毛细管电泳法检测肝细胞癌微卫星不稳定性的影响因素  

Microsatellite instability genotyping in hepatocellular carcinoma by capillary electrophoresis system and its effective factors

作　　者：冼志红[1] 丛文铭[1] 张树辉[1] 

机构地区：[1]中国人民解放军第二军医大学东方肝胆外科医院病理科,上海市200438

出　　处：《世界华人消化杂志》2004年第8期1781-1784,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30370645上海市卫生系统百名优秀跨世纪学科带头人培养计划资助项目.No.98BR007~~

摘　　要：目的:探索适用于肝癌大规模基因组扫描的自动化微卫星不稳定性基因分型实验方法. 方法:应用6对高度多态性微卫星标记物对56例肝细胞癌基因组进行PCR扩增,产物通过MegaBACE 500毛细管电泳测序仪进行电泳,采用Genetic Profiler软件进行基因分型,摸索不同PCR条件以及PCR体系残留混合物对分型结果的影响. 结果:通过1次PCR和电泳能得到48个样品的分型结果, 常见的等位基因表型包括杂合子、纯合子、杂合子丢失、微卫星不稳定性和等位基因失衡等几种模式,其中微卫星不稳定性基因表型呈多种特征;MSI的频率为32.1%(18/56), 10例(18.2%)出现高频MSI;PCR反应体系残留混合物(dNTP、引物和盐离子的浓度等)对基因分型的结果有明显不利影响,可使等位基因片段大小分析结分错误导致结论错误;样品浓度对分析结分影响较小,在50 mg/L时比较合适. 结论:少数肝细胞癌存在MSI,表明其在HCC发生中作用较小.应用MegaBACE 500毛细管电泳测序仪和Genetic Profiler分析软件进行微卫星基因组扫描效率高,结果可靠, 但测序体系中必须维持最佳盐离子的浓度和样品浓度.AIM: To search for a large-scale automated microsatellite instability genotyping method for genomic scanning in liver neoplasms. METHODS: Fluorescent PCR products of 6 microsatellite polymorphic markers in 56 cases of hepatocellular carcinoma were separated on the MegaBACE-500 capillary array elec-trophoresis (CAE) instrument and analyzed with MegaBACE Genetic Profiler software. The effects of different reaction residues concentration in PCR reactive system on genotyping were investigated. RESULTS: Genetyping of forty-eight specimens was achieved in parallel by using PCR and capillary electrophoresis system after one cycle. The genotyping profiles included heterpzygosity, homozygosity, loss of heterozygosity, microsatellite instability and allelic imbalance, in which the phenotypes of microsatellite instability had a lot of characteristics. MSI was found in 18 of 56 HCC (32.1%) at one or several loci. Ten of 56 (18.2%) HCCs had MSI-H. The concentration of reaction residues (including dNTP, primers, and salt ion) in PCR system effected badly on scoring errors in allele size, which might result in misleading conclusions. The concentration of PCR reaction products affected little on scoring errors, and about 50 ng/μL was suitable for analysis. CONCLUSION: The frequency of microsatellite instability is very low in HCC and it is an uncommom event during hepatocarcinogenesis. MegaBACE-500 CAE instrument and MegaBACE Genetic Profiler software is a high-throughput, accurate and reliable method for genomic scanning, but products and salt ion concentration in PCR reactive system is one of the key factors altering genotyping results.

关 键 词：微卫星不稳定性 肝细胞癌 基因分型 表型 基因组扫描 测序 样品浓度 结论 体系 实验方法 

分 类 号：R735.7[医药卫生—肿瘤] G633[医药卫生—临床医学]