免疫磁珠与荧光定量逆转录聚合酶链反应在胃癌患者外周血癌细胞检测中的应用价值  

Study of the method of using immunomagnetic bead and fluorescence quantitative reverse transcriptase polymerase chain reaction to detect the cancer cells in peripheral blood of patients with gastric carcinoma

作　　者：郭俊明[1] 肖丙秀[1] 金之瑾 秦丽君[3] 陈健 戴德坚[4] 

机构地区：[1]宁波大学医学院,315211 [2]宁波市医学分子诊断中心 [3]宁波大学医学院附属医院消化内科 [4]浙江大学医学院附属第二医院普外科

出　　处：《中华检验医学杂志》2004年第11期796-798,共3页Chinese Journal of Laboratory Medicine

基　　金：浙江省医药卫生科研基金资助项目(2002A073);宁波市重点博士基金资助项目(02J2010107);宁波大学重点科研项目(z0214015)

摘　　要：目的研究免疫磁珠和荧光定量逆转录聚合酶链反应(RTPCR)检测胃癌患者外周血癌细胞的应用价值。方法将不同浓度的人胃腺癌细胞系MGC803细胞加入到健康人血中,经淋巴细胞分离液收集单核细胞后,等分4份。其中1份直接用于提取RNA(方法Ⅰ)、其他分别用阴性磁珠(方法Ⅱ)、阳性磁珠(方法Ⅲ)及联用阴性和阳性磁珠(方法Ⅳ)富集癌细胞。然后,以癌胚抗原(CEA)mRNA作为分子标志物进行荧光定量RTPCR检测其相对拷贝数;并检测45例胃癌、30例胃溃疡患者和30名健康人外周血CEAmRNA。结果方法Ⅱ、Ⅲ和Ⅳ测得的CEAmRNA相对拷贝数与癌细胞数间有良好的相关性(方法Ⅱ和ⅢP<005;方法ⅣP<001),尤以Ⅳ方法为佳,可检测出每毫升血中含有的1个胃癌细胞。Ⅰ～Ⅳ方法检测胃癌患者阳性率分别为4222%、5333%、5111%和6444%;方法Ⅳ与方法Ⅰ比较,差异有显著意义(P<005)。用方法Ⅳ检测30例胃溃疡患者均为阴性,而其他3种方法检出2例阳性;30名健康人中4种方法均未检出扩增产物。结论免疫磁珠与荧光定量RTPCR法可提高胃癌患者外周血CEAmRNA的检出率。Objective To study the methodology of using immunomagnetic bead and quantitative reverse transcriptase polymerase chain reaction (RT PCR) to detect the cancer cells in peripheral blood of patients with gastric carcinoma Methods Different concentrations of human gastric adenocarcinoma MGC 803 cells were spiked into the blood from health persons Then, mononuclear cells (MNC) were collected through Ficoll Each sample was divided into four parts and used by four methods: Method Ⅰ, MNC were directly used to extract RNA; Method Ⅱ, negative immuno bead method; Method Ⅲ, positive immuno bead method; Method Ⅳ, negative and positive immuno bead method Finally, carcinoembryonic antigen (CEA) mRNA were detected by RT PCR The CEA mRNA in peripheral blood from 45 patients with gastric cancer, 30 patients with gastric ulcer and 30 health persons were detected Results A highly significant correlation between the cancer cell number and relative CEA mRNA levels was found only when CD45 and Ber EP4 immuno magnetic beads were used successively ( P <0 01) The detection sensitive was one cancer cell per ml blood 42 22%, 53 33%, 51 11% and 64 44% of 45 patients with gastric cancer were detected the present of CEA mRNA by methodⅠ,Ⅱ, Ⅲ and Ⅳ, respectively (Ⅳ vs Ⅰ, P <0 05) None was detected the presence of CEA mRNA among 30 patients with gastric ulcer by method Ⅳ, and two cases were detected by other methods The CEA mRNA was not detected among 30 health persons by all methods ConclusionThe use of immunomagnetic bead method and quantitative RT PCR may raise the detective rate of CEA mRNA in peripheral blood of patients with gastric carcinoma

关 键 词：免疫磁珠 荧光定量逆转录聚合酶链反应 胃癌 外周血 癌细胞检测 

分 类 号：R735.2[医药卫生—肿瘤] R730.43[医药卫生—临床医学]