应用RT-PCR并Southern杂交技术检测乳腺癌骨髓微转移

Detection of Micrometastases in the Bone Marrow of Patients with Breast Cancer Using Reverse Transcriptase Polymerase Chain Reaction and Southern Hybridization.

目的 检测原发性乳腺癌骨髓微转移的发生及与其它临床参考指标的关系。方法 应用逆转录 -聚合酶链反应 (RT -PCR)并Southern杂交技术 ,检测骨髓单个核细胞中细胞角蛋白 19(CK - 19)基因表达。结果 5 2例骨髓标本共检出微转移 19例 ( 3 6.5 % ) ,其中 17例淋巴结有转移者 ,9例CK - 19表达阳性 ( 5 2 .9% ) ;而无淋巴结转移的 3 5例中 ,10例CK - 19表达阳性 ( 2 8.6% )。微转移检出率与肿瘤大小有关 (P <0 .0 5 )。结论 以CK -19为标志物 ,RT -PCR并Southern杂交方法检测原发性乳腺癌骨髓微转移灵敏、特异 ,可做为临床判断预后的参考指标。

Objective The aim of this study was to detect micrometastases in the bone marrow of patients with breast cancer using reverse transcriptase polymerase chain reaction and southern hybridization,and compare with other clinical parameters.Methods Abstract total RNA from samples of bone marrow and lymph nodes(as positive control),then Reverse Transcription and amplification through polymerase chain reaction (PCR) using cytokeratin 19 (CK-19) gene specific primers.PCR products were analysised by agarose electrophoresis and were checked further by Southern blot hybridization.Human breast canceer cell line T47D was mixed with bone marrow cells at different proportions.The positive detection rate was compared between RT-PCR,Southern blotting and immunohistochemistary (IHC) methods.Results Cytokeratin 19 gene was expressed in all 6 pathological metastasis positive lymph nodes,while the expression wasn't seen in 8 bone marrow samples of normal and haemato-malignant disease.Nineteen cases were CK-19 positive in all 52 patients (36.5%),in which 9 cases came from 17 lymph node metastases positive patients (52 9%) and 10 from 35 lymph node metastases negative patients (28 6%).CK-19 positive cells can be detected by RT-PCR and Southern blotting method at the proportion of 1:10 6,while IHC method at 1:5×10 4,demonstrated that the former method was at least 20 times more sensitive than that of later.The micrometastases positive rate of the larger tumor size group (>5 0cm) was significantly (P<0 05) greater than that of the smaller group (≤2 0cm).There were no significant differences between micrometastasis positive and negative group among any of other clinical parameters.Conclusion RT-PCR and Southern blotting methods,using CK-19 as a biological marker,was sensitive and spectfic to detect bone marrow micrometastases of primary breast cancer.It can be as the prognostic parameter for clinical treatment.