SAGE网站的蛋白质分析中，髓母细胞瘤主要发育调控基因zic1与cMYC呈双向表达，在预后不良的髓母细胞瘤组织中高表达。因此，我们正在利用针对myc的条件耗尽系统的siRNA来分析myc在髓母细胞瘤发生中的作用。稳定的转化子还没有获得，这项实验正在进行中。我们已经建立了针对恶性脑瘤、胶质母细胞瘤和髓母细胞瘤的干细胞疗法。在此过程中，在通常的间充质干细胞(MSC)培养中发现了永生化细胞。分离、培养了多个MSC永生化克隆，并对其生物学特性进行了分析。结果表明，细胞增殖能力、运动能力增强，细胞凋亡率和全能性降低。对永生化的MSC的表达谱也进行了分析。结果显示永生化MSC中Wnt、Shh、Notch组分表达上调，干细胞标记物CD133表达显著降低。这些结果得到了定量RT-PCR、Western分析和免疫组织化学的证实。将永生化的MSC接种到大鼠脑内的同种异体移植分析表明，这些细胞可以植入，而原代MSC无法存活。较少

Recently, it has been clarified that Wnt and sonic hedgehog signals are involved in medulloblastoma oncogenesis. Beside some other signals, such as Notch, which is known to also have roles in this phenomenon, these signals have cross talk with each other, the molecular mechanisms underlying the oncogenesis have not been fully understood. The other hand, with achievement of the Human Genome Project, human genomic database has been established and several methods, DNA microarray and SAGE, with which most of transcripts can be analyzed at once, have been developed. Moreover the concept of cancer stem cell has been suggested. The evidences of cancer stem cell theory and roles in proliferation, recurrence and/or metastasis were recently reported by some investigators.We have analyzed and identified many developmentally regulated genes in medulloblastoma oncogenesis in human and rodents model animals. To clarify the molecular mechanisms underlying this phenomenon and the effect against the b … More iological properties of medulloblastoma cancer stem cell, we performed further analyses. FACS analyses using anti-CD133 mAb, as a cancer stem cell marker, in medulloblastoma cell lines, Daoy and D286MED. A few cells revealed positive immunoreactivity against the antibody. Furthermore, the expression profile was analyzed in CD133 positive cells enriched medulloblastoma cell line.In silicon analyses using SAGE web site, medulloblastoma predominant developmentally regulated gene, zic1, showed reciprocal expression pattern against cmyc, which highly expressed in medulloblastoma tumor tissue of poor prognosis cases. Therefore we are analyzing role of myc in medulloblastoma oncogenesis using conditional deplete system of siRNA against myc. The stable transformant still have not acquired, this experiment is now on going.We have been establishing stem cell therapy for malignant brain tumor, glioblastoma and medulloblastoma. In that precess, the immortalized cells were found in usual mesenchymal stem cell (MSC) culture. Some clones of immortalized MSC were isolated and developed, and the biological properties were analyzed. It was found that the ability of proliferation, mobility were increased, however, apoptosis and totipotency were decreased. The expression profile was also analyzed in this immortalized MSC. It was revealed that constituents of Wnt, Shh, Notch were up-regulated in immortalized MSC and the expression of stem cell marker, CD133 was dramatically decreased. These results were confirmed using quantitative RT-PCR, western analyses and immunohistochemistry. Allograft analyses, inoculation of the immortalized MSC into the rat brain, revealed that these cells could be engrafted whereas primary MSC could not survived. Less