Role of developmental regulated genes in biological properties of medulloblastoma

近年来，研究表明Wnt和超音hedgehog信号参与成神经管细胞瘤的发生。除了一些其他信号，如Notch，已知也在这一现象中起作用，这些信号之间相互串扰，肿瘤发生的分子机制尚未完全了解。另一方面，随着人类基因组计划的实现，人类基因组数据库的建立和DNA微阵列和SAGE等能够一次分析大部分转录本的方法的发展。此外，还提出了癌症干细胞的概念。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。我们分析并鉴定了人类和啮齿类模型动物髓母细胞瘤肿瘤发生过程中许多发育调控基因。为了阐明这一现象背后的分子机制以及对髓母细胞瘤肿瘤干细胞的作用，我们进行了进一步的分析。使用抗cd133单抗作为癌症干细胞标记物，在髓母细胞瘤细胞系，day和D286MED中进行FACS分析。少数细胞对抗体显示阳性免疫反应。进一步，分析了CD133阳性细胞富集的成神经管细胞瘤细胞系的表达谱。SAGE网站的硅分析显示，髓母细胞瘤主要发育调控基因zic1与cmyc呈互反表达模式，cmyc在预后不良的髓母细胞瘤肿瘤组织中高表达。因此，我们正在分析myc在成神经管细胞瘤肿瘤发生中的作用，使用siRNA的条件耗尽系统来对抗myc。稳定的转变还没有获得，这个实验正在进行中。我们已经建立了干细胞治疗恶性脑肿瘤、胶质母细胞瘤和髓母细胞瘤的方法。在此过程中，在常规间充质干细胞（MSC）培养中发现永生化细胞。分离并培养了一些永生化MSC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、移动性增强，但细胞凋亡和全能性降低。在永生化MSC中也分析了表达谱。结果表明，在永生化MSC中，Wnt、Shh、Notch等成分表达上调，而干细胞标志物CD133的表达显著降低。这些结果经定量RT-PCR、western分析和免疫组织化学证实。同种异体移植分析，将永生化间充质干细胞接种到大鼠脑中，发现这些细胞可以移植，而原代间充质干细胞不能存活。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