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目的探讨荧光定量聚合酶链反应(FQ-PCR)检测大肠癌患者外周血癌细胞角蛋白20 mRNA(CK20 mRNA)的表达水平及临床价值。方法采用FQ-PCR法测定136例结直肠癌及其肝脏转移患者外周血癌细胞CK20 mRNA的表达水平,系统研究了FQ-PCR法测定CK20 mRNA的重复性、定量范围和扩增效率,分析比较其与传统RT-PCR、免疫组化法检测结果的一致性。结果FQ- PCR法测定CK20 mRNA的批内CV为3.6%,批间CV为5.3%,定量范围为10~3～10~8 copies/ml,扩增效率为87.4%。与传统RT-PCR和免疫组化法相比较,Kappa值分别为0.87和0.83。结直肠癌患者外周血癌细胞CK20 mRNA的表达水平为(3.52×10~4±1.47×10~4)copies/ml,表达阳性率为48.5%;75例对照者均无CK20 mRNA表达。Dukes A、B、C、D期大肠癌患者外周血癌细胞CK20 mRNA的阳性率分别为9.5%、25.0%、48.8%和87.5%,各组间差异有统计学意义(P<0.05)。肝脏转移者CK20 mRNA阳性率为87.5%,显著高于肝脏无转移者(32.3%,P<0.05)。35例结直肠癌患者术后1、3、5周内CK20 mRNA表达水平呈下降趋势,但术后1、3周与术前比较,差异无统计学意义(P>0.05),术后5周显著低于术前(P<0.05)。结论FQ-PCR是较灵敏的快速定量检测CK20 mRNA的方法。大肠癌患者外周血癌细胞CK20 mRNA的表达与肿瘤的复发和转移有关,CK20 mRNA检测将有助于大肠癌血源性播散的监测。
Objective To investigate the expression of cytokeratin 20 mRNA (CK20 mRNA) in peripheral blood of patients with colorectal cancer by fluorescence quantitative polymerase chain reaction (FQ-PCR) and its clinical value. Methods The expression of CK20 mRNA in peripheral blood of 136 patients with colorectal cancer and its liver metastasis was detected by FQ-PCR. The repeatability, quantitation range and amplification efficiency of CK20 mRNA were determined by FQ-PCR. Consistency with traditional RT-PCR and immunohistochemistry. Results The intra-assay CV of CK20 mRNA by FQ-PCR was 3.6%, the CV between batches was 5.3%, and the quantification range was 10 ~ 3 ~ 10 ~ 8 copies / ml. The amplification efficiency was 87.4%. Compared with traditional RT-PCR and immunohistochemistry, Kappa values were 0.87 and 0.83, respectively. The expression of CK20 mRNA in peripheral blood of colorectal cancer patients was (3.52 × 10 ~ 4 ± 1.47 × 10 ~ 4) copies / ml, the positive expression rate was 48.5%. No CK20 mRNA expression was detected in 75 cases of colorectal cancer patients. The positive rates of CK20 mRNA in peripheral blood of Dukes A, B, C, D patients were 9.5%, 25.0%, 48.8% and 87.5%, respectively. There was significant difference between the two groups (P <0.05). The positive rate of CK20 mRNA in liver metastasis was 87.5%, which was significantly higher than that in liver without metastasis (32.3%, P <0.05). The expression of CK20 mRNA in 35 patients with colorectal cancer showed a decreasing trend in 1, 3 and 5 weeks after operation, but there was no significant difference between 1 and 3 weeks after operation (P> 0.05) Significantly lower than preoperative (P <0.05). Conclusion FQ-PCR is a more sensitive method for rapid and quantitative detection of CK20 mRNA. The expression of CK20 mRNA in peripheral blood cancer cells of patients with colorectal cancer is related to the recurrence and metastasis of tumor, and the detection of CK20 mRNA will be helpful for the monitoring of blood-borne dissemination in colorectal cancer.