应用实时定量RT-PCR方法检测子宫内膜癌患者盆腔淋巴结中微转移的探讨

doi:10.3877/cma.j.issn.1673-5250.2006.06.103

目的

建立一种应用实时定量RT-PCR技术，检测子宫内膜癌患者淋巴结微转移的方法。

方法

以细胞角蛋白19(cytokeratin 19，CK₁₉)为标志基因，对14例患者的218枚淋巴结进行实时定量RT-PCR。

结果

本方法能够在10⁶个外周血单核细胞中测到10个MCF-7细胞。本组10名患者的64枚淋巴结有CK₁₉ mRNA表达，6名患者的33枚淋巴结为PCR阳性。临床病理阳性患者的淋巴结PCR阳性率高于临床病理阴性但PCR阳性的患者；临床病理阴性但PCR阳性患者的淋巴结中，CK₁₉ mRNA的背景表达率高于临床病理和PCR均为阴性的患者。PCR阳性淋巴结在盆、腹腔内的分布无解剖规律可循。

结论

实时定量RT-PCR检测子宫内膜癌患者淋巴结微转移的敏感度高、特异性强、速率快、操作简便，适于临床推广应用。

关键词: 子宫内膜癌, 聚合酶链反应, 微转移, 细胞角蛋白

Objective

To develop a quantitative method for the detection of lymph node micrometastases of patients with endometrial cancer.

Methods

218 lymph nodes from 14 patients were studied.Real-time quantitative analysis was performed using cytokeratin 19(CK₁₉)as a gene marker.

Results

This method resulted in the reproducible quantification of 10 to 10⁶ MCF-7 cells per 10⁶ peripheral blood nucleated cells.CK₁₉ mRNA was detected in 62 lymph node from 10 patients.33 lymph node from 6 patients were PCR positive.The PCR-positive rate of lymph node was higher in patients with pathologically metastatic lymph node than in patients with path-negative but PCR-positive lymph node.Furthermore, the CK₁₉ mRNA background expression rate was higher in the lymph node of path-negative but PCR-positive patients than in lymph nodes of path-negative and PCR-negative patients.

Conclusions

Real-time quantitative RT-PCR with TaqMan probes is a sensitive, specific and rapid method for the detection of micrometastases in lymph node of patients with endometrial cancer.

Key words: endometrial cancer, polymerase chain reaction, micrometastasis, cytokeratin

图1　质粒(PCR4-T0P0 CK₁₉)实时定量PCR的扩增曲线(A)和标准曲线。(A)质粒的浓度自4 × 10⁵拷贝/ μl至40拷贝/ μl按10倍稀释的PCR曲线。(B)循环阈值与质粒浓度对数关系的标准曲线

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Real-time quantitative RT-PCR detection of lymph node micrometastases of patients with endometrial cancer

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Real-time quantitative RT-PCR detection of lymph node micrometastases of patients with endometrial cancer