肝癌凋亡细胞cDNA文库构建及其凋亡相关基因的初步筛选  

Construction of cDNA library and its preliminary screening of associated genes of human hepatoma apoptotic cells

作　　者：王文亮[1] 杨帆[1] 张晓东[1] 马运国[1] 王冰枫[1] 

机构地区：[1]第四军医大学病理学教研室,西安710032

出　　处：《临床与实验病理学杂志》1999年第6期477-479,共3页Chinese Journal of Clinical and Experimental Pathology

基　　金：国家自然科学基金

摘　　要：目的：通过肝癌凋亡细胞。DNA文库的构建，进而筛选人肝癌细胞凋亡相关基因。方法：用阿霉素诱导人肝癌HCC－9204细胞凋亡，磁珠法提取mRNA，然后将其反转录为cDNA，将合成的cDNA加上EcoR1接头，并与λgt11载体臂连接，构建为表达的cDNA文库．PCR法鉴定插入片断长度，T载体法克隆插入片断。消减杂交和点杂交法相结合筛选。DNA文库。结果：肝癌凋亡细胞CDNA文库容量为1×106pfu／μgDNA，平均片断长度为1～2kb．筛选CDNA文库得到一长度为1．5kb的DNA片段，并已测其部分序列。结论：构建的肝癌凋亡细胞CDNA文库对筛选和鉴定人肝癌细胞凋亡相关基因具有重要作用。筛选的基因可能是人肝癌细胞凋亡调控基因中的一个。Purpose To construct cDNA library of human hepatoma apoptotic cells, then screening the asso-ciated genes of the hepatoma apoptotic cells. Methods The hepatoma apoptotic cells in HCC-9204 hepatoma cellline were induced by adriamycin, and its mRNA were extracted by polyAT tract system 1000. After the mRNAsynthesized cDNA by DNA polymerase (reverse transcriptase), the synthetic cDNA was added on EcoRIadapters, and they were linked with phosphorylated λgtll vector arms, constructing expression vector of cDNAlibrary. The length of inserted DNA fragment was determined with PCR and the inserted DNA fragment wascloned with T vector method. Constructed cDNA library was screened by subtractive and spot blot hybridizationmethods. Results The account of cDNA library was 1 x 106 pm/ug DNA. The average length of DNA fragmentin cDNA library was 1 ～ 2 kb, and the length of DNA fragment in screening cDNA library was 1. 5 kb. Some ofDNA sequence was detected. Conclusion The constructed cDNA library of hepatoma apoptotic cells has an im－portant role for screening and identifying the genes related to apoptosis in hepatoma cells. The screened 1. 5 kbDNA fragment could be one of the regulation genes of hepatorna cell apoptosis.

关 键 词：肝肿瘤 细胞凋亡 CDNA文库 克隆 

分 类 号：R735.702[医药卫生—肿瘤]