本文選題：二去水衛(wèi)矛醇 + 肺癌��； 參考：《廣西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:評價(jià)二去水衛(wèi)矛醇(dianhydrogalactitol,DAG)對肺癌NCI-H460細(xì)胞的抗腫瘤作用,探討其抗腫瘤作用機(jī)制。方法:(1)DAG對NCI-H460細(xì)胞增殖的影響。(1)采用CCK-8法、細(xì)胞克隆形成實(shí)驗(yàn),計(jì)算增殖抑制率及半數(shù)抑制濃度IC50,評價(jià)DAG對NCI-H460細(xì)胞的增殖抑制作用。(2)采用顯微拍照觀察不同濃度DAG給藥48 h后,肺癌NCI-H460細(xì)胞形態(tài)的改變。(2)DAG對NCI-H460細(xì)胞遷移的影響。(1)采用劃痕實(shí)驗(yàn)評價(jià)DAG對NCI-H460細(xì)胞橫向遷移的影響。(2)采用Transwell-migration小室模型評價(jià)DAG對NCI-H460細(xì)胞縱向遷移的影響。(3)DAG對NCI-H460細(xì)胞DNA、Ca~(2+)和線粒體膜電位的影響。(1)采用Hoechst 33342染色檢測DAG對細(xì)胞核染色質(zhì)的影響。(2)采用單細(xì)胞凝膠電泳檢測DAG對細(xì)胞DNA的損傷作用。(3)采用Fluo-3 AM為Ca~(2+)熒光探針,檢測DAG對細(xì)胞內(nèi)Ca~(2+)的影響。(4)采用Rhodamine123(Rhm 123)為線粒體膜電位熒光探針,檢測DAG對細(xì)胞線粒體膜電位的影響。(4)DAG對Topo Ⅱα、Topo Ⅱβ的影響。(1)采用Real-time PCR法檢測拓?fù)洚悩?gòu)酶Ⅱα(Topo Ⅱα)、拓?fù)洚悩?gòu)酶Ⅱβ(Topo Ⅱβ)mRNA的表達(dá)水平。(2)采用Western blot法檢測Topo Ⅱα、Topo Ⅱβ蛋白表達(dá)水平。(3)應(yīng)用計(jì)算機(jī)模擬分子對接技術(shù)預(yù)測DAG與Topo Ⅱα、Topo Ⅱβ的相互作用。結(jié)果:(1)CCK-8法檢測結(jié)果顯示,DAG對NCI-H460細(xì)胞的體外抗腫瘤活性顯著,與對照組比較有顯著性差異,其48 h的IC50為9.68±1.02μg/mL。細(xì)胞克隆形成實(shí)驗(yàn)結(jié)果表明,DAG能持續(xù)抑制腫瘤細(xì)胞的增殖。(2)劃痕實(shí)驗(yàn)和Transwell-migration小室檢測結(jié)果表明,DAG能抑制腫瘤細(xì)胞的遷移。(3)Hoechst 33342檢測細(xì)胞凋亡結(jié)果顯示,DAG使細(xì)胞核染色質(zhì)發(fā)生明顯改變,與空白對照組比較,各給藥組細(xì)胞均不同程度出現(xiàn)細(xì)胞變圓縮小、貼壁能力減弱、胞漿內(nèi)顆粒邊緣化甚至細(xì)胞破碎等損傷現(xiàn)象;此外,單細(xì)胞凝膠電泳檢測出DAG可導(dǎo)致DNA損傷。Fluo-3 AM熒光染色結(jié)果顯示,與空白對照組比較,各給藥組熒光強(qiáng)度增強(qiáng),即細(xì)胞內(nèi)Ca~(2+)濃度增加,說明DAG誘導(dǎo)細(xì)胞凋亡與細(xì)胞內(nèi)Ca~(2+)濃度增加有關(guān);Rhm 123熒光染色結(jié)果顯示,與空白對照組比較,各給藥組熒光強(qiáng)度減弱,表明DAG使細(xì)胞線粒體膜電位下降,即DAG誘導(dǎo)細(xì)胞凋亡與線粒體膜電位下降有關(guān)。(4)Real-time PCR檢測結(jié)果顯示不同濃度的DAG均能明顯降低Topo Ⅱα、Topo ⅡβmRNA表達(dá)量降低;Western blot檢測表明,DAG明顯使Topo Ⅱα、Topo Ⅱβ蛋白表達(dá)量降低,提示DAG誘導(dǎo)細(xì)胞凋亡與下調(diào)Topo Ⅱα、Topo Ⅱβ有關(guān);計(jì)算機(jī)模擬分子對接顯示DAG與Topo Ⅱα、Topo Ⅱβ有相互結(jié)合作用。結(jié)論:(1)DAG能顯著抑制NCI-H460細(xì)胞的增殖。(2)DAG能顯著抑制NCI-H460細(xì)胞的遷移。(3)DAG誘導(dǎo)細(xì)胞損傷與其改變細(xì)胞內(nèi)Ca~(2+)濃度、線粒體膜電位及DNA損傷有關(guān)。(4)作用機(jī)制研究表明DAG能降低Topo Ⅱα、Topo ⅡβmRNA和蛋白水平,并與Topo Ⅱα、Topo Ⅱβ結(jié)合,最終可能導(dǎo)致DNA雙鏈斷裂,引起細(xì)胞死亡。
[Abstract]:Aim: to evaluate the antitumor effect of dianhydrogalactil DAG on NCI-H460 cells of lung cancer and its mechanism. Methods the effect of DAG on the proliferation of NCI-H460 cells was studied by CCK-8 assay. The cell clone formation assay was used to calculate the inhibitory rate of proliferation and IC50, and to evaluate the inhibitory effect of DAG on the proliferation of NCI-H460 cells. The effects of DAG on the proliferation of NCI-H460 cells were evaluated by microphotography for 48 h after administration of different concentrations of DAG. Morphological changes of NCI-H460 cells in lung cancer. Effect of DAG on the migration of NCI-H460 cells. (1) the effect of DAG on the lateral migration of NCI-H460 cells was evaluated by scratch assay. (2) Transwell-migration chamber model was used to evaluate the effect of DAG on the longitudinal migration of NCI-H460 cells. Hoechst 33342 staining was used to detect the effect of DAG on nuclear chromatin. The single cell gel electrophoresis (SCGE) was used to detect the damage of DAG to cell DNA. Fluo-3 AM was used as the Ca~(2) fluorescent probe. The effect of DAG on intracellular Ca~(2. (4) Rhodamine123(Rhm 123) was used as fluorescence probe of mitochondrial membrane potential. Detection of the effect of DAG on mitochondrial membrane potential. The effect of Topo 鈪，

本文編號(hào)：1968151