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AZD9291單獨(dú)以及聯(lián)合LC對PC-9-IR細(xì)胞的放療增敏作用及其機(jī)制研究

發(fā)布時(shí)間:2018-01-08 01:04

  本文關(guān)鍵詞:AZD9291單獨(dú)以及聯(lián)合LC對PC-9-IR細(xì)胞的放療增敏作用及其機(jī)制研究 出處:《浙江中醫(yī)藥大學(xué)》2017年博士論文 論文類型:學(xué)位論文


  更多相關(guān)文章: AZD9291 細(xì)梗香草總皂苷 放療增敏 PC-9-IR T790M


【摘要】:目的:AZD9291單獨(dú)以及聯(lián)合LC對PC-9-IR細(xì)胞的放療增敏作用及其機(jī)制研究。方法:采用MTS方法檢測AZD9291及細(xì)梗香草總皂苷(LC)單獨(dú)或聯(lián)合使用對非小細(xì)胞肺癌細(xì)胞株H460,PC-9,PC-9-IR,H1975的體外抑制作用。采用克隆形成試驗(yàn)了解AZD9291單獨(dú)以及聯(lián)合LC對肺癌細(xì)胞株的放療增敏作用,計(jì)算放療增敏比率(SER)。通過Westemblot 了解AZD9291對PC-9-IR細(xì)胞放療后EGFR,ERK,AKT蛋白及其磷酸化水平的變化。采用流式細(xì)胞技術(shù)和免疫熒光技術(shù)檢測γH2AX水平,了解AZD9291作用于PC-9-IR細(xì)胞后,放療對細(xì)胞發(fā)生凋亡的影響。通過干擾RNA技術(shù),沉默PC-9-IR細(xì)胞的DNA-PKcs基因,了解沉默前后AZD9291對PC-9-IR細(xì)胞的放療增敏作用變化。采用BALB/C nu/nu裸鼠移植瘤模型,驗(yàn)證AZD9291的體內(nèi)放療增敏作用。采用免疫組化檢測腫瘤組織的EGFR的磷酸化水平以及Ki67,DNA-PKcs,γ-H2AX和CC3蛋白的表達(dá)情況。結(jié)果:AZD9291對人肺癌細(xì)胞株H460,PC-9,PC-9-IR和H1975的IC50分別為 1.27μM,8.36nM,7.04nM 和 10.62nM。LC 對人肺癌細(xì)胞株 H460,PC-9,PC-9-IR和 H1975 的 IC50 分別為 4.190μg/ml,1.658μg/ml,1.017μg/ml 和 5.577μg/ml。不同濃度LC和AZD9291對PC-9-IR細(xì)胞的抑制作用聯(lián)合指數(shù)(Combine Index,CI)從0.16到0.65不等,分布于CI=1.0下方,當(dāng)LC濃度為0.5096μg/ml時(shí),與一定濃度的AZD9291(1.76nM)的聯(lián)合效應(yīng)最好,CI為0.16。在肺癌H460細(xì)胞中AZD9291濃度為1.271μM和6.355μM時(shí)放射增敏指數(shù)分別為0.87(P0.05)、1.03(P0.05)。人肺癌 PC-9 細(xì)胞中 AZD9291 濃度為 8.38nM 和41.81nM 時(shí)放射增敏指數(shù)分別為 1.07(P0.05)、1.12(P0.05)。在 PC-9-IR細(xì)胞中 AZD9291 濃度為 7.04nM 和 35.19nM 時(shí),SER 值為 1.16(P0.005)、1.29(P0.0001)。在 H1975 細(xì)胞中 AZD9291 濃度為 10.62nM 和 53.10nM 時(shí),SER 值為1.03(P0.05)、1.18(P0.005)。AZD9291 預(yù)處理能顯著降低 PC-9-IR 細(xì)胞放療后的EGFR、AKT和ERK蛋白磷酸化水平。然而,AZD9291預(yù)處理能顯著降低H1975細(xì)胞放療后的EGFR、和AKT蛋白磷酸化水平但ERK蛋白磷酸化水平無明顯變化。PC-9-IR經(jīng)AZD9291和放療處理后的細(xì)胞凋亡率為50.4±5.8%,明顯高于對照組的12.4±1.2%(P0.01),也高于AZD9291或放療處理組的 34.6±3.9%、25.2±2.5%(P0.05)。經(jīng) AZD9291 處理,PC-9-IR 細(xì)胞在經(jīng)過6Gy放療8小時(shí)后,yH2AX陽性細(xì)胞達(dá)到最高,為85.3±4.8%,然后逐漸下降。但放療后24hγH2AX陽性細(xì)胞(63.4±4.3%)和48hyH2AX陽性細(xì)胞(36.1±2.7%)與對照組差異顯著(P0.05)。siRNA-DNA-PKcs 處理后 AZD9291對PC-9-IR細(xì)胞的放療增敏比1.09(P0.05)。單獨(dú)使用AZD9291(1.76nM)或LC(0.51μg/ml)的放療增敏指數(shù)為1.02和0.98,與對照組差異不顯著(P0.05),二者聯(lián)合使用的放療增敏指數(shù)為1.28,與對照組和單獨(dú)用藥組比較差異顯著(P0.05)。對照組相對腫瘤體積達(dá)到2063.5 ± 149.3,單純放療組為1483.7 ±96.8,較對照組差異具有統(tǒng)計(jì)學(xué)意義,(P0.05)。AZD9291治療組移植瘤體積為1117.5±68.0,較對照組差異具有統(tǒng)計(jì)學(xué)意義,(P0.01)。AZD9291聯(lián)合放療治療組645.7±77.9,與對照組比較差異顯著(P0.001)。AZD9291治療后能明顯降低EGFR的磷酸化水平,盡管放療對EGFR的磷酸化水平影響不大,但聯(lián)合AZD9291治療能顯著降低EGFR磷酸化水平。與對照組比較,AZD9291治療一方面能夠減低Ki67的表達(dá),另一方面增加CC3蛋白的表達(dá)�?瞻捉M和AZD9291組γH2AX蛋白的表達(dá)無明顯增高且變化不明顯,但放療組γH2AX蛋白的表達(dá)增高明顯,聯(lián)合AZD9291后可以顯著減低γH2AX蛋白的表達(dá)。同樣,與單獨(dú)放療比較,AZD9291后可以顯著減低DNA-PKcs蛋白的表達(dá)。結(jié)論:AZD9291和LC對肺癌H460,PC-9,PC-9-IR及H1975細(xì)胞系具有增殖抑制的作用。LC和AZD9291聯(lián)合使用對PC-9-IR細(xì)胞具有較好的增值抑制協(xié)同作用。AZD9291通過抑制PC-9-IR細(xì)胞的增值和促進(jìn)凋亡,減緩放療導(dǎo)致的細(xì)胞DNA損傷的修復(fù)進(jìn)程,從而可以發(fā)揮對PC-9-IR細(xì)胞的放療增敏作用。
[Abstract]:Objective: To study the sensitization effects and its mechanism of AZD9291 LC alone and combined with radiotherapy on PC-9-IR cells. Methods: MTS method was used to detect AZD9291 and total saponin (LC) alone or in combination on the cell lines H460, PC-9 non-small cell lung cancer, PC-9-IR, inhibition of H1975 in vitro. The clone formation test AZD9291 LC alone and combined with radiotherapy on lung cancer cell line sensitizing effect, calculation of radiosensitization ratio (SER). AZD9291 of PC-9-IR cells after radiotherapy EGFR, ERK by Westemblot, the changes of AKT protein and phosphorylation. Using flow cytometry and immunofluorescence detection of gamma H2AX the level of understanding of the role of AZD9291 in PC-9-IR cells, the effects of radiation on apoptosis of cells. By RNA interference technology, DNA-PKcs gene silencing in PC-9-IR cells, understanding the silencing of PC-9-IR cells before and after AZD9291 radiotherapy sensitization. The BALB/C nu/nu xenograft model of nude mice, in vivo sensitization of radiotherapy verification AZD9291. Immunohistochemical detection of tumor tissue the phosphorylation level of EGFR and Ki67, DNA-PKcs, -H2AX and CC3 protein expression of gamma. Results: AZD9291 on human lung cancer cell line H460, PC-9, PC-9-IR and H1975 IC50 were 1.27 M, 8.36nM, 7.04nM and 10.62nM.LC on human lung cancer cell lines H460, PC-9, PC-9-IR and H1975 IC50 were 4.190 g/ml, 1.658 g/ml, inhibition of different concentrations of LC and AZD9291 1.017 g/ml and 5.577 g/ml. of PC-9-IR cells combined with index (Combine Index CI) ranging from 0.16 to 0.65 located in CI=1.0, below, when the LC concentration is 0.5096 g/ml, with a certain concentration of AZD9291 (1.76nM) the best combined effect, CI 0.16. AZD9291 in lung cancer H460 cells at the concentration of 1.271 M and 6.355 M radiation sensitivity index were 0.87 (P0. 05), 1.03 (P0.05). The concentration of AZD9291 in human lung cancer PC-9 cells 8.38nM and 41.81nM radiosensitization index were 1.07 (P0.05), 1.12 (P0.05) in PC-9-IR cells. The concentration of AZD9291 was 7.04nM and 35.19nM, SER = 1.16 (P0.005), 1.29 (P0.0001) in H1975 cells. The concentration of AZD9291 in 10.62nM and 53.10nM, SER = 1.03 (P0.05), 1.18 (P0.005).AZD9291 pretreatment can significantly reduce the radiation cell after PC-9-IR EGFR, AKT and ERK phosphorylation. However, AZD9291 pretreatment can significantly reduce the radiotherapy after H1975 cells EGFR, and phosphorylation of AKT protein but the ERK protein phosphorylation level of.PC-9-IR had no obvious change after AZD9291 cell apoptosis and radiotherapy after treatment rate was 50.4 + 5.8%, significantly higher than the control group 12.4 + 1.2% (P0.01), also higher than the AZD9291 or radiotherapy treatment groups 34.6 + 3.9%, 25.2 + 2.5% (P0.05) by AZD9291. Treatment of PC-9-IR cells in 6Gy after radiotherapy after 8 hours, yH2AX positive cells reached a maximum of 85.3 + 4.8%, and then decreased gradually. But after radiotherapy 24h gamma H2AX positive cells (63.4 + 4.3%) and 48hyH2AX positive cells (36.1 + 2.7%) significant difference with the control group (P0.05) after the treatment of.SiRNA-DNA-PKcs AZD9291 radiation on PC-9-IR cell sensitivity ratio of 1.09 (P0.05). Using AZD9291 alone (1.76nM) or LC (0.51 g/ml) the radiosensitization index was 1.02 and 0.98, no significant difference with control group (P0.05), radiotherapy combined with the use of two party sensitivity index was 1.28, compared with the control group and single medication the control group (P0.05 group). The relative tumor volume reached 2063.5 + 149.3, 1483.7 + 96.8 radiotherapy group, with statistically significant difference compared with the control group,.AZD9291 treatment group (P0.05) tumor volume was 1117.5 + 68, with statistically significant difference compared with the control group, (P0.01).AZD9 291 combined with radiotherapy group 645.7 + 77.9, compared with the control group (P0.001) after.AZD9291 treatment can significantly reduce the level of EGFR phosphorylation, although radiotherapy on the phosphorylation of EGFR is not affected, but the combined AZD9291 treatment can significantly reduce the phosphorylation levels of EGFR. Compared with the control group, the expression of AZD9291 in the treatment of hand to reduce Ki67, on the other hand, the increased expression of CC3 protein. The expression of blank group and AZD9291 group gamma H2AX protein was not increased and did not change significantly, but the expression of H2AX protein gamma radiation group increased significantly, combined with AZD9291 can significantly reduce the expression of H2AX protein after gamma. Similarly, compared with radiotherapy alone, the expression of AZD9291 can significantly reduce DNA-PKcs protein. Conclusion: AZD9291 and LC on PC-9, lung cancer H460, PC-9-IR and H1975 cell lines with.LC and AZD9291 inhibited the proliferation of the combined use of good growth of PC-9-IR cells Synergistic effect of.AZD9291 inhibits PC-9-IR cell proliferation and promotes apoptosis, slowing down the repair process of DNA damage induced by radiotherapy, thereby playing a radiosensitizing effect on PC-9-IR cells.

【學(xué)位授予單位】:浙江中醫(yī)藥大學(xué)
【學(xué)位級別】:博士
【學(xué)位授予年份】:2017
【分類號(hào)】:R734.2

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