CAJ | 학술논문

背景与目的：EGFR-TKI治疗NSCLC失败后，化疗仍可取得一定的治疗效果，是可选择的治疗方案之一。核苷酸还原酶(ribonucleotide reductase，RR)、胸苷酸合成酶(thymidylate synthase，TS)、核苷酸切除修复交叉互补基因1(excision repair cross complementstion group 1，ERCC1)、3型β微管蛋白(β-tubulin-Ⅲ，TUBB3)分别与吉西他滨、培美曲塞、铂类药物及微管类药物的化疗药物敏感性存在相关性，可以通过这些分子标志物的表达水平来预测化疗药物的敏感性。RRMI、TS、ERCC1和TUBB3高表达患者化疗药物的敏感性降低，低表达患者化疗药物敏感性增高。本研究拟探讨EGFR-T790M突变所致吉非替尼耐药肺腺癌细胞对顺铂、吉西他滨、长春瑞滨、紫杉醇、多西他赛和培美曲塞化疗药物敏感性的变化。方法：通过MTT法检测PC9及PC9/GR细胞对顺铂、吉西他滨、长春瑞滨、紫杉醇、多西他赛和培美曲塞的IC50，探讨其对上述药物的化疗敏感性。采用液相芯片法，检测PC9及PC9/GR细胞ERCC1 mRNA、RRM1 mRNA、TUBB3 mRNA和TSmRNA的表达水平。通过蛋白质印迹法(Western blot)检测PC9及PC9/GR细胞ERCC1、RRM1、TUBB3和TS蛋白的表达水平。结果：与PC9细胞株相比较，PC9/GR细胞株对吉非替尼、顺铂、吉西他滨和培美曲塞的IC50明显增高(P＜0.05)；对长春瑞滨、紫杉醇和多西他赛的IC50明显降低(P＜0.05)。PC9/GR细胞对吉非替尼、顺铂、吉西他滨、长春瑞滨、紫杉醇、多西他赛和培美曲塞的耐药指数分别为70、1.56、1.61、0.34、0.39、0.14和1.71。与PC9细胞株mRNA的表达量相比较，PC9/GR细胞株ERCC1 mRNA、RRM1 mRNA和TS mRNA的表达量明显增高(P＜0.05)，TUBB3的mRNA的表达量明显降低，差异均有统计学意义(P＜0.05)。与PC9细胞株蛋白的表达量相比较，PC9/GR细胞株ERCC1、RRM1和TS的蛋白表达量明显增高，TUBB3蛋白的表达量明显降低，差异均有统计学意义(P＜0.05)。结论：肺腺癌细胞株发生EGFR-T790M突变后对化疗药物敏感性发生变化，对顺铂、吉西他滨和培美曲塞的敏感性降低，对长春瑞滨、紫杉醇和多西他赛的敏感性增高；其化疗药物敏感性发生变化的原因可能与肺腺癌细胞株发生EGFR-T790M突变后ERCC1mRNA、RRM1 mRNA、TSmRNA及其蛋白表达量发生变化相关。揹景與目的：EGFR-TKI治療NSCLC失敗後，化療仍可取得一定的治療效果，是可選擇的治療方案之一。覈苷痠還原酶(ribonucleotide reductase，RR)、胸苷痠閤成酶(thymidylate synthase，TS)、覈苷痠切除脩複交扠互補基因1(excision repair cross complementstion group 1，ERCC1)、3型β微管蛋白(β-tubulin-Ⅲ，TUBB3)分彆與吉西他濱、培美麯塞、鉑類藥物及微管類藥物的化療藥物敏感性存在相關性，可以通過這些分子標誌物的錶達水平來預測化療藥物的敏感性。RRMI、TS、ERCC1和TUBB3高錶達患者化療藥物的敏感性降低，低錶達患者化療藥物敏感性增高。本研究擬探討EGFR-T790M突變所緻吉非替尼耐藥肺腺癌細胞對順鉑、吉西他濱、長春瑞濱、紫杉醇、多西他賽和培美麯塞化療藥物敏感性的變化。方法：通過MTT法檢測PC9及PC9/GR細胞對順鉑、吉西他濱、長春瑞濱、紫杉醇、多西他賽和培美麯塞的IC50，探討其對上述藥物的化療敏感性。採用液相芯片法，檢測PC9及PC9/GR細胞ERCC1 mRNA、RRM1 mRNA、TUBB3 mRNA和TSmRNA的錶達水平。通過蛋白質印跡法(Western blot)檢測PC9及PC9/GR細胞ERCC1、RRM1、TUBB3和TS蛋白的錶達水平。結果：與PC9細胞株相比較，PC9/GR細胞株對吉非替尼、順鉑、吉西他濱和培美麯塞的IC50明顯增高(P＜0.05)；對長春瑞濱、紫杉醇和多西他賽的IC50明顯降低(P＜0.05)。PC9/GR細胞對吉非替尼、順鉑、吉西他濱、長春瑞濱、紫杉醇、多西他賽和培美麯塞的耐藥指數分彆為70、1.56、1.61、0.34、0.39、0.14和1.71。與PC9細胞株mRNA的錶達量相比較，PC9/GR細胞株ERCC1 mRNA、RRM1 mRNA和TS mRNA的錶達量明顯增高(P＜0.05)，TUBB3的mRNA的錶達量明顯降低，差異均有統計學意義(P＜0.05)。與PC9細胞株蛋白的錶達量相比較，PC9/GR細胞株ERCC1、RRM1和TS的蛋白錶達量明顯增高，TUBB3蛋白的錶達量明顯降低，差異均有統計學意義(P＜0.05)。結論：肺腺癌細胞株髮生EGFR-T790M突變後對化療藥物敏感性髮生變化，對順鉑、吉西他濱和培美麯塞的敏感性降低，對長春瑞濱、紫杉醇和多西他賽的敏感性增高；其化療藥物敏感性髮生變化的原因可能與肺腺癌細胞株髮生EGFR-T790M突變後ERCC1mRNA、RRM1 mRNA、TSmRNA及其蛋白錶達量髮生變化相關。
배경여목적：EGFR-TKI치료NSCLC실패후，화료잉가취득일정적치료효과，시가선택적치료방안지일。핵감산환원매(ribonucleotide reductase，RR)、흉감산합성매(thymidylate synthase，TS)、핵감산절제수복교차호보기인1(excision repair cross complementstion group 1，ERCC1)、3형β미관단백(β-tubulin-Ⅲ，TUBB3)분별여길서타빈、배미곡새、박류약물급미관류약물적화료약물민감성존재상관성，가이통과저사분자표지물적표체수평래예측화료약물적민감성。RRMI、TS、ERCC1화TUBB3고표체환자화료약물적민감성강저，저표체환자화료약물민감성증고。본연구의탐토EGFR-T790M돌변소치길비체니내약폐선암세포대순박、길서타빈、장춘서빈、자삼순、다서타새화배미곡새화료약물민감성적변화。방법：통과MTT법검측PC9급PC9/GR세포대순박、길서타빈、장춘서빈、자삼순、다서타새화배미곡새적IC50，탐토기대상술약물적화료민감성。채용액상심편법，검측PC9급PC9/GR세포ERCC1 mRNA、RRM1 mRNA、TUBB3 mRNA화TSmRNA적표체수평。통과단백질인적법(Western blot)검측PC9급PC9/GR세포ERCC1、RRM1、TUBB3화TS단백적표체수평。결과：여PC9세포주상비교，PC9/GR세포주대길비체니、순박、길서타빈화배미곡새적IC50명현증고(P＜0.05)；대장춘서빈、자삼순화다서타새적IC50명현강저(P＜0.05)。PC9/GR세포대길비체니、순박、길서타빈、장춘서빈、자삼순、다서타새화배미곡새적내약지수분별위70、1.56、1.61、0.34、0.39、0.14화1.71。여PC9세포주mRNA적표체량상비교，PC9/GR세포주ERCC1 mRNA、RRM1 mRNA화TS mRNA적표체량명현증고(P＜0.05)，TUBB3적mRNA적표체량명현강저，차이균유통계학의의(P＜0.05)。여PC9세포주단백적표체량상비교，PC9/GR세포주ERCC1、RRM1화TS적단백표체량명현증고，TUBB3단백적표체량명현강저，차이균유통계학의의(P＜0.05)。결론：폐선암세포주발생EGFR-T790M돌변후대화료약물민감성발생변화，대순박、길서타빈화배미곡새적민감성강저，대장춘서빈、자삼순화다서타새적민감성증고；기화료약물민감성발생변화적원인가능여폐선암세포주발생EGFR-T790M돌변후ERCC1mRNA、RRM1 mRNA、TSmRNA급기단백표체량발생변화상관。
Background and purpose:Chemotherapy is an alternative treatment option, which could still get a therapeutic effect, when the EGFR-TKI treatment of non-small cell lung cancer failed. Studies have shown that RR, TYMS, ERCC1 and TUBB3 have respectively relationship with chemosensitivity of gemcitabine, pemetrexed, platinum-based drugs and microtubule-based chemotherapy drugs.The expression levels of these molecular markers can predict the sensitivity of these chemotherapy drugs. The patients with RRMI, TS, ERCC1 and TUBB3 higher expression have reduced chemosensitivity, and lower expression have increased sensitivity. The purpose of this study was to explore the sensitivity of tumor cell lines with acquired resistance to geiftinib caused byEGFR-T790M mutation to cisplatin, gemcitabine, pemetrexed, vinorelbine, paclitaxel and docetaxel.Methods:MTT assay was used to detect the IC50 values of cisplatin, gemcitabine, vinorelbine, paclitaxel and docetaxel, pemetrexed to PC9 and PC9/GR cells, and to explore the chemosensitivity of lung adenocarcinoma cells to these chemotherapy drugs; Luminex method was used respectively to detect the expression levels of ERCC1 mRNA, TUBB3 mRNA, TS mRNA, and RRM1 mRNA in PC9 and PC9/GR cells. Western blot was used to detect the protein expression levels of ERCC1, TUBB3, TS and RRM1 in PC9 and PC9/GR cells.Results: The IC50 values of cisplatin, gemcitabine and pemetrexed to PC9/GR cells were signiifcantly higher than those to PC9 cells (P<0.05), while the IC50 values of vinorelbine, paclitaxe and docetaxel to PC9/GR cells were signiifcantly decreased (P<0.05). Luminex method showed the expressions of ERCC1 mRNA, TS mRNA and RRM1 mRNA in PC9/GR cells were signiifcantly increased than those in PC9 cells (P<0.05), while the expression of TUBB3 mRNA was signiifcantly decreased (P<0.05). Western blot method showed the expressions of TUBB3, TS and RRM1 protein in PC9/GR cells were signiifcantly increased than those in PC9 cells (P<0.05), while TUBB3 protein expression in PC9/GR cells was signiifcantly decreased (P<0.05). Western blot method analysis result showed that the expressions of TUBB3, TS and RRM1 protein in PC9/GR cells were significantly increased than those in PC9 cells (P<0.05), while TUBB3 protein expression in PC9/GR cells was signiifcantly decreased (P<0.05). Conclusion:The chemosensitivity of lung adenocarcinoma with EGFR-T790M mutation is changed. It has decreased sensitivity to cisplatin, gemcitabine, pemetrexed and increased sensitivity to vinorelbine, paclitaxel and docetaxel. The reason of the change of chemosensitivity of geiftinib-resistant lung adenocarcinoma cell maybe related to the changes of ERCC1 mRNA, RRM1 mRNA and TS mRNA and their protein expressions.