AZD9291单独以及联合LC对PC-9-IR细胞的放疗增敏作用及其机制研究

发布时间：2018-01-08 01:04

本文关键词：AZD9291单独以及联合LC对PC-9-IR细胞的放疗增敏作用及其机制研究　出处：《浙江中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:AZD9291单独以及联合LC对PC-9-IR细胞的放疗增敏作用及其机制研究。方法:采用MTS方法检测AZD9291及细梗香草总皂苷(LC)单独或联合使用对非小细胞肺癌细胞株H460,PC-9,PC-9-IR,H1975的体外抑制作用。采用克隆形成试验了解AZD9291单独以及联合LC对肺癌细胞株的放疗增敏作用,计算放疗增敏比率(SER)。通过Westemblot 了解AZD9291对PC-9-IR细胞放疗后EGFR,ERK,AKT蛋白及其磷酸化水平的变化。采用流式细胞技术和免疫荧光技术检测γH2AX水平,了解AZD9291作用于PC-9-IR细胞后,放疗对细胞发生凋亡的影响。通过干扰RNA技术,沉默PC-9-IR细胞的DNA-PKcs基因,了解沉默前后AZD9291对PC-9-IR细胞的放疗增敏作用变化。采用BALB/C nu/nu裸鼠移植瘤模型,验证AZD9291的体内放疗增敏作用。采用免疫组化检测肿瘤组织的EGFR的磷酸化水平以及Ki67,DNA-PKcs,γ-H2AX和CC3蛋白的表达情况。结果:AZD9291对人肺癌细胞株H460,PC-9,PC-9-IR和H1975的IC50分别为 1.27μM,8.36nM,7.04nM 和 10.62nM。LC 对人肺癌细胞株 H460,PC-9,PC-9-IR和 H1975 的 IC50 分别为 4.190μg/ml,1.658μg/ml,1.017μg/ml 和 5.577μg/ml。不同浓度LC和AZD9291对PC-9-IR细胞的抑制作用联合指数(Combine Index,CI)从0.16到0.65不等,分布于CI=1.0下方,当LC浓度为0.5096μg/ml时,与一定浓度的AZD9291(1.76nM)的联合效应最好,CI为0.16。在肺癌H460细胞中AZD9291浓度为1.271μM和6.355μM时放射增敏指数分别为0.87(P0.05)、1.03(P0.05)。人肺癌 PC-9 细胞中 AZD9291 浓度为 8.38nM 和41.81nM 时放射增敏指数分别为 1.07(P0.05)、1.12(P0.05)。在 PC-9-IR细胞中 AZD9291 浓度为 7.04nM 和 35.19nM 时,SER 值为 1.16(P0.005)、1.29(P0.0001)。在 H1975 细胞中 AZD9291 浓度为 10.62nM 和 53.10nM 时,SER 值为1.03(P0.05)、1.18(P0.005)。AZD9291 预处理能显著降低 PC-9-IR 细胞放疗后的EGFR、AKT和ERK蛋白磷酸化水平。然而,AZD9291预处理能显著降低H1975细胞放疗后的EGFR、和AKT蛋白磷酸化水平但ERK蛋白磷酸化水平无明显变化。PC-9-IR经AZD9291和放疗处理后的细胞凋亡率为50.4±5.8%,明显高于对照组的12.4±1.2%(P0.01),也高于AZD9291或放疗处理组的 34.6±3.9%、25.2±2.5%(P0.05)。经 AZD9291 处理,PC-9-IR 细胞在经过6Gy放疗8小时后,yH2AX阳性细胞达到最高,为85.3±4.8%,然后逐渐下降。但放疗后24hγH2AX阳性细胞(63.4±4.3%)和48hyH2AX阳性细胞(36.1±2.7%)与对照组差异显著(P0.05)。siRNA-DNA-PKcs 处理后 AZD9291对PC-9-IR细胞的放疗增敏比1.09(P0.05)。单独使用AZD9291(1.76nM)或LC(0.51μg/ml)的放疗增敏指数为1.02和0.98,与对照组差异不显著(P0.05),二者联合使用的放疗增敏指数为1.28,与对照组和单独用药组比较差异显著(P0.05)。对照组相对肿瘤体积达到2063.5 ± 149.3,单纯放疗组为1483.7 ±96.8,较对照组差异具有统计学意义,(P0.05)。AZD9291治疗组移植瘤体积为1117.5±68.0,较对照组差异具有统计学意义,(P0.01)。AZD9291联合放疗治疗组645.7±77.9,与对照组比较差异显著(P0.001)。AZD9291治疗后能明显降低EGFR的磷酸化水平,尽管放疗对EGFR的磷酸化水平影响不大,但联合AZD9291治疗能显著降低EGFR磷酸化水平。与对照组比较,AZD9291治疗一方面能够减低Ki67的表达,另一方面增加CC3蛋白的表达。空白组和AZD9291组γH2AX蛋白的表达无明显增高且变化不明显,但放疗组γH2AX蛋白的表达增高明显,联合AZD9291后可以显著减低γH2AX蛋白的表达。同样,与单独放疗比较,AZD9291后可以显著减低DNA-PKcs蛋白的表达。结论:AZD9291和LC对肺癌H460,PC-9,PC-9-IR及H1975细胞系具有增殖抑制的作用。LC和AZD9291联合使用对PC-9-IR细胞具有较好的增值抑制协同作用。AZD9291通过抑制PC-9-IR细胞的增值和促进凋亡,减缓放疗导致的细胞DNA损伤的修复进程,从而可以发挥对PC-9-IR细胞的放疗增敏作用。
[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.

【学位授予单位】：浙江中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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