【作者】 王蔚；

【导师】 钱江； 黄倩； 袁一飞；

【作者基本信息】 复旦大学 ， 眼科学， 2008， 硕士

【摘要】 目的:以变性高效液相色谱(denaturing high performance liquidchromatography,DHPLC)检测视网膜母细胞瘤(retinoblastoma,RB)的Rh1基因外显子,探讨其突变情况以及DHPLC的检测情况,建立应用DHPLC检测Rb1基因的技术平台。方法:收集25例行眼球摘除的视网膜母细胞瘤患者的肿瘤组织,和3例健康对照者的外周血,进行基因组提取,以多聚酶链反应(polymerase chain reaction,PCR)扩增Rb1基因的第3,7,9,12和26个外显子片段。以DHPLC技术检测其突变情况,再采用DNA直接测序法进行进一步分析。结果:全部25例患者和3例健康对照者均扩增出5个外显子片段。进行变性高效液相色谱分析,第3,7,9,12,26个外显子的最优化检测注温分别为54.4,55.2,53.4,53.4和55.2℃。行变性高效液相色谱分析结果,第3,7,9,12个外显子中,25例患者和3例健康对照者均呈现同样峰形;而第26个外显子中,有2例患者呈现不同峰形。对所有片段进行测序,第3,7,9,12外显子的扩增片段中未发现突变。而外显子26的扩增片段中,DHPLC峰形和其他片段不同的2个样品也未出现突变。结论:DHPLC能快速检测出视网膜母细胞瘤Rb1外显子片段的突变位点,可以用于视网膜母细胞瘤的基因检测。更多还原

【Abstract】 Objective:To investigate the exon mutation of Rb1 gene in retinoblastoma by using denaturing high performance liquid chromatography,and to establish the detecting technique.Methods:Twenty-five patients with retinoblastoma were performed eyeball enucleation.The human genomic DNA of the tumor tissue was isolated.The peripheral venous blood of 3 healthy person was set as the control group.The 3^rd,7^th,9^th,12^th and 26^th exon fragments were amplificated by the way of PCR.And then,the exon mutation of Rb1 gene was detected by DHPLC.The method of direct sequencing was used to further study.Results:All 25 patients and the control group had been amplificated 5 exon fragments.By the way of DHPLC,the optimizing column temperature of the 3^rd,7^th,9^th,12^th and 26^th exon were different from 54.4,55.2,53.4, 53.4 and 55.2℃.The 3^rd,7^th,9^th and 12^th exon in all 25 patients and the control group showed the same peak form with DHPLC,while the 26^th exon in 2 patients appeared not.Sequencing all the fragments,the mutation could not be found in the 3^rd,7^th,9^th and 12^th exon fragments.And the same as the 26^th exon in 2 patients which had the different peak form of DHPLC.Conclusions:The exon mutation of the retinoblastoma could be detected quickly by the method of DHPLC,which could be used in gene detection.更多还原