二烯丙基二硫诱导人胃癌细胞差异表达基因cDNA文库的建立

目的　建立二烯丙基二硫 (DADS)诱导人胃癌细胞MGC80 3细胞差异表达基因的cDNA文库。方法　采用抑制消减杂交技术 ,筛选DADS处理人胃癌细胞MGC80 3细胞后差异表达的相关基因。经过抑制消减杂交和巢式聚合酶链反应扩增 ,获得富集的差异表达的cDNA文库 ,PCR检测插入片断的阳性克隆。结果　建立DADS诱导人胃癌MGC80 3细胞差异表达基因的cDNA文库 ,并从消减文库中随机挑取的 10 0个白色克隆中筛选个阳性克隆 ,发现插入片断为 10 0～ 6 0 0bp。结论　应用抑制消减杂交方法可有效筛选DADS处理人胃癌细胞MGC80 3细胞后差异表达的相关基因 ,其消减cDNA文库的建立 ,为进一步寻找胃癌发生相关新基因 ,深入揭示DADS对肿瘤细胞的作用机制提供了新的重要线索。

Construction of a subtracted cDNA library of differentially expressed genes in human gastric carcinoma induced by diallyl disulfide

Aim To construct a subtracted cDNA library o f differentially expressed genes in human gastric carcinoma induced by diallyl dis ulfide(DADS). Methods Differentially expressed cDNA species induc ed by DADS in MGC 803 human gastric carcinoma cell line was determined by using suppression subtractive hybridization (SSH). Then these cDNA species were direct ly inserted into T/A cloning vector to set up the subtractive library. Amplification of th e library was carried out with transformation of E.coli by high voltage electrop erforation. One hundred positive bacteria clones were randomly picked and identi fied using PCR method. Results The amplified library contained more than 1,000 positive bacteria clones. Random analysis of 100 clones with PCR m ethod showed that all clones contained 100～600 bp inserts.Conclusions A subtracted cDNA library of differentially expressed genes in MGC 803 hum an gastric carcinoma cell line induced by DADS is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundati on for screening and cloning new and specific tumor correlative genes of human g astric carcinoma, and provides a new idea for further exploring the mechanism of DADS effects on carcinoma cells.