Abstract
背景与目的在肺腺癌靶向治疗中的吉非替尼继发性耐药是临床遇到的重要问题。本研究旨在探讨吉非替尼是否可诱导肺腺癌PC9细胞发生继发性耐药，以及表皮生长因子受体（epidermal growth factor receptor, EGFR）启动子甲基化在耐药过程中的作用，拟为肺腺癌耐药提供新的治疗靶点。方法体外培养肺腺癌吉非替尼敏感细胞株PC9，应用不同浓度吉非替尼进行干预。MTT法检测PC9细胞株对吉非替尼的敏感性。亚硫酸氢盐处理后测序法（bisulfite sequencing polymerase chain reaction, BSP）检测肺腺癌PC9细胞株EGFR启动子甲基化水平。使用5-氮杂-2’-脱氧胞苷（5-Aza-dc）对吉非替尼耐药细胞株PC9/GR细胞株进行干预。MTT法检测耐药株PC9/GR对吉非替尼敏感性的变化。结果经过从0.01 μmol/L逐渐提升吉非替尼诱导浓度至3 μmol/L之后，MTT显示耐药细胞株PC9/GR细胞株半抑制浓度（half maximal inhibitory concentration, IC50）[（3.95±0.23）μmol/L]较之前敏感株PC9[(0.01±0.002) μmol/L]明显升高（P < 0.05），BSP显示发生异常甲基化的位点结果比较：PC9/GR甲基化水平明显升高（74% vs 59%, P < 0.05）。RT-PCR显示PC9/GR细胞株较PC9细胞株EGFR mRNA表达量升高（P < 0.05）。使用5-Aza-dc处理PC9/GR细胞后，PC9/GR细胞株IC50较对照组降低[（2.55±0.14）μmol/L vs（3.87±0.034）μmol/L, P < 0.05）]。结论吉非替尼体外浓度递增诱导法可诱导PC9细胞株产生吉非替尼继发性耐药，成功构建PC9/GR耐药细胞株。PC9细胞EGFR启动子甲基化异常可能参与了吉非替尼继发性耐药机制。
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