mTOR抑制剂AZD8055对胆管癌细胞生物学行为的影响及分子机制研究

发布时间：2018-01-14 06:15

  本文关键词：mTOR抑制剂AZD8055对胆管癌细胞生物学行为的影响及分子机制研究　出处：《延边大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： AZD8055 自噬 胆管癌细胞 Akt/mTOR信号通路

【摘要】：目的:探讨mTOR抑制剂AZD8055对胆管癌细胞生物学行为的影响及其分子机制。材料和方法:MTT法检测不同浓度的AZD8055对HuCCT1胆管癌细胞体外增殖能力的影响;平板克隆形成实验检测不同浓度的AZD8055对HuCCT1细胞集落形成能力的影响;划痕愈合实验检测AZD8055对胆管癌细胞HuCCT1横向迁移能力的影响;流式细胞仪检测AZD8055对细胞凋亡的影响;蛋白印迹实验检测AZD8055对胆管癌细胞自噬和凋亡相关蛋白表达的影响及Akt/mTOR信号通路相关蛋白表达水平。结果:1.MTT 检测实验结果显示:经 25nM、50nM、100nM、200nM、400nM 的 AZD8055处理胆管癌细胞24h、48h、72h后,与对照组相比其增殖率明显降低,呈剂量依赖性(P0.05);2.平板克隆形成实验结果显示:与对照组相比,25nM、50nM和100nM作用于HuCCT1细胞14天后,均显著抑制细胞集落形成能力(P0.05);3.细胞划痕实验检测结果显示:经100nM和200nM浓度的AZD08055处理细胞后,与对照组相比胆管癌细胞迁移距离明显缩短(P0.05);4.流式细胞仪检测结果显示:50nM、100nM、200nM浓度AZD8055处理胆管癌细胞48h后,与对照组相比,其诱导凋亡效果不明显;5.蛋白印迹实验结果显示:与对照组相比,Cleaved caspase 3和促凋亡蛋白Bax下调,抗凋亡蛋白Bcl-2上调,且呈浓度依赖性(P0.05);与对照组相比,经100nM、200nM、400nM浓度AZD8055处理胆管癌细胞24h后,自噬相关标记物BeclinⅠ蛋白的表达增高,LC3Ⅱ/LC3Ⅰ比例也增高(P0.05);与对照组相比,Akt、S6、4EBP1的磷酸化蛋白表达水平明显降低(P0.05)。结论:1.AZD8055可抑制胆管癌细胞增殖能力,其机制与诱导细胞自噬和下调Akt/mTOR信号通路相关;2.AZD8055可抑制胆管癌细胞的横向迁移能力,其机制与下调Akt/mTOR信号通路相关。
[Abstract]:Objective: to investigate the effect of mTOR inhibitor AZD8055 on the biological behavior of cholangiocarcinoma cells and its molecular mechanism. The effect of different concentrations of AZD8055 on the proliferation of HuCCT1 cholangiocarcinoma in vitro was detected by MTT assay. The effects of different concentrations of AZD8055 on the colony forming ability of HuCCT1 cells were detected by plate clone forming assay. The effect of AZD8055 on the transversal migration of cholangiocarcinoma cell line HuCCT1 was detected by scratch healing test. The effect of AZD8055 on apoptosis was detected by flow cytometry. The effect of AZD8055 on the expression of autophagy and apoptosis-related protein and the expression level of Akt/mTOR signal pathway related protein in cholangiocarcinoma cells were detected by Western blot assay. The test results showed that: after 25 nm. Compared with the control group, the proliferation rate of cholangiocarcinoma cells treated with 50nM / 100nM / 200nM / 400nM AZD8055 for 24 h or 48 h / 72 h was significantly lower than that of the control group. In a dose-dependent manner, P0.05; 2. The results of plate clone formation test showed that 25 nM 50nM and 100nM were treated with HuCCT1 cells for 14 days. The ability of colony formation was significantly inhibited (P 0.05). 3. The results of cell scratch test showed that the cells were treated with 100nm and 200nM AZD08055. Compared with the control group, the migration distance of cholangiocarcinoma cells was significantly shorter than that of the control group. 4. The results of flow cytometry showed that the apoptosis of cholangiocarcinoma cells treated with AZD8055 for 48 h was not obvious compared with the control group. 5. The results of Western blot showed that Cleaved caspase _ 3 and pro-apoptotic protein Bax were down-regulated, and anti-apoptotic protein Bcl-2 was up-regulated. And the concentration dependence was P0.05A; Compared with control group, the expression of autophagy related marker Beclin 鈪，

本文编号：1422373