發(fā)布時間：2018-01-14 06:15

  本文關(guān)鍵詞：mTOR抑制劑AZD8055對膽管癌細胞生物學(xué)行為的影響及分子機制研究　出處：《延邊大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： AZD8055 自噬 膽管癌細胞 Akt/mTOR信號通路

【摘要】：目的:探討mTOR抑制劑AZD8055對膽管癌細胞生物學(xué)行為的影響及其分子機制。材料和方法:MTT法檢測不同濃度的AZD8055對HuCCT1膽管癌細胞體外增殖能力的影響;平板克隆形成實驗檢測不同濃度的AZD8055對HuCCT1細胞集落形成能力的影響;劃痕愈合實驗檢測AZD8055對膽管癌細胞HuCCT1橫向遷移能力的影響;流式細胞儀檢測AZD8055對細胞凋亡的影響;蛋白印跡實驗檢測AZD8055對膽管癌細胞自噬和凋亡相關(guān)蛋白表達的影響及Akt/mTOR信號通路相關(guān)蛋白表達水平。結(jié)果:1.MTT 檢測實驗結(jié)果顯示:經(jīng) 25nM、50nM、100nM、200nM、400nM 的 AZD8055處理膽管癌細胞24h、48h、72h后,與對照組相比其增殖率明顯降低,呈劑量依賴性(P0.05);2.平板克隆形成實驗結(jié)果顯示:與對照組相比,25nM、50nM和100nM作用于HuCCT1細胞14天后,均顯著抑制細胞集落形成能力(P0.05);3.細胞劃痕實驗檢測結(jié)果顯示:經(jīng)100nM和200nM濃度的AZD08055處理細胞后,與對照組相比膽管癌細胞遷移距離明顯縮短(P0.05);4.流式細胞儀檢測結(jié)果顯示:50nM、100nM、200nM濃度AZD8055處理膽管癌細胞48h后,與對照組相比,其誘導(dǎo)凋亡效果不明顯;5.蛋白印跡實驗結(jié)果顯示:與對照組相比,Cleaved caspase 3和促凋亡蛋白Bax下調(diào),抗凋亡蛋白Bcl-2上調(diào),且呈濃度依賴性(P0.05);與對照組相比,經(jīng)100nM、200nM、400nM濃度AZD8055處理膽管癌細胞24h后,自噬相關(guān)標(biāo)記物BeclinⅠ蛋白的表達增高,LC3Ⅱ/LC3Ⅰ比例也增高(P0.05);與對照組相比,Akt、S6、4EBP1的磷酸化蛋白表達水平明顯降低(P0.05)。結(jié)論:1.AZD8055可抑制膽管癌細胞增殖能力,其機制與誘導(dǎo)細胞自噬和下調(diào)Akt/mTOR信號通路相關(guān);2.AZD8055可抑制膽管癌細胞的橫向遷移能力,其機制與下調(diào)Akt/mTOR信號通路相關(guān)。
[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，

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