一氧化氮诱导食管癌细胞过表达基因的分离与鉴定  

Identification of Overexpressed Genes Induced by Nitric Oxid in Human Esophageal Carcinoma Cell Line

作　　者：李恩民[1] 陈玮莹[1] 陈爱云[1] 吴健谊[1] 许丽艳[2] 

机构地区：[1]汕头大学医学院生物化学与分子生物学教研室,广东汕头515031 [2]汕头大学医学院肿瘤病理研究室,广东汕头515031

出　　处：《癌症》2002年第6期610-614,共5页Chinese Journal of Cancer

基　　金：广东省自然科学基金博士启动基金资助项目(984068);广东省自然科学基金资助项目(990799);广东省医学科学技术研究基金资助项目(B1999105)

摘　　要：背景与目的:在一氧化氮(nitricoxid,NO)对食管癌细胞的诱导作用中,一些基因可能会出现过表达,但究竟哪些基因过表达至今尚不清楚。本研究拟对NO诱导食管癌细胞中的过表达基因进行分离鉴定。方法:以人食管癌细胞系EC109作为驱赶方(driver),以被NO诱导的EC109作为实验方(tester),应用抑制消减杂交(suppressionsubtractivehybridization,SSH)等技术手段研究NO诱导的食管癌细胞中基因的过表达情况。在获得6个SSH阳性克隆的基础上,通过反向mRNA斑点印迹阵列实验进行过表达基因的筛选,通过Northernblot对过表达基因进行验证,并对过表达基因的表达标志序列(expressedsequencetag,EST)实施序列测定,然后与GenBank进行BLAST同源性比较和序列突变分析。结果:鉴定出了两个线粒体DNA(mitochondrialDNA,mtDNA)编码的基因,即ND-4L和ND-4,并在ND-4L/ND-4基因片段(10736-11449)上发现了三处点突变(10872T→C,11001A→G,1134A→G)。结论:NO可以诱导线粒体DNA编码的ND-4L和ND-4等基因过表达,这将为深入揭示NO对肿瘤细胞的作用机制提供新的重要线索。Background &Objective:It is possible that some important genes were overexpressed in esophageal carcinoma cells induced by nitric oxid(NO), but it is not yet clear which the genes are. The objective of this study was to separate and identify the overexpressive genes induced by NO in EC109 esophageal carcinoma cell line. Methods:Overexpression of the genes induced by nitric oxid(NO) in EC109 esophageal carcinoma cell line was determined by using suppression subtractive hybridization(SSH). Sixty nine positive clones were obtained by EcoR I digesting, and overexpressive genes were screened by reverse mRNA dot blot and further confirmed by Northern blot analysis. The nucleotide of their expressed sequence tag(EST) was sequenced and analyzed by using NCBI database. Results:The two mitochondrial DNA coding genes, ND 4L and ND 4, were identified. The ND 4L and ND 4 genes were overexpressed distinctly in the EC109 cell induced by NO. The three point mutations were discovered in a fragment(10736 11449) of ND 4L and ND 4 genes. Conclusion:NO can induce overexpression of the ND 4L and ND 4 genes which is coded by mitochondrial DNA. These results provide a new idea for further exploring the mechanism of NO effecting on carcinoma cells.

关 键 词：一氧化氮 诱导 食管癌 线粒体DNA编码基因 基因过表达 抑制消减杂交 

分 类 号：R735.1[医药卫生—肿瘤]