反转录聚合酶链反应对Dukes A、B期大肠癌淋巴结微转移的检测_论文.docxVIP

反转录聚合酶链反应对Dukes A、B期大肠癌淋巴结微转移的检测 【摘要】 目的 探讨反转录聚合酶链反应（Reverse Transcriptase Polymerase Chain Reaction，RT-PCR）检测Dukes A、B期大肠癌淋巴结微转移的应用。方法 40例行根治性手术的Dukes A、B期大肠癌患者，应用RT-PCR法检测切除的492个淋巴结中细胞角蛋白（Cytokeratin，CK）20 mRNA的表达；另取2例Dukes C期大肠癌的8个HE染色阳性淋巴结作为阳性对照，2例良性胃肠道疾病的15个淋巴结作为阴性对照；并采用系列稀释试验检测本法的敏感性。 结果 40例Dukes A、B期大肠癌患者的492个淋巴结中有17例的53个检测到CK20 mRNA的表达；2例Dukes C期大肠癌的8个HE染色阳性淋巴结RT-PCR结果均为阳性，2例良性胃肠道疾病的15个淋巴结RT-PCR结果均为阴性。系列稀释试验提示CK20 RT-PCR的敏感性为105个正常淋巴细胞中检测到1个肿瘤细胞存在。结论 CK20 RT-PCR是检测Dukes A、B期大肠癌淋巴结微转移灵敏而又特异的方法。 【关键词】 大肠肿瘤；淋巴转移；微转移；基因诊断；反转录聚合酶链反应；角蛋白20 【Abstract】 Objective This study was undertaken to investigate the clinical implication of reverse transcriptase polymerase chain reaction (RT-PCR) to detect lymph nodes micrometastases in Dukes A and B colorectal cancer patients.Methods We examined 492 lymph nodes obtained from 40 colorectal cancer patients of Dukes A and B who underwent curative operation，using RT-PCR to detect the expression of cytokeratin 20(CK20) mRNA. Eight lymph nodes proved pathologic positive were served as positive control and 15 lymph nodes obtained from 2 patients with benign disease as negative control. We also used serial dilution experiment to determine the sensitivity of this method.Results Among 492 lymph nodes，CK20 mRNA expression was detected in 53 of 17 cases. CK20 mRNA expression was detected in all positive control and undetected in all negative control. The results of serial dilution experiment indicated that the limit of sensitivity was 1 tumor cell per 105 normal lymphocytes.Conclusion CK20 RT-PCR is a sensitive and specific method to detect lymph node micrometastases in colorectal cancer patients of Dukes A and B. 【Key words】 colorectal neoplasms；lymphatic metastases；micrometastase；genetic diagnosis；reverse transcriptase polymerase chain reaction (RT-PCR)；keratin 20 大肠癌术后转移和复发是患者的主要死亡原因。行根治术的患者约有20%～40%在5年内出现转移或复发［1］，但这些患者在手术前后的常规检查中（包括手术探查、B超、CT等）并未发现显性转移，而微小的转移灶是客观存在的。我们将这些微小的存在于血循环、淋巴道、骨髓和其它各组织脏器中临床常规检查难以发现的转移灶称