了解散发性大肠癌微卫星不稳定及其hMSH2基因突变情况。方法　用6个微卫星位点标记，PCR法检测微卫星不稳定性(microsatellite instability, MI)，银染PCR-SSCP法检测hMSH2基因5、7、8、12、13、15外显子突变。结果　60例大肠癌中，微卫星改变总的发生率为50%(30/60)；20例(33.33%)表现微卫星不稳定性，其中4例同时有杂合性缺失(loss of heterozygosity, LOH)，DNA复制错误(RER)阳性11例(18.33%)；14例(23.3%)检测到LOH。8例微卫星不稳定性大肠癌组织检测到第5外显子杂合性突变，而未发现胚系突变。结论　散发性大肠癌中微卫星不稳定是一个常见的分子事件，与hMSH2基因胚系突变无关，可能为体细胞性突变或/和缺失所致。

Study on microsatellite instability and hMSH2 gene mutation in sporadic colorectal cancer

XIONG Bin, ZHENG Shu, CAI Xinhan.

　　Cancer Institute, Department of General Surgery, The Second Affiliated Hospital of Hubei Medical University, Wuhan 430071

　　【Abstract】Objective To study the microsatellite instability (MI) and hMSH2 gene mutation in sporadic colorectal cancer.Method MI was detected by using D2S123, D2S119,D13S160, D8S282, D3S1293 and D18S58 microsatellite markers. Silver staining PCR-SSCP method was used to detect mutation of hMSH2 gene exons 5, 7, 8, 12, 13, 15 in the MI cases.Results Among the 60 cases, 33.3% of the cases showed MI, 18.3% of the cases RER positive, 23.3% of the cases loss of heterozygosity. hMSH2-somatic heterozygosity mutation was found on the exon 5 in sporadic colorectal cancer tissues in 8 MI cases.Conclosions MI phenotype might be present ubiquitously in sporadic colorectal cancer in China. Its positive ratio was consistent with that of other reports. Germ-line mutation of hMSH2 was not related with the occurrence of MI in sporadic colorectal cancer. The sporadic colorectal cancer with MI might have alterations of unknown genes related to the mismatch repair system.

　　【Key words】 Microsatellite instability Colorectal cancer hMSH2 Gene Mutation

　　我们对60例散发性大肠癌组织的微卫星不稳定性进行研究，结合临床病理资料，探讨其临床意义，并研究微卫星不稳定性散发大肠癌hMSH2突变。

　　材料和方法

　　1、标本收集和基因组DNA提取　经病理证实的大肠癌60例，其中男37例，女23例，年龄32～78岁，平均56岁。标本随机取自浙江医科大学附二医院1996年3月～1997年5月间的手术新鲜组织，基因组DNA提取采用蛋白酶K消化，酚-氯仿抽提法。

　　2、微卫星不稳定性检测　选用D2S119、D13S160、D8S282、D3S1293、D2S123和D18S58六个微卫星位点进行检测。PCR反应体系为50 mmol/L KCl，10 mmol/L Tris-Cl, 0.1%Trito X-100, 1.5 mmol/L MgCl2，200 μmol/L dNTPS，模板DNA 200 ng，上下游引物各50 pmol,Taq酶1 U，加去离子水至50 μl。PCR反应参数为：94 ℃变性5分钟，然后进入35个循环(94 ℃45秒，退火温度30秒，72 ℃1分钟)，最后70 ℃延伸10分钟，4 ℃保存。PCR产物用8%变性聚丙烯酰胺凝胶电泳，电泳结束后进行银染。

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