本文關(guān)鍵詞：mTOR抑制劑AZD8055對(duì)膽管癌細(xì)胞生物學(xué)行為的影響及分子機(jī)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(xì)胞 Akt/mTOR信號(hào)通路

【摘要】：目的:探討mTOR抑制劑AZD8055對(duì)膽管癌細(xì)胞生物學(xué)行為的影響及其分子機(jī)制。材料和方法:MTT法檢測(cè)不同濃度的AZD8055對(duì)HuCCT1膽管癌細(xì)胞體外增殖能力的影響;平板克隆形成實(shí)驗(yàn)檢測(cè)不同濃度的AZD8055對(duì)HuCCT1細(xì)胞集落形成能力的影響;劃痕愈合實(shí)驗(yàn)檢測(cè)AZD8055對(duì)膽管癌細(xì)胞HuCCT1橫向遷移能力的影響;流式細(xì)胞儀檢測(cè)AZD8055對(duì)細(xì)胞凋亡的影響;蛋白印跡實(shí)驗(yàn)檢測(cè)AZD8055對(duì)膽管癌細(xì)胞自噬和凋亡相關(guān)蛋白表達(dá)的影響及Akt/mTOR信號(hào)通路相關(guān)蛋白表達(dá)水平。結(jié)果:1.MTT 檢測(cè)實(shí)驗(yàn)結(jié)果顯示:經(jīng) 25nM、50nM、100nM、200nM、400nM 的 AZD8055處理膽管癌細(xì)胞24h、48h、72h后,與對(duì)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(shí)驗(yàn)結(jié)果顯示:與對(duì)照組相比,25nM、50nM和100nM作用于HuCCT1細(xì)胞14天后,均顯著抑制細(xì)胞集落形成能力(P0.05);3.細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)結(jié)果顯示:經(jīng)100nM和200nM濃度的AZD08055處理細(xì)胞后,與對(duì)照組相比膽管癌細(xì)胞遷移距離明顯縮短(P0.05);4.流式細(xì)胞儀檢測(cè)結(jié)果顯示:50nM、100nM、200nM濃度AZD8055處理膽管癌細(xì)胞48h后,與對(duì)照組相比,其誘導(dǎo)凋亡效果不明顯;5.蛋白印跡實(shí)驗(yàn)結(jié)果顯示:與對(duì)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(duì)照組相比,經(jīng)100nM、200nM、400nM濃度AZD8055處理膽管癌細(xì)胞24h后,自噬相關(guān)標(biāo)記物BeclinⅠ蛋白的表達(dá)增高,LC3Ⅱ/LC3Ⅰ比例也增高(P0.05);與對(duì)照組相比,Akt、S6、4EBP1的磷酸化蛋白表達(dá)水平明顯降低(P0.05)。結(jié)論:1.AZD8055可抑制膽管癌細(xì)胞增殖能力,其機(jī)制與誘導(dǎo)細(xì)胞自噬和下調(diào)Akt/mTOR信號(hào)通路相關(guān);2.AZD8055可抑制膽管癌細(xì)胞的橫向遷移能力,其機(jī)制與下調(diào)Akt/mTOR信號(hào)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，

本文編號(hào)：1422373