【摘要】：目的：對(duì)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(jìn)行體外抗腫瘤活性篩選,初步探討其抗腫瘤活性的作用機(jī)制。方法：1.采用CCK-8法測(cè)定MAPAT對(duì)人肺癌細(xì)胞株A549、人胰腺癌細(xì)胞株panc-1、人子宮癌細(xì)胞株Hela、人腦膠質(zhì)瘤細(xì)胞株SN-K、人鼻咽癌細(xì)胞株CNE-2細(xì)胞的增值抑制作用。2.HE染色法觀察MAPAT作用后細(xì)胞形態(tài)的變化。3.透射電鏡觀察腫瘤細(xì)胞的亞顯微結(jié)構(gòu)變化。4.Annexin FITC/PI標(biāo)記法檢測(cè)細(xì)胞凋亡的影響。5.細(xì)胞周期實(shí)驗(yàn)分析DNA倍體變化。6.熒光定量PCR技術(shù)分析MAPAT對(duì)拓?fù)洚悩?gòu)酶(TopoⅠ、Ⅱα、Ⅲβ) mRNA表達(dá)的影響。7.蛋白印跡法分析MAPAT對(duì)拓?fù)洚悩?gòu)酶(TopoⅠ、Ⅱα、Ⅲβ)蛋白表達(dá)的影響。結(jié)果：1. CCK-8實(shí)驗(yàn)結(jié)果顯示,MAPAT對(duì)體外培養(yǎng)的5種腫瘤細(xì)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(xì)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(duì)A549細(xì)胞的IC50最小,其IC50=8.90μmol/L,當(dāng)26.77μmol/L時(shí)抑制率達(dá)到70%,故選取A549作為后續(xù)研究的細(xì)胞模型,設(shè)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(jié)果可見(jiàn)隨著MAPAT的作用濃度增加,給藥組細(xì)胞生長(zhǎng)密度較對(duì)照組依次降低,細(xì)胞間隙增大,并可見(jiàn)細(xì)胞收縮體積變小,核固縮藍(lán)色深染的凋亡形態(tài)學(xué)改變。3.透射電鏡觀察結(jié)果顯示,MAPAT可誘導(dǎo)A549細(xì)胞的體積縮小、核固縮、染色質(zhì)聚集的凋亡形態(tài)產(chǎn)生。細(xì)胞器空泡變形和細(xì)胞自噬,細(xì)胞器與細(xì)胞核的腫脹變形,使核膜受損,細(xì)胞變形壞死,細(xì)胞膜受損,細(xì)胞崩裂。4.細(xì)胞凋亡實(shí)驗(yàn)結(jié)果顯示,MAPAT對(duì)人肺癌細(xì)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(duì)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(xì)胞周期實(shí)驗(yàn)結(jié)果,與空白對(duì)照組相比,MAPAT對(duì)A549細(xì)胞作用48小時(shí)后,S期及G2/M期細(xì)胞明顯增加。6.熒光定量PCR和Western Blot實(shí)驗(yàn)結(jié)果顯示,MAPAT作用于A549后,與空白對(duì)照組相比,上調(diào)Topo II a在mRNA及蛋白水平的表達(dá),下調(diào)Topo Ⅰ、Topo Ⅱβ在mRNA及蛋白水平的表達(dá)。結(jié)論：吖啶類衍生物MAPAT可抑制CNE-2、panc-1、SN-K、A549和Hela細(xì)胞的細(xì)胞增殖,其中對(duì)A549的作用最顯著；MAPAT對(duì)腫瘤細(xì)胞的抑制作用可能與影響細(xì)胞周期分布和誘導(dǎo)細(xì)胞凋亡有關(guān)；MAPAT抑制人肺癌A549細(xì)胞Topo Ⅰ、Topo Ⅱβ在mRNA及蛋白水平的表達(dá),這有可能與MAPAT抑制A549細(xì)胞增值的作用機(jī)制有關(guān)。
[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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