应用分支DNA技术及SqRT-PCR方法检测结直肠癌患者腹腔冲洗液中游离癌细胞

Detecting Free Cancer Cells in Peritoneal Cavity of Colorectal Cancer Patients by Branched-Chain DNA and SqRT-PCR

目的探讨分支DNA(b-DNA)及半定量RT-PCR(SqRT-PCR)在结直肠癌术中腹腔冲洗液中游离癌细胞检测中的应用。方法分别采用基于b-DNA信号放大的基因表达定量检测技术及SqRT-PCR方法检测48例结直肠癌患者术中腹腔冲洗液中CEA mRNA的表达,同时行腹腔冲洗液细胞学检查(peritoneal lavage cytolo-gy,PLC),收集12例结直肠良性病变患者的腹腔冲洗液为阴性对照,GAPDH mRNA为内参对照。结果b-DNA技术和SqRT-PCR方法检测游离癌细胞的阳性率(43.8%,31.3%)较PLC的检出率(4.2%)高(P<0.01)。结直肠癌患者腹腔冲洗液中CEA mRNA的相对表达量均与肿瘤分化程度、浆膜侵犯程度和Dukes分期有关(P<0.05),而与肿瘤大小、患者性别、年龄无关(P>0.05)。结论b-DNA技术和SqRT-PCR方法检测游离癌细胞各有优缺点;腹腔内游离癌细胞的存在与结直肠癌临床病理因素有关。

Objective To evaluate branched-chain DNA （b-DNA） signal amplification and semi-quantitative （Sq） RT-PCR in detection of free cancer cells in peritoneal flushing fluid of colorectal cancer patients during surgery. Methods The CEA mRNA in peritoneal flushing fluid in 48 cases of colorectal cancer were detected by b-DNA and SqRT-PCR. Peritoneal flushing fluid cytology （PLC） was conformed simultaneously to detect the free cancer cells. The peritoneal flushing fluid of 12 cases with colorectal benign disease were taken as negative control,GAPDH mRNA as internal control. Results In colorectal cancer patients,positive rate of free cancer cells by b-DNA and SqRT-PCR （43.8%,31.3%） was higher than that by PLC （4.2%）. The relative quantitative expressions of CEA mRNA were related to the Dukes staging,depth invasion and differentiation degree （P0.05）,but irrelevant to tumor size,the patients＇ age and gender （P0.05）. Conclusion Both b-DNA and SqRT-PCR technologies have advantages and disadvantages to detect free cancer cells in peritoneal flushing fluid,which are related to clinicopathological factors.