中国人民解放军第四军医大学病理学教研室 陕西省西安市 710032

    曾建新，男，1968-9-17生，湖南邵东人，汉族. 博士后，主要从事 肝癌分子病理研究.

    项目负责人 王文亮，710032，陕西省西安市，第四军医大学病理学 教研室.wlwang@fmmu.edu.cn

    Telephone: 029-3284284 Fax: 029-3284284

    收稿日期 2001-08-09 接受日期 2001-09-04

    Construction of differentially expres sed cDNA library in human hepat o cellular carcinoma apoptotic cells with suppression subtractive hybri dization

    Jian-Xin Zeng, Wen-Liang Wang, Wen-Jing Luo and Zhi -Li Wang

    Department of Pathology, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China.

    Correspondence to: Wen-Liang Wang, Department of Pathology, Fo urth Military Medical University, 17 Changle West Road, Xi’an 710032, Shaanxi Province, China.

    Received 2001-08-09 Accepted 2001-09-04

    Abstract

    AIM To construct apoptotic subtractive cDNA library of human hepatocellular carcinoma cells using suppression subtractive hybridization (SSH ) technique and to clone apoptosis associated genes in hepatoma.

    METHODS Poly A⁺ RNAs were isolated from apoptotic HCC-9204 ce lls induced by arsenic trioxide and were reversely transcripted into double stra nd cDNAs (tester). After the cDNAs were digested into short cDNAs with blunt end s, they were divided into two groups and were ligated to the special adaptor1 an d adaptor 2R, respectively. The tester cDNAs were then hybridized with driver cD NA from normal HCC-9204 cells and the products were amplified twice by nested PC R technique. The PCR products were connected with pT-Adv plasmid vectors and wer e transformed into E. coli JM109 cells. The inserts of cDNAs were analyzed b y restrictive enzyme EcoR I and were identified by reverse Northern blot. ......