应用抑制消减杂交(SSH)克隆肺腺癌多药耐药细胞特异表达基因

Suppression subtractive hybridization identified genes differentially expressed in a human lung adenocarcinoma multidrug resistance cell line

目的 克隆和筛选肺腺癌多药耐药细胞特异表达基因。方法 将肺腺癌多药耐药细胞 (SPC A 1/CDDP)作为实验组 ,肺腺癌细胞 (SPC A 1)作为对照组 ,应用抑制消减杂交技术 ,构建实验组特异表达cDNA消减文库 ;用斑点杂交初步筛选cDNA消减文库后 ,将获得的阳性克隆进行测序和同源性分析 (Genebank)。结果 建立了一个肺腺癌多药耐药细胞 (SPC A 1/CDDP)特异表达cDNA消减文库 ,斑点杂交初步筛选显示 2 3个克隆中有SPC A 1/CDDP特异表达cDNA片断 ,测序和同源性分析表明 2个cDNA片断为新序列 ,其余cDNA片断与已知基因有 96%～ 10 0 %的同源性。结论 2个新的cDNA序列可能为未知肺腺癌多药耐药相关基因序列 ;抑制消减杂交是克隆特异表达基因的有效方法。

Objective To clone and screen multidrug resistance related gene of human adenocarcinoma cell. Methods Suppression subtractive hybridization (SSH) was performed on human adenocarcinoma multidrug resistance cell line (SPC A 1/CDDP, as tester, which was established from cell line SPC A 1 under the inducement of cisplatin) and human adenocarcinoma cell line (SPC A 1, as driver). After the construction of subtracted cDNA library, dot blot was used to screen the subtracted cDNA library with forward and reverse subtracted cDNA probes. The differentially expressed cDNA fragments in SPC A 1/CDDP was sequenced and analyzed in Genebank with Blast search. Results A subtracted cDNA library of high quality was constructed. Twenty three differentially expressed cDNA fragments in SPC A 1/CDDP were identified. Two of them were novel cDNA sequences and the others shared 96%～100% homology with the known sequence. Conclusion The novel cDNA sequences might be the human lung adenocarcinoma multidrug resistance related genes. SSH is an effective approach to identify differentially expressed genes.