一条肺癌多药耐药相关基因的功能鉴定及染色体定位

Appraisal of multi-drug resistance function of a new gene cloned from human lung cancer cell line SPC-A-1/CDDP and identify its chromosome′s locus

目的 进一步分析从肺癌多药耐药细胞株 (SPC A 1/CDDP)克隆出的差异表达基因染色体的定位 ,并鉴定其耐药相关功能。方法 登录美国国家生物技术信息中心网 (www ncbi nlm nih gov) ,根据克隆的目的基因序列数据 ,查询人类基因库 ,分析确定该基因在人类染色体上的位置。以DNA重组技术构建该目的基因的反义表达载体 ,电穿孔法将其转染多药耐药细胞株SPC A 1/CDDP ,采用半定量RT PCR研究该基因表达受抑情况 ,并以MTT法分析转染细胞对几种化疗药物的敏感性改变。结果 电子信息学确定该目的基因定位在人类染色体的 19q13 3～19q13 4位点上。成功构建该基因的反义表达载体 ,转染反义表达载体的SPC A 1/CDDP细胞的目的基因mRNA含量明显减少 ,即基因表达受抑。MTT药敏测试发现转染了反义表达载体的SPC A 1/CDDP细胞对顺铂、阿霉素、5 氟尿嘧啶、长春新碱、足叶乙苷和丝裂霉素 6种化疗药物的耐药指数分别是 3 87、3 2 8、6 71、2 6 5、2 11、5 0 2 ;对前五种化疗药物的耐药指数与对照细胞相比分别下降了 2～3倍。表明该基因与肿瘤细胞的化疗敏感性密切相关。结论 该差异表达基因是一条肺癌耐药相关新基因 ,其在人类染色体的位置为 19q13 3～ 19q13 4。

objective In the former research work, a differential-expressed gene was cloned from multi-drug resistance lung cancer cell line (SPC-A-1/CDDP) with suppression substractive hybridization, and in this study we further analyze the site of this gene on the chromosome, and appraise its function related to multi-drug resistance in lung cancer cells. Methods The cDNA sequence data of the gene was input to computer and analyzed to ascertain its site on human chromosome by screening the gen bank on the www.ncbi.nlm.nih.gov. With DNA recombination technique, the gene was reversedly inserted to the vector pLXSN to get an antisense expression recombinant vector pLXSN-R, which was then transfected into SPC-A-1/CDDP cells with the aid of electroporation technique. And the semi-quantitative RT-PCR technique was used to quantify the mRNA content of gene in the transfected cells. Finally, the chemosensitivity of the transfected cells was tested with MTT method. Results The gene was located on the human chromosome 19q13.3-19q13.4 locus. The antisense gene recombinant vector was successfully constructed and transfected into SPC-A-1/CDDP cells as shown by its inhibitory activity on the mRNA expression of the gene. The drug-resistance indexes of transfected SPC-A-1/CDDP cells for cisplatin, doxorubicin, 5-fluorouracil, vincristin, hydroxycamptothecine and etoposide were obviously decreased. Conclusion The function of this gene is related to multi-drug resistance in human lung cancer cell, and its locus on the human chromosome is at 19q13.3-1913.4.