摘要
癌基因C-myc激活和突变在胃癌形成过程中起着重要作用。通过毛细管电泳(CE)方法检测50例胃癌患者中C-myc基因突变,建立一种准确、快速诊断早期胃癌的方法。本实验采用PCR扩增胃癌及癌旁正常组织中C-myc基因第二外显子易发突变的部位基因序列,扩增样品分别经96℃变性和EcoRⅤ酶切处理,以PAGE-SSCP,CE-SSCP,CE-RFLP分别对其突变情况进行检测。优化的CE检测条件:筛分介质PEO浓度3.0%,pH 8.2,电压15 kV,温度15℃;荧光检测:λex=488 nm,λem=520 nm。检测结果:C-myc基因总突变率为20.0%(10/50)。测序分析结果显示C-myc基因第二外显子第53密码子存在点突变,碱基A变为碱基T(GAT→GTT),碱基的改变使氨基酸由亮氨酸替代为谷氨酰胺。本研究数据证实C-myc基因突变与胃癌的形成紧密相关,CE检测C-myc突变基因可作为胃癌早期诊断的简便可靠的方法。
Activation of C myc oncogene by mutation has been reported to play an important role in gastric cancer tumorigenesis. We determined C-myc mutation in 50 patients with gastric cancer by capillary electrophoresis （CE） to establish an exactly and volant clinical diagnostic method in early gastric cancer. In this experiment, genomic DNA was extracted from normal tissue and gastric cancer tissue and the sequence of C myc oncogene on exon 2 （possible with high mutation frequency） in the extracted genomic DNA was amplified by polymerase chain reaction （PCR）. Then amplified DNA samples were denatured at 96 oC and digested by EcoRV and detected by PAGE-single strand conformation polymorphism （SSCP）, CE-SSCP, CE-restriction fragment length polymorphism （RFLP）. The optimum CE detection conditions including 3.0% sieving medium poly（ethylene oxide） （PEO）, pH 8.2, separation voltage of 15 kV and temperature of 15 oC were adopted. The laser-induced fluorescence detector was set at λex=488 nm, λcm=520 nm. The results reveal that the mutation frequen- cy of C-myc gene is 20.0% （10/50）. Sequence analysis further identified a point mutation of A to T in exon 2 codon 53 （GAT→GTT）, causing an amino acid substitution of leucine to glutamine. This study may suggest a correlation of the mutation of C myc oncogene with gastric cancer progression. This work also demonstrates that CE can offer a convenient and reliable method to early diagnosis of gastric cancer.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2011年第11期1695-1700,共6页
Chinese Journal of Analytical Chemistry
基金
国家自然基金(No.20775089)
甘肃省自然基金(No.ZS031-A25-70-E)
中国博士后基金(No.2005037576)资助项目
关键词
胃癌
C-myc
突变
毛细管电泳
单链构象多态性
限制性片段长度多态性
Gastric cancer
C-myc
Mutation
Capillary electrophoresis
Single strand conformationpolymorphism
Restriction fragment length polymorphism
