联合免疫磁化分选技术和实时荧光定量RT-PCR检测结直肠癌外周血肿瘤细胞及其临床意义  

Quatitative detection of peripheral circulating tumor cells by combination of immunomagnetic separation technique and real-time quantitative RT-PCR and its clinical implication in colorectal cancer

作　　者：王进[1] 汪彬哲[1] 胡伟民[1] 

机构地区：[1]广州医学院附属肿瘤医院胃肠肿瘤外科,广州510095

出　　处：《消化肿瘤杂志（电子版）》2011年第4期240-245,共6页Journal of Digestive Oncology(Electronic Version)

基　　金：广东省科技计划项目(编号2009B030801361)

摘　　要：目的建立联合应用免疫磁化分选技术和实时荧光定量RT-PCR检测循环肿瘤细胞的方法,并探讨定量检测结直肠癌患者外周血肿瘤细胞的临床意义。方法体外培养结肠癌HCT-116细胞,与5 ml健康人外周静脉血混合,采用密度梯度离心法分离外周血单个核细胞。分离后的标本分为免疫磁珠富集组(A组)和非富集组(B组),比较实时荧光定量RT-PCR方法检测其中人端粒酶逆转录酶(hTERT)mRNA的表达水平。抽取22例初治的结直肠癌患者和22例健康人外周静脉血,标本分为免疫磁珠富集组(C组)和非富集组(D组),健康者标本设为E组,比较三组hTERT-mRNA的表达水平,并探究与临床病理参数的关系。结果 A组hTERT-mRNA表达水平显著高于B组,差异有统计学意义(P<0.05)。22例结直肠癌患者中,C组hTERT-mRNA表达水平显著高于D组(P<0.05)。结直肠癌患者C组hTERT-mRNA表达水平显著高于健康对照E组(P<0.05)。结直肠癌患者hTERT-mRNA表达水平与患者性别、年龄、肿瘤大小、部位和肿瘤浸润深度无显著相关(P>0.05),与患者淋巴结有无转移和TNM分期呈显著相关(P<0.05)。结论联合应用免疫磁化分选技术和RT-PCR的检测方法 ,通过测定hTERT-mRNA表达水平可定量检测外周血肿瘤细胞,检测灵敏度优于单纯RT-PCR检测方法。结直肠癌患者外周血hTERT-mRNA的表达水平与淋巴结转移和TNM分期显著相关。Objective To establish a method for detection of circulating colorectal tumor cells(CTCs)by combination of immunomagnetic separation technology with real-time RT-PCR,and investigate the clinical significance of this method.Methods The colon cancer HCT-116 cells were mixed with 5ml whole blood from healthy donors,and peripheral blood mononuclear cells(PBMC) mixed with tumor cells was separated by density gradient centrifugation.PBMC in enrichment group(Group A) were co-cultured with CD326 monoclonal antibody-coated beads and enriched with immunomagnetic sorting method,while non-enrichment group(Group B) co-cultured with PBS.RNA extraction and quantitative real-time RT-PCR were performed to assess the expression level of human telomerase reverse transcriptase(hTERT).The same experiments were performed in 22 newly diagnosed colorectal cancer patients and 22 healthy donors.The relationships of clinicopathological parameters with hTERT expression were analyzed.Results hTERT-mRNA expression level of enrichment group A was significantly higher than that of non-enrichment group B(P<0.05).hTERT-mRNA expression level of enrichment group was significantly higher than those of non-enrichment group in 22 colorectal patients and the healthy control group.hTERT-mRNA expression was not significantly correlated with gender,age,tumor size,location and depth of invasion(all P>0.05),but significantly correlated with lymph node metastasis and TNM staging(P<0.05).Conclusions Combination of immunomagnetic separation technology with real-time RT-PCR of hTERT-mRNA expression is more sensitive to detect CTCs than RT-PCR only.hTERT-mRNA expression level is correlated with lymph node metastasis and TNM staging in colorectal cancer.

关 键 词：结直肠癌 循环肿瘤细胞 人端粒酶逆转录酶 免疫磁化分选技术 实时荧光定量逆转录聚合酶链反应 

分 类 号：R735.3[医药卫生—肿瘤]