【作者】 杨冰；

【导师】 徐启武； 黄强； 于佶； 车晓明；

【作者基本信息】 复旦大学 ， 外科学， 2005， 博士

【摘要】 第一部分:胶质瘤相关基因的反向多点杂交研究 目标 利用反向多点杂交技术检测胶质瘤相关基因谱在胶质瘤组织中的表达情况,为这些基因在胶质瘤恶性进展中的作用提供依据,并找出具有进一步研究价值的基因。 方法 分别提取正常脑和胶质瘤标本中的总RNA,并反转录为cDNA,随机引物法标记探针,与点于特殊尼龙膜上的基因片断进行反向杂交。用复日SmartView图像分析软件进行处理,将各个基因的表达强度和β—actine进行比对,根据比值作相关基因的半定量分析。 结果 与正常脑组织比较,2级胶质瘤中表达上调的基因38条,下调24条;3级胶质瘤中有45条基因上调,17条基因下调;4级胶质瘤中有32条上调,29条下调,1条差异不明显。经方差分析,按病理级别分组,差异具有统计学意义(p<0.05)的有Caspasel、CDK4、c13、A20、EWS、Caspase3和EPS8。将恶性胶质瘤(WHOⅢ级和WHOⅣ级胶质瘤)基因表达水平与WHOⅡ级胶质瘤相比较,我们发现CDK4、MMP2、TOP1、c13和RAD17表达水平存在统计学差异(p<0.05)。 结论 反向多点杂交可以同时检测数十个基因的表达,并能对表达量高低进行半定量分析。我们通过这种方法发现,我们发现两条(A20、eps8)与胶质瘤相关的新基因。 第二部分:A20、EPS8、RAD17在胶质瘤中表达的研究 目的:第一部分中我们已经利用反向多点杂交技术筛选到A20、EPS8、RAD17等有研究价值的基因。我们利用RT-PCR技术对A20、EPS8、RAD17进行进一步的研究。一方面在更大的样本范围内明确以上基因的表达改变情况,另一方面通过对这些基因在不同级别星形胶质细胞瘤标本中表达水平的半定量分析为进一步的功能研究提供依据。 方法:提取6例正常脑组织和16例中枢神经系统胶质瘤标本的总RNA,并反转录为cDNA。加入相应的引物后PCR扩增。扩增产物电泳后,按预期产物条带更多还原

【Abstract】 Objective: To analyze the glioma relative gene expressions states and find potential gene target used in the future by reverse dot-blot.Method: total RNA was extracted from gilomas and normal brain tissues, and reversely transcribed into cDNA. The probe was marked randomly and the dot blot was performed with the gene fragment which had been added onto nylon membrane. Using smart view image analysis system we contrasted the glioma development relative genes with β - actine, and made semiquantitation analysis by their ratios. Result: Contrast with the normal brain, there were 38 genes up-regulated and 24 genes down-regulated in grade two gliomas, 45 genes up-regulated and 17 genes down-regulated in grade three gliomas, 32 genes up-regulated -. 29 genes down-regulated and one unchanged in grade four glioblastomas. Grouped by pathology classification, Caspasel CDK4 c13 A20 EWS Caspase3 and EPS8 were significant in statistic. Compared maglinant gliomas with grade 2 gliomas, CDK4 MMP2 TOP1 c13 and RAD 17 were singificant in statistic. Conclusion: The expressions of genes in different tissues can be analysed semiquantitively by reverse dot blot. Using this way ,the overexpressions of two novel glioma relative genes (A20, EPS8) were found in glioma.Part II: Measure EPS8 A20 RAD17 expressions states in gliomasObjective: In the first part, we find three novel giloma relative genes (A20 EPS8 RAD 17) by reverse dot blot. Then, We will use RT-PCR to do more research about these genes expression states in different grade gliomas, which can confirm EPS8更多还原