肺腺癌多药耐药细胞特异表达基因的克隆与鉴定

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）作为实验方，肺腺癌细胞（SPC－A－1）作为对照方，应用抑制消减杂交技术，构建实验方特异表达cDNA消减文库；用斑点杂交法初步筛选cDNA消减文库后，将获得的阳性克隆进行测序和同源性分析（Genbank)，对新的cDNA序列进行Northern blot杂交验证。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，斑点杂交法初步筛选显示23个个克隆中有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片段与已知基因有93％－100％的同源性，Northern blot杂交结果表明2个新的cDNA片断来自SPC－A－1／CDDP细胞。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；抑制消减杂交法是克隆特异表达基因的有效方法。

Suppression Subtractive Hybridization Identified Genes Differentially Expressed in a Multidrug Resistance Cell Line of Human Lung Adenocarcinoma

Objective: The aim of this study was to clone and screen multidrug resistance related gene of human adenocarcinoma cell. Methods: The suppression subtractive hybridization (SSH) was performed on human adenocarcinoma multidrug resistance cell line (SPC-A-1/CDDP, as tester) and human adenocarcinoma cell line (SPC-A-1, as driver). After the subtracted cDNA library being constructed, the dot blots 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 through Genbank with Blast search. The novel cDNA sequences were analyzed by Northern blots. Results: A high quality subtracted cDNA library 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 had 93％－100％ homology with the known genes respectively. Northern blots indicated the novel cDNA sequences only expressed in SPC-A-1/CDDP cell. Conclusion: The novel cDNA sequences might be multidrug resistance related genes in human lung adenocarcinoma. SSH is a powerful technique to identify differentially expressed genes.