摘要
目的:建立多重RT-PCR同步检测技术,联合检测黑色素瘤抗原MAGEA3基因、人类斯钙素hsTC1基因、上皮细胞角质蛋白CK20、CK19基因及癌胚抗原CEA基因mRNA在食管癌患者外周血中的表达。方法:在分析MAGEA3、hSTC1、CK20、CK19、CEA和β-actin六种基因mRNA全长序列资料的基础上,自行设计并筛选出6对引物及6条寡核苷酸探针,利用多重RT-PCR技术并结合基因芯片对样本进行检测。结果:全部样品均可检出β-actin,55例食管癌患者外周静脉血中MAGEA3、hSTC1、CK20、CK19和CEA的阳性表达率分别为25.45%、47.27%、49.09%、34.55%和52.73%;多重RT-PCR技术同时检测MAGEA3、hSTC1、CK20、CK19和CEA基因,如果至少表达其中一种即可作为阳性表达者,其阳性表达率可达72.73%。而10例食管癌组织中均为阳性,28例正常健康捐献者的外周血中有2例CEA表达阳性,27例非肿瘤病人的外周血中有3例CK20阳性,3例CK19阳性,3例CEA阳性。测序结果证实RT-PCR产物确为MAGEA3、hSTC1、CK20、CK19和CEA基因。结论:联合检测MAGEA3、hSTC1、CK20、CK19和CEA可以增加外周血循环癌细胞检测的敏感性,本研究建立的多重RT-PCR技术可以做为一种快速、灵敏的检测手段。
Objective： We establish a multiplex reverse transcription-polymerase chain reaction method to evaluate the co-expression of MAGEA3, hSTC1, CK20, CK19 and CEA genes mRNA in peripheral blood of patients with esophageal carcinoma. Methods： 12 oligonudeotide primers were screened to use, which were type-specific for MAGEA3, hSTC1, CK20, CK19, CEA and β-actin mRNA genotype of homo sapiens. A multiplex RT-PCR based assay was developed and the amplified product was detected by gene chip Technique. Housekeeping genes β-actin mRNA was analyzed to check the stability of mRNA in sam- pies. Results： All samples showed detectable quantities of β-actin mRNA and 10 tumor tissues all showed the five target gene mRNA. MAGEA3, hSTC1, CK20, CK19 and CEA mRNA were detected in 25.45%, 47.27%, 49.09%, 34.55% and 52.73% blood of 55 cancer patients, respectively, while CEA mRNA were detected in 3 blood of 28 healthy donors and CK20, CK19, CEA mRNA were detected respectively in 3 blood of 27 non-cancer. When all five target genes were examined, the positive rate of expression of one gene was 72.73%. Conclusions： It indicates that the combined detecaon of MAGEA3, hSTC1, CK20, CK19 and CEA mRNA could enhance the ability of detection of tumor cells in peripheral blood of esophageal carcinoma. And the multiplex RTPCR may be a rapid and sensitive method for sdecting treatment, estimating clinical prognosis and surveilleing curative effect.
出处
《现代生物医学进展》
CAS
2007年第7期1007-1009,1001,共4页
Progress in Modern Biomedicine
基金
江苏省卫生厅重点资助项目(H200116)
江苏省教育厅科研基金(HJ04-010)
南京市科技发展计划项目(2005019-1)
