發(fā)布時間：2018-12-06 16:03

【摘要】：目的：對本課題組合成的吖啶酰胺基硫脲類衍生物MAPAT(2-甲氧基-9-吖啶(吡啶酰胺基)硫脲)進行體外抗腫瘤活性篩選,初步探討其抗腫瘤活性的作用機制。方法：1.采用CCK-8法測定MAPAT對人肺癌細胞株A549、人胰腺癌細胞株panc-1、人子宮癌細胞株Hela、人腦膠質瘤細胞株SN-K、人鼻咽癌細胞株CNE-2細胞的增值抑制作用。2.HE染色法觀察MAPAT作用后細胞形態(tài)的變化。3.透射電鏡觀察腫瘤細胞的亞顯微結構變化。4.Annexin FITC/PI標記法檢測細胞凋亡的影響。5.細胞周期實驗分析DNA倍體變化。6.熒光定量PCR技術分析MAPAT對拓撲異構酶(TopoⅠ、Ⅱα、Ⅲβ) mRNA表達的影響。7.蛋白印跡法分析MAPAT對拓撲異構酶(TopoⅠ、Ⅱα、Ⅲβ)蛋白表達的影響。結果：1. CCK-8實驗結果顯示,MAPAT對體外培養(yǎng)的5種腫瘤細胞CNE-2、 panc-1、SN-K、A549以及Hela均有一定的抑制作用, MAPAT處理A549、CNE-2、panc-1、SN-K及Hel a細胞48h的ICso分別是(8.90±0.49)μmol/L、 (44.44±0.02)μmol/L、(133.54±0.87)μmol/L、(80.16±0.18)μmol/L、(92.77±0.12)μmol/L,其中對A549細胞的IC50最小,其IC50=8.90μmol/L,當26.77μmol/L時抑制率達到70%,故選取A549作為后續(xù)研究的細胞模型,設置低劑量組為4.45μmol/L、中劑量組為8.90μmol/L,高劑量組為26.77μmol/L。2.HE染色結果可見隨著MAPAT的作用濃度增加,給藥組細胞生長密度較對照組依次降低,細胞間隙增大,并可見細胞收縮體積變小,核固縮藍色深染的凋亡形態(tài)學改變。3.透射電鏡觀察結果顯示,MAPAT可誘導A549細胞的體積縮小、核固縮、染色質聚集的凋亡形態(tài)產生。細胞器空泡變形和細胞自噬,細胞器與細胞核的腫脹變形,使核膜受損,細胞變形壞死,細胞膜受損,細胞崩裂。4.細胞凋亡實驗結果顯示,MAPAT對人肺癌細胞株A549作用48h后,低、中、高劑量組的壞死率分別為(2.63+0.21)%、(2.70±1.19)%、(38.80-±0.10)%；與空白對照相比較,加藥組隨著MAPAT濃度增加,壞死率顯著增加(P0.01)；低劑量組、中劑量組凋亡率逐漸增大(P0.05)。5.細胞周期實驗結果,與空白對照組相比,MAPAT對A549細胞作用48小時后,S期及G2/M期細胞明顯增加。6.熒光定量PCR和Western Blot實驗結果顯示,MAPAT作用于A549后,與空白對照組相比,上調Topo II a在mRNA及蛋白水平的表達,下調Topo Ⅰ、Topo Ⅱβ在mRNA及蛋白水平的表達。結論：吖啶類衍生物MAPAT可抑制CNE-2、panc-1、SN-K、A549和Hela細胞的細胞增殖,其中對A549的作用最顯著；MAPAT對腫瘤細胞的抑制作用可能與影響細胞周期分布和誘導細胞凋亡有關；MAPAT抑制人肺癌A549細胞Topo Ⅰ、Topo Ⅱβ在mRNA及蛋白水平的表達,這有可能與MAPAT抑制A549細胞增值的作用機制有關。
[Abstract]:Aim: to screen the antitumor activity of acridine aminothiourea derivative MAPAT (2-methoxy-9-acridine (pyridyl) thiourea) in vitro and to explore the mechanism of its antitumor activity. Method 1: 1. CCK-8 assay was used to determine the effect of MAPAT on human lung cancer cell line A549 and human pancreatic cancer cell line panc-1, human uterine carcinoma cell line Hela, human glioma cell line SN-K, The proliferation inhibition of human nasopharyngeal carcinoma (NPC) cell line CNE-2 cells. The morphological changes of MAPAT cells after MAPAT treatment were observed by 2.HE staining. 3. The ultrastructural changes of tumor cells were observed by transmission electron microscope (TEM). The effect of apoptosis was detected by 4.Annexin FITC/PI labeling method. 5. 5. Cell cycle analysis of DNA ploidy. 6. The effect of MAPAT on the expression of topoisomerase (Topo 鈪，

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