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[目的]建立稳定表达ABCG2蛋白的A549-ABCG2细胞系并观察其生物学特性变化,为选择敏感化疗药物及制定临床治疗方案提供研究平台。[方法]非小细胞肺癌(NSCLC)组织标本中提取总RNA,RT-PCR获得ABCG2全序列基因后插入pcDNA3.1质粒,转染A549细胞系,挑取克隆稳定筛选并命名为A549-ABCG2细胞。通过流式细胞术、MTT法及ATP激酶活性检测,分析目的基因与耐药之间的相关性。[结果]RT-PCR及Western blot证明A549-ABCG2细胞目的基因表达稳定。流式细胞仪检测A549凋亡细胞明显多于A549-ABCG2,且使用多柔比星后G1峰前出现明显凋亡峰,使用羟基喜树碱后A549-ABCG2 S期细胞减少,G1期增加;A549 S期增加细胞,G1期减少,A549-ABCG2细胞药物敏感性较A549细胞明显降低,而ATP激酶活性明显升高。药物剂量和作用时间与ATP激酶活性之间具有正相关性。[结论]成功地建立的ABCG2阳性表达细胞系,为细胞研究耐药现象提供了良好的实验平台。
[Objective] To establish A549-ABCG2 cell line stably expressing ABCG2 protein and to observe the changes of its biological characteristics, and to provide a research platform for selecting sensitive chemotherapeutics and developing clinical treatment plan. [Methods] Total RNA was extracted from tissue samples of non-small cell lung cancer (NSCLC). The full length ABCG2 gene was amplified by RT-PCR and inserted into pcDNA3.1 plasmid. The plasmid was transfected into A549 cell line and cloned into A549-ABCG2 cells . The correlation between the target gene and drug resistance was analyzed by flow cytometry, MTT assay and ATP kinase activity assay. [Result] RT-PCR and Western blot showed that the gene expression of A549-ABCG2 cells was stable. The apoptotic cells in A549 cells were significantly higher than that in A549-ABCG2 cells by flow cytometry, and obvious apoptosis peak appeared in front of G1 peak after using doxorubicin. The cells in A549-ABCG2 S phase decreased after treated with hydroxycamptothecin and increased in G1 phase. A549 S phase increased cells, G1 phase decreased, A549-ABCG2 cell drug sensitivity was significantly lower than A549 cells, and ATP kinase activity was significantly increased. There is a positive correlation between drug dosage and duration of action and ATP kinase activity. [Conclusion] The successfully established ABCG2 positive cell lines provide a good experimental platform for cell research on drug resistance.