吉非替尼耐药人肺腺癌细胞的建立及耐药机制的研究  

Establishment of a gefitinib-resistant cell line of human lung adenocarcinoma and its drug resistant mechanism

作　　者：宁兰兰[1] 马为[1] 刘俊[1] 王汉平[2] 李裕军[1] 

机构地区：[1]广州医科大学附属广州市第一人民医院呼吸科,广东广州510180 [2]广州医科大学附属广州市第一人民医院中心实验室,广东广州510180

出　　处：《中国热带医学》2014年第9期1045-1049,共5页China Tropical Medicine

基　　金：广州市科技计划项目(No.2011J4100027)

摘　　要：目的建立吉非替尼耐药的人肺腺癌细胞株A549/GR,并初步探讨其生物学特性及其耐药机制。方法采用逐步增加吉非替尼剂量法诱导人肺腺癌细胞A549形成吉非替尼耐药的细胞株A549/GR;CCK-8法检测细胞的半数抑制浓度(IC50)及绘制细胞生长曲线,并计算耐药指数;显微镜下观察吉非替尼作用前后A549、A549/GR细胞形态学的改变;直接测序法检测EGFR基因在酪氨酸激酶区的基因突变;q RT-PCR法检测EGFR基因在m RNA水平表达的改变。结果 CCK-8法测得A549/GR细胞的耐药指数为6.00,显示成功建立了吉非替尼耐药的细胞模型,其倍增时间较亲代细胞缩短;形态学观察显示A549/GR细胞极性消失,有变长梭形的趋势;A549/GR细胞系EGFR基因酪氨酸激酶区未发现基因突变;q RT-PCR法显示A549/GR细胞EGFR基因在m RNA水平较亲代细胞轻度上调。结论成功建立吉非替尼耐药人肺腺癌A549/GR细胞模型,A549/GR细胞耐药性的产生与EGFR基因18-21外显子区域突变无关。Objective To establish a gefitinib-resistant A549/GR cell line of human lung adenocarcinoma, and explore its biological characteristics and drug resistance mechanisms. Methods C, efitinib-resistant A549/GR was induced by stepwise selection after exposure to increasing doses of gefitinib. CCK-8 assay was applied to determinate IC50, draw cell growth curve and calculate resistance index. The morphologic changes of A549/GR were observed trader ordinary optical microscope; mutation analysis of EGFR was performed via direct sequencing; expression levels in mRNA of EGFR were determined by Q-RT-PCR method. Results A gefitinib-resistant lung adenocarcinoma cell line A549/GR were successfully established with resistance index of 6.00, which showed a markedly higher proliferation rate than parental cells. Loss of polarity and elongated spindle-shaped changes were observed under ordinary optical microscope. No gene mutations of EGFR in the tyrosine kinase were detected; and Q-RT-PCR results indicated that expression levels of mRNA in EGFR gene of A549/GR cell line were a little up-regulated as compared with parental cells （P〈0.05）. Conclusions A gefifinib-resistant lung adenocarcinoma cell line A549/GR have been successfully established, and the development of drug resistance is not related with mutations in exons 18 - 21 of EGFR.

关 键 词：吉非替尼 耐药性 表皮生长因子受体 非小细胞肺癌 

分 类 号：R734.2[医药卫生—肿瘤]