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[目的]建立细胞角蛋白18(CK18)实时荧光定量PCR(RTFQ-PCR)检测系统,定量检测CK18在胃癌淋巴结中的表达水平,发现淋巴结微转移,从而提高胃癌淋巴结转移诊断的阳性率。[方法]应用TaqMan探针实时荧光定量PCR方法,以CK18作为淋巴结微转移的标志基因,制备CK18表达序列的标准品,并建立标准曲线。定量检测24例病理证实无淋巴结转移的胃癌患者199枚淋巴结的CK18mRNA拷贝数。[结果]RTFQ-PCR检测CK18mRNA的95%CV值为2.38%,敏感性达10个拷贝。24例病理无淋巴结转移的胃癌患者,15例CK18mRNA阳性。阳性率为62.5%(15/24)。RTFQ-PCR检测CK18mRNA的量值水平,最小值为2.57×102,最大值1.08×108。[结论]应用RTFQ-PCR方法,以CK18作为淋巴结微转移的标志基因,可提高胃癌淋巴结转移的检出率,能够发现组织病理学检查不能发现的微转移灶,并可能对微转移分级有益。
[Objective] To establish a real-time fluorescence quantitative PCR (RTFQ-PCR) detection system for cytokeratin 18 (CK18) to quantitatively detect the expression of CK18 in lymph node of gastric cancer and to find out the micrometastasis of lymph nodes and to improve the positive rate of lymph node metastasis in gastric cancer. [Method] With TaqMan probe real-time fluorescence quantitative PCR method and CK18 as the marker gene of lymph node micrometastasis, the standard of CK18 expression sequence was prepared and a standard curve was established. Quantitative detection of CK18 mRNA copies in 199 lymph nodes from 24 pathologically confirmed gastric cancer patients without lymph node metastasis. [Results] The 95% CV value of CK18 mRNA detected by RTFQ-PCR was 2.38% and the sensitivity was 10 copies. Twenty-four patients with gastric cancer without lymph node metastasis had positive CK18 mRNA in 15 cases. The positive rate was 62.5% (15/24). The level of CK18 mRNA was detected by RT-PCR. The minimum value was 2.57 × 102 and the maximum was 1.08 × 108. [Conclusion] Using RTFQ-PCR method and CK18 as markers of lymph node micrometastasis can improve the detection rate of lymph node metastasis in gastric cancer, find micrometastases that can not be found by histopathological examination, and may be helpful for micrometastasis.