肿瘤敏感相关基因克隆cDNA消减文库的构建

Cloning of Tumor Sensitive Related Genes:Construction of a cDNA Library of Suppression Subtractive Hybtridization

目的 :烷基磷脂类化合物 (十六烷基磷酸胆碱 ,HePC)诱导人上皮性肿瘤细胞系产生交叉耐药。方法 :用抑制消减杂交技术 ,构建KB和KBrcDNA消减文库。KB为testercDNA与过量的KBrcDNA消减杂交 ,非特异性cDNA片段被有效消减 ,特异表达的文库得到富集。结果 :扩增后得到 12 7个克隆 ,挑取质粒 ,酶切分析均得到 4 0 0～ 80 0bp插入片段。在获得可分析的 78个克隆中 ,2 7%没有同源基因片段或同源性很低 ,暂定为“新基因” ;14 %具有染色体明确定位 ,应该认为是化疗敏感肿瘤KB细胞所特有的cDNA片段 ;19%在人类EST文库MGC和CGAP中出现 ,可能是肿瘤细胞所特有基因 ;发现与耐药性相关的已知功能基因高达 4 0 %。结论 :探索伴随肿瘤细胞耐药发生基因丢失 ,以开拓抗性逆转措施的新途径。

Abstruct:Objective To study the new cross muliresist mechanism for HePC Methods The human tumor epitheliacress resistance cell strain KBr that induced by HePC from KB was established and suppression subtractive hybtridization(SSH) was used to screen out differently expressed genes in KB cells in KB cell After enzyme restriction shows that all plasmids in the clones contain 400-800 bp inserts KBr Subtrctive contain library with high subtractive efficency was set up successfully Results The amplified contains 127 positive clones About 78 cDNA clone were obtained form this library by sequencing 27% genes were not find homologus fragment or lower than homologous fragment,may be new genes 14% genes have chromosome locations We think that exclusively chemistry sensitive tumor cDNA fragment KB cell 19% genes MGC and CGAP appear to human EST library May be tumor cell have characterize by genes We find more than 40% genes are known multiresistance genes Conclusions The propose is to explore the of tumor multiresist genes and find new way to reverse multiresistance