【作者】 赵大卫；

【导师】 张进华；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:通过体外实验研究表皮生长因子受体抑制剂吉非替尼诱导肺癌细胞A549、H460凋亡的作用,并检测其下游信号通路,了解靶向药物的抗肿瘤活性,探讨其中存在机制。方法:1本实验选用两个肺癌细胞株A549、H460为研究对象,采用四甲基偶氮唑蓝(MTT)法测定两株细胞对吉非替尼的敏感性。取对数生长期上述细胞接种96孔板,加入相应浓度吉非替尼,72小时后加入MTT,通过多通道酶标仪检测各孔的吸光值A(波长=490nm,以波长520nm为参考)。计算细胞IC50,为下一步选择合适药物浓度诱导上述细胞凋亡提供参考。2取20μM吉非替尼作用于A549、H460两种肺癌细胞,加药24小时后,甲醇固定细胞,DAPI染色后,置倒置荧光显微镜下观察,观察吉非替尼的诱导肺癌细胞凋亡作用。3取对数生长期细胞,铺25mm培养瓶,待细胞完全贴壁后加入不同浓度0、0.1、1.0、8.0μM吉非替尼,药物处理细胞2h后,再加入终浓度30ng/mlEGF,刺激细胞表皮生长因子受体(EGFR)及相应下游信号的磷酸化,15min后消化细胞,用含磷酸酶抑制剂的冷PBS洗涤3次。RIPA试剂盒抽提细胞总蛋白。用Bradford蛋白分析试剂盒测量所提取蛋白的浓度。应用Western-Blotting方法检测细胞加药后EGFR及磷酸化EGFR(p-EGFR)、Akt及磷酸化Akt(p-Akt)蛋白水平变化,以GAPDH为内参照。结果:1吉非替尼浓度为3、6、8、10、12.5、25、50μM时,A549细胞的增殖抑制率分别是8.26%、40.76%、45.87%、45.72%、55.69%、86.79%、95.67%;H460细胞的增殖抑制率分别是0.72%、3.44%、17.4%、12.52%、22.71%、68.91%、94.05%。该组数据表明靶向EGFR的酪氨酸激酶抑制剂吉非替尼对A549、H460细胞的增殖均有明显的抑制作用,并且这种作用为剂量依赖性,随药物浓度的升高抑制率逐渐升高。吉非替尼抑制A549、H460细胞增殖的IC50分别为10.81μM,18.44μM,与相关文献报道的结果相似。2取20μM吉非替尼作用于A549、H460两种肺癌细胞,24小时后经DAPI染色,在倒置荧光显微镜下观察,光镜下可见凋亡细胞,细胞核内可见浓染致密的颗粒荧光,典型凋亡细胞核出现新月型改变,核固缩或片段化。本实验在体外模拟了吉非替尼的作用,观察了靶向药物吉非替尼诱导肺癌细胞凋亡的作用。3 Western-Blotting方法检测EGFR、Akt蛋白磷酸化的结果显示:EGFR在肺癌A549和H460细胞系均有表达,在A549细胞系表达相对较高。在吉非替尼浓度为1.0μM时,两细胞系中EGFR蛋白的磷酸化开始受到抑制,并随着药物浓度的增加受抑制程度逐渐明显,当吉非替尼浓度为8.0μM时,两个细胞系EGFR蛋白的磷酸化几乎完全被抑制。两细胞系中磷酸化的Akt蛋白在吉非替尼浓度较低时即能够受到一定程度的抑制,并随着浓度的增加受抑制程度逐渐明显。结论:本研究选取肺癌细胞株A549、H460为研究对象,采用MMT法观察两个细胞株对吉非替尼的药物敏感性,结果表明:吉非替尼对A549、H460细胞的增殖均有明显的抑制作用,并且这种作用呈剂量依赖性。吉非替尼抑制A549、H460细胞增殖的IC50分别为10.81μM,18.44μM。取接近IC50值20μM吉非替尼诱导A549、H460细胞凋亡,经DAPI染色后观察到细胞核内可见浓染致密的颗粒荧光,典型凋亡细胞核出现新月型改变,固缩或片段化的核,显示了其诱导细胞凋亡的作用。MTT实验和凋亡实验在细胞水平上体外观察了吉非替尼抑制细胞增殖和诱导细胞凋亡的作用,观察了靶向药物吉非替尼的抗肿瘤活性。运用Wester-blotting方法在蛋白水平上通过EGFR和Akt磷酸化抑制实验发现吉非替尼能够抑制Akt的磷酸化,而Akt磷酸化与细胞抗凋亡能力有关,因此认为EGFR抑制剂吉非替尼诱导肺癌A549、H460细胞凋亡,其分子机制可能是通过抑制Akt蛋白的磷酸化来实现的。本研究有助于我们更加清楚的了解靶向治疗药物吉非替尼对肺癌细胞的杀伤作用及可能机制,为靶向治疗病人的筛选和新的靶向治疗药物的开发提供理论依据。更多还原

【Abstract】 Objective: To study aim to investigate the anti-tumor effect of small molecular tyrosine kinase inhibitor that targeted epidermal growth factor receptor gefitinib in lung cancer cell lines A549and H460 in vitro, and to explore its possible mechanisms in signal transduction pathway.Methods:1 Two cell lines A549 and H460 were selected as the study object, and MTT method was used to detect the sensitivity of the two cell lines to gefitinib. While the two cell lines, namely A549 and H460 in their well status, following studies were carried out; respectively, After the cells had adhered diverse concentrations of gefitinib were added to the medium of cells, and incubated for 72 hours. After the incubation MTT was added to each well and the absorbance was detected (at the wave-length of 490nm, and 520nm as reference). So the concentration of inhibition rate of 50 percent was calculated, namely, the cell line’s IC50, and to provide reference for selecting an appropriate concentration to induce apoptosis of the two cell lines.2 The concentration of 20μM gefitinib was selected to treat A549 and H460 cells, disposed cells to gefitinib for 24 hours, the cells were dyed with DAPI, in order to observe the apoptosis induced by gefitinib under the fluorescence microscope.3 Selecting the above cells with well status, respectively seeded the cells in 25mm culture flask. After the cells totally adhereed to the well, exposed the cells to gefitinib at the following concentrations 0μM, 0.1μM, 1.0μM, 8.0μM and 2 hours later, 30ng/ml EGF was added to the medium, in order to activate the phosphorylation of EGFR and downstream signals, then incubated for 15 minutes, then cells were digested. In the cold PBS containing phosphatase inhibitor. The whole protein were distract with RIPA kit. Measure protein concentration with Bradford protein-analysis kit. Western-Blotting was carried out with the protein obtained above to determine the protein level of EGFR, p-EGFR, Akt and p-Akt. GAPDH was used as a loading control.Results:1 When A549 cells were treated with gefitinib at the concentration of 3μM, 6μM, 8μM, 10μM, 12.5μM, 25μM and 50μM, the inhibition rate of A549 were 8.26%, 40.76%, 45.87%, 45.72%, 55.69%, 86.79%, 95.67% respectively, and the rates of H460 were 0.72%, 3.44%, 17.4%, 12.52%, 22.71%, 68.91%, 94.05%. The proliferation of lung cancer cell line A549 and H460 was inhibited by gefitinib, and the effect was dose-dependent, the inhibition rate increased with the augmentation of gefitinib concentration. The IC50 of gefitinib on lung cancer cell line A549 and H460 was 10.81μM and 18.44μM respectively. It was similar to the results reported.2 Disposing the two lung cancer lines to 20μM gefitinib to induse apoptosis, and after the cells were dyed with DAPI. Grain fluorescence with dense-dyed appeared in the nucleolus, and concavo-convex’s, fastened shrink or segmented nucleolus appeared in the emblematical apoptosis nucleolus. It simulated the effect of gefitinib in vitro, and observed gefitinib as a targeted therapy could induce lung cancer cells apoptosis.3 Western-blotting was used to determine EGFR and Akt phosphorylation inhibited by gefitinib: EGFR expressed in both of the two cell lines, and higher expression level in A549 cell line. EGFR phosphorylation was to some degree inhibited in the two lung cancer cell lines with 0.1μM gefitinib, and more obvious inhibition was observed with the increase of gefitinib concentration, EGFR protein phosphorylated was almost totally inhibited with mediun containing 8.0μM gefitinib in the two lung cancer cell lines. Akt phosphorylation was inhibited in both lung cancer cell lines can be observed, and the degree of inhibition was more obvious with the increase dose of gefitinib.Conclusion:1 The study selected two cell lines: A549 and H460, and MTT method was used to determine the sensitivity of the lung cancer cell line to gefitinib. The proliferation of lung cancer cell line A549 and H460 were inhibited by gefitinib, and the effect was dose-dependent. The concentration of IC50 for the effect of gefitinib on lung cancer cell line A549 and H460 was 10.81μM and 18.44μM. A concentration adjacent of gefitinib (20μM) to IC50 was selected to dispose the two lung cancer lines, and observed after dyed with DAPI, grain fluorescence with dense-dyed appeared in the nucleolus, concavo-convex’s, fastened shrink or segmented nucleolus appeared in the emblematical apoptosis nucleolus. It shows the cell apoptosis. This observed the effect of gefitinib by cell experiment and apoptosis experiment in cellular level. And it showed that gefitinib is a useful drug in inducing apotosis in lung cancer cell lines.2 The experiment of EGFR and Akt phosphorylation by Western-blotting from detected Akt phosphorylation can be controlled by gefitinib, and the Akt phosphorylation is related with anti-apoptosis, so we believe the molecular mechanism on the apoptosis of lung cancer cell caused by gefitinib may be at the bottom of the Akt phosphorylation controlled. This study help us to realize the effect of gefitinib as an anti-tumor drug and the possible molecular mechanism as a targeted therapy, and provided theoretic base for the patients selection to determine whom will receive the targeted therapy and meaningful for in new targeted therapy development.更多还原