逆转录多聚酶链反应检测角蛋白19mRNA表达方法的建立

DEVELOPMENT OF RT PCR METHOD TO DETECT KERATIN 19 mRNA EXPRESSION

目的：探讨角蛋白１９（Ｋ１９）逆转录多聚酶链反应（ＲＴＰＣＲ）方法检测肿瘤隐匿性微小转移灶。方法：检测Ｌ７８、ＳＷ４８０、Ｋ５６２细胞株，正常人外周血单个核细胞（ＰＢＭＣ）及肺癌患者骨髓细胞（ＢＭＭＣ）中特异性的Ｋ１９ｍＲＮＡ表达，并与病理学和免疫组织化学方法进行比较。结果：可在１０６个ＰＢＭＣ中检测到１个Ｋ１９阳性细胞的Ｋ１９ｍＲＮＡ表达，而正常人ＰＢＭＣ未见表达。在４例肺癌患者ＢＭＭＣ中，３例Ｋ１９呈阳性，与病理学及免疫组化结果相同。１例Ｋ１９呈阳性而免疫组化为阴性。结论：本方法特异、敏感、快速，可用于检测外周血、骨髓及淋巴结中鳞癌。

Objective:To develope an assay of method reverse transcriptase polymerase chain reaction(RT PCR)with keratin 19(K 19) asa marker for diagnosis of micrometastases.Methods:The expression of K 19 mRNA on L 78,SW 480,K 562 tumor cell lines,normal peripheral blood mononuclear cells(PBMC) and bone marrow mononuclear cells(BMMC) of lung cancer patients were examined,which was compared with pathology and immunohistochemical staining.Results:The detection sensitivity of K 19 RT PCR was 1/10 6 PBMC.No expression of K 19 mRNA was found in normal PBMC.Three of 4 patients with lung cancer had detectable K 19 in BMMC which were similar in pathology and immunohistochemical staining.One of 4 patients had positive K 19 RT PCR assay,but negative immunohistochemical staining.Conclusion:This assay method may be useful to investigate occult micrometastases of squamous cell carcinoma and adenocarcinoma in PBMC or bone marrow and lymph nodes with its high sensitivity,speed,and specificity.