塞来昔布联合吉非替尼在非小细胞肺癌中的实验研究

发布时间：2018-04-08 12:07

本文选题：环氧化酶-2　切入点：吉非替尼　出处：《石河子大学》2015年硕士论文

【摘要】：目的:本研究通过观察塞来昔布联合吉非替尼分别对表皮生长因子受体(EGFR)突变细胞株(HCC827)和EGFR野生型细胞株(A549)的增殖和凋亡的影响,探讨塞来昔布联合吉非替尼是否对非小细胞肺癌细胞系具有协同杀伤作用及与EGFR基因状态之间的相关性;并通过Western blot检测COX-2和p-EGFR相关蛋白的表达,对其机制进行初步探讨;从而为临床上塞来昔布与吉非替尼的联合使用提供依据,为晚期非小细胞肺癌患者的个体化治疗提供更完善的方案。方法:塞来昔布、吉非替尼单独及联合干预两种细胞系48h后,四甲基偶氮唑盐(MTT)法测定细胞生长抑制率;Alinexin V/PI法检测细胞凋亡率;Western blot法检测p-EGFR和COX-2蛋白表达。实验数据以x±S表示,用SPSS 17.0软件进行方差分析,检验水准α=0.05。结果:1.四甲基偶氮唑盐(MTT法)检测药物分别对两种细胞生长的抑制作用:不同浓度的塞来昔布和吉非替尼单独或联合干预两种细胞48h后,MTT法检测细胞生长抑制率提示两者对HCC827、A549细胞的杀伤效应呈剂量依赖性。HCC827细胞不同浓度联合用药组的细胞生长抑制率均大于单独用药组的细胞生长抑制率(P0.05);但A549细胞低浓度联合用药组的细胞生长抑制率与单独用药组的细胞生长抑制率比较差异无统计学意义(P0.05),但高浓度联合时联合用药组的细胞生长抑制率大于单独用药组的细胞生长抑制率(P0.05)。2.流式细胞术检测药物对细胞凋亡的影响:HCC827细胞正常对照组的早期凋亡率为(0.26±0.10)%,10umol/L塞来昔布联合10umol/L吉非替尼处理细胞48h后,可见(39.22±3.67)%的细胞凋亡,明显高于10umol/L塞来昔布、10umol/L吉非替尼单独用药的细胞凋亡率(9.71±0.94、21.54±1.98)%;但对于A549细胞,正常对照组的早期凋亡率为(0.84±0.10)%,10umol/L塞来昔布联合10umol/L吉非替尼处理细胞48h后,可见(4.92±0.36)%的细胞凋亡,与10umol/L塞来昔布、10umol/L吉非替尼单独用药的细胞凋亡率(1.88±0.53、4.77±0.55)%之间差异无显著性(P0.05)。提高用药浓度后,我们发现80umol/L塞来昔布联合40umol/L吉非替尼处理细胞48h后,可见(25.91±4.08)%的细胞凋亡,明显高于80umol/L塞来昔布、40umol/L吉非替尼单独用药的细胞凋亡率(6.30±1.02、18.46±1.92)%。3.Western blot法检测药物对细胞COX-2、p-EGFR蛋白的影响:两种细胞中COX-2和p-EGFR均有表达。80umol/L塞来昔布、40umol/L吉非替尼及两药联合分别作用于两种细胞后,与单药组相比,两药联合组可显著下调COX-2、p-EGFR蛋白的表达(P0.05)。结论:1.在EGFR基因突变型细胞株HCC827中,塞来昔布与吉非替尼联合应用可以更显著的抑制细胞生长、介导细胞凋亡;2.在EGFR基因野生型细胞株A549中,只有高浓度的塞来昔布与吉非替尼联合应用才具有协同抑制细胞生长、介导细胞凋亡的作用;3.其机制可能与诱导凋亡、抑制EGFR的活化及下调COX-2蛋白的表达有关。
[Abstract]:Aim: to investigate the effects of celecoxib combined with gefitinib on the proliferation and apoptosis of epidermal growth factor receptor (EGFR) mutant cell line HCC827) and EGFR wild-type cell line A549).To investigate the synergistic killing effect of celecoxib combined with gefitinib on non-small cell lung cancer cell lines and its correlation with EGFR gene status, and to explore the mechanism of Celecoxib combined with gifitinib in detecting the expression of COX-2 and p-EGFR related proteins by Western blot.The results provide evidence for the clinical use of celecoxib and gefitinib, and provide a more complete scheme for individualized treatment of advanced non-small cell lung cancer (NSCLC).Methods: after 48 h of Celecoxib and Gifitinib alone and combined intervention, the cell growth inhibition rate was determined by MTT assay and the apoptosis rate was detected by Alinexin V/PI method. The expression of p-EGFR and COX-2 protein was detected by Western blot method.The experimental data are expressed as x 卤S, and SPSS 17.0 software is used to carry out ANOVA, and the test level is 伪 0. 05.The result is 1: 1.MTT assay (MTT method) was used to detect the inhibitory effects of different concentrations of celecoxib and gefitinib on the growth of two kinds of cells. After 48 hours of intervention, MTT assay showed that the inhibition rate of cell growth was determined by MTT method.In a dose-dependent manner, the cell growth inhibition rate of HCC827 cells combined with different concentrations of HCC827 cells was higher than that of single drug group (P 0.05), but the cell growth inhibition rate of low concentration A549 cell combination group was higher than that of control group (P 0.05), but the cell growth inhibition rate of low concentration A549 cell combination group was higher than that of control group (P < 0.05).There was no significant difference between the inhibition rate of cell growth and the rate of cell growth inhibition in the single drug group, but the inhibitory rate of cell growth in the combined treatment group was higher than that in the single drug group at high concentration.The effect of drugs on apoptosis was detected by flow cytometry. The early apoptotic rate in the normal control group was 0.26 卤0.10 渭 m / L celecoxib combined with 10umol/L gefitinib for 48 h, and the apoptosis rate was 39.22 卤3.67%.There was no significant difference between the apoptotic rate (1.88 卤0.53 卤4.77 卤0.55%) and that of 10umol/L cebuxime 10 umol / L gefitinib alone (P 0.05).After increasing the concentration of 80umol/L, we found that after 48 hours of treatment with 80umol/L and 40umol/L, apoptosis was found in 25.91 卤4.08% of the cells.After acting on two kinds of cells,Compared with the single drug group, the expression of COX-2 p-EGFR protein was significantly down-regulated in the combined group.Conclusion 1.In EGFR mutant cell line HCC827, the combination of celecoxib and gefitinib can significantly inhibit cell growth and mediate apoptosis.In EGFR gene wild type cell line A549, only the combination of celecoxib and gefitinib can synergistically inhibit cell growth and mediate apoptosis.The mechanism may be related to inducing apoptosis, inhibiting the activation of EGFR and down-regulating the expression of COX-2 protein.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

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