【作者】 周明锐；

【导师】 任祖渊； 黄秉仁； 王欣；

【作者基本信息】 中国协和医科大学 ， 神经外科， 2000， 博士

【摘要】 脑胶质瘤是危害人类健康的常见恶性肿瘤之一，在各种原发性中枢神经系统肿瘤中发病率最高。目前，胶质瘤特别是恶性胶质瘤的治疗还是一个非常棘手的问题。与正常胶质细胞相比，胶质瘤细胞中许多基因发生了结构和表达的改变，其中主要有癌基因的激活和抑癌基因的失活以及大量相关基因的表达变化。这些基因改变是胶质瘤恶性生物学行为的基础，但对此的了解还不是很清楚。体外培养的肿瘤细胞系的刺激、诱导常能提供有关肿瘤发生发展及药物、生物治疗的有用信息。分离、克隆胶质瘤发生发展和分化相关的cDNA，能够丰富人们对胶质瘤的分子机理认识，可以提供诊断、治疗的新线索，在理论和应用上具有重要的价值。 脑胶质瘤，特别是胶质母细胞瘤最常见而突出的分子特征为表皮生长因子受体(EGFR)的突变和(或)过表达，并与胶质瘤的恶性程度和生物学行为密切相关。为此，我们用其相应的配基EGF刺激诱导人脑胶质母细胞瘤BT325细胞，分析这一过程中所发生的基因表达谱变化；探寻胶质瘤发生发展的相关基因，以期发现可用作胶质瘤治疗的靶基因和(或)分期分型的分子标志。 分别给予每毫升0.1ng，1ng，10ng，100ng EGF刺激BT325细胞，发现并证实随着剂量的增大，BT325细胞生长增殖明显的抑制。EGF可抑制胶质瘤细胞生长这一现象，尚未引起人们的关注，文献中主要是EGF刺激胶质瘤细胞增殖和浸润的报道，但也仍未对其分子机制进行深入的研究。 应用新近发展的mRNA差异显示分析(DDRT-PCR)技术，分析BT325细胞生长抑制后的差异表达基因。进行不同的锚定引物和随机引物配对的DDRT-PCR反应，经变性聚丙烯酰胺长凝胶电泳，PCR产物的回收、扩增，分离了61个差异片段，大小介于200-500bp之间。为进一步鉴定分离出的片段所代表的基因的差异表达情况，把这些片段点膜制成cDNAarray，采用生长抑制组和对照组RNA反转录标记的cDNA第一条链的混合探针，分别同时对所得的差异片段进行反向Northern斑点杂交。根据斑点杂交的结果挑选出6个表达明显上调的差异表达片段进行克隆和测序。更多还原

【Abstract】 The glioblastoma is one of the common malignant tumors endangering human health and it has the highest incidence among the primary tumors in center nervous system. At present time, the therapy of glioblastoma, especially the malignant glioblastoma is very difficulty. Compared with those in normal glial cell, many genes in glioblastoma cell have been changed in structure and expression, including activated oncogenes, inactivated tumor suppressor genes and many associating genes. Such gene expression changes are the molecular basis of biological behaviors of malignant glioblastoma, but still not well known. The stimulation and induction of in vitro tumor cell culture can provide useful information about the cancer genesis, drug and biological therapy. The isolation and cloning of cDNA associating with glioblastoma developing and differentiation can enrich our understanding molecular mechanism of glioblastoma formation, provide the clue to diagnosis and treatment and have important theoretical and practical values.The most common and outstanding molecular characteristic of Glioma, especially the glioblastoma, is the mutation and /or over-expression of Epidermal Growth Factor Receptor (EGFR), closely associating with malignancy and biological behavior. So we stimulate and induce human glioblastoma BT325 cells with Epidermal Growth Factor to analyses and detect the gene spectrum changes related to glioma growth and progression, anticipating to find target moleculars which can be use as glioma gene therapy and/or molecular markers of glioma staging and classification.We use the EGF 0.1ng/ml, 1ng/ml, 10ng/ml; 100ng/ml to stimulate BT325 cells separatively and find that with increased dosage, the growth and proliferation of BT325 was apparently inhibited. EGF inhibiting glioma cellgrowth has not been caused to attention. There were some papers更多还原