本文選題：環(huán)氧化酶-2　切入點(diǎn)：吉非替尼　出處：《石河子大學(xué)》2015年碩士論文

【摘要】：目的:本研究通過觀察塞來昔布聯(lián)合吉非替尼分別對表皮生長因子受體(EGFR)突變細(xì)胞株(HCC827)和EGFR野生型細(xì)胞株(A549)的增殖和凋亡的影響,探討塞來昔布聯(lián)合吉非替尼是否對非小細(xì)胞肺癌細(xì)胞系具有協(xié)同殺傷作用及與EGFR基因狀態(tài)之間的相關(guān)性;并通過Western blot檢測COX-2和p-EGFR相關(guān)蛋白的表達(dá),對其機(jī)制進(jìn)行初步探討;從而為臨床上塞來昔布與吉非替尼的聯(lián)合使用提供依據(jù),為晚期非小細(xì)胞肺癌患者的個(gè)體化治療提供更完善的方案。方法:塞來昔布、吉非替尼單獨(dú)及聯(lián)合干預(yù)兩種細(xì)胞系48h后,四甲基偶氮唑鹽(MTT)法測定細(xì)胞生長抑制率;Alinexin V/PI法檢測細(xì)胞凋亡率;Western blot法檢測p-EGFR和COX-2蛋白表達(dá)。實(shí)驗(yàn)數(shù)據(jù)以x±S表示,用SPSS 17.0軟件進(jìn)行方差分析,檢驗(yàn)水準(zhǔn)α=0.05。結(jié)果:1.四甲基偶氮唑鹽(MTT法)檢測藥物分別對兩種細(xì)胞生長的抑制作用:不同濃度的塞來昔布和吉非替尼單獨(dú)或聯(lián)合干預(yù)兩種細(xì)胞48h后,MTT法檢測細(xì)胞生長抑制率提示兩者對HCC827、A549細(xì)胞的殺傷效應(yīng)呈劑量依賴性。HCC827細(xì)胞不同濃度聯(lián)合用藥組的細(xì)胞生長抑制率均大于單獨(dú)用藥組的細(xì)胞生長抑制率(P0.05);但A549細(xì)胞低濃度聯(lián)合用藥組的細(xì)胞生長抑制率與單獨(dú)用藥組的細(xì)胞生長抑制率比較差異無統(tǒng)計(jì)學(xué)意義(P0.05),但高濃度聯(lián)合時(shí)聯(lián)合用藥組的細(xì)胞生長抑制率大于單獨(dú)用藥組的細(xì)胞生長抑制率(P0.05)。2.流式細(xì)胞術(shù)檢測藥物對細(xì)胞凋亡的影響:HCC827細(xì)胞正常對照組的早期凋亡率為(0.26±0.10)%,10umol/L塞來昔布聯(lián)合10umol/L吉非替尼處理細(xì)胞48h后,可見(39.22±3.67)%的細(xì)胞凋亡,明顯高于10umol/L塞來昔布、10umol/L吉非替尼單獨(dú)用藥的細(xì)胞凋亡率(9.71±0.94、21.54±1.98)%;但對于A549細(xì)胞,正常對照組的早期凋亡率為(0.84±0.10)%,10umol/L塞來昔布聯(lián)合10umol/L吉非替尼處理細(xì)胞48h后,可見(4.92±0.36)%的細(xì)胞凋亡,與10umol/L塞來昔布、10umol/L吉非替尼單獨(dú)用藥的細(xì)胞凋亡率(1.88±0.53、4.77±0.55)%之間差異無顯著性(P0.05)。提高用藥濃度后,我們發(fā)現(xiàn)80umol/L塞來昔布聯(lián)合40umol/L吉非替尼處理細(xì)胞48h后,可見(25.91±4.08)%的細(xì)胞凋亡,明顯高于80umol/L塞來昔布、40umol/L吉非替尼單獨(dú)用藥的細(xì)胞凋亡率(6.30±1.02、18.46±1.92)%。3.Western blot法檢測藥物對細(xì)胞COX-2、p-EGFR蛋白的影響:兩種細(xì)胞中COX-2和p-EGFR均有表達(dá)。80umol/L塞來昔布、40umol/L吉非替尼及兩藥聯(lián)合分別作用于兩種細(xì)胞后,與單藥組相比,兩藥聯(lián)合組可顯著下調(diào)COX-2、p-EGFR蛋白的表達(dá)(P0.05)。結(jié)論:1.在EGFR基因突變型細(xì)胞株HCC827中,塞來昔布與吉非替尼聯(lián)合應(yīng)用可以更顯著的抑制細(xì)胞生長、介導(dǎo)細(xì)胞凋亡;2.在EGFR基因野生型細(xì)胞株A549中,只有高濃度的塞來昔布與吉非替尼聯(lián)合應(yīng)用才具有協(xié)同抑制細(xì)胞生長、介導(dǎo)細(xì)胞凋亡的作用;3.其機(jī)制可能與誘導(dǎo)凋亡、抑制EGFR的活化及下調(diào)COX-2蛋白的表達(dá)有關(guān)。
[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 鹵S, and SPSS 17.0 software is used to carry out ANOVA, and the test level is 偽 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 鹵0.10 渭 m / L celecoxib combined with 10umol/L gefitinib for 48 h, and the apoptosis rate was 39.22 鹵3.67%.There was no significant difference between the apoptotic rate (1.88 鹵0.53 鹵4.77 鹵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 鹵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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R734.2

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