本文關(guān)鍵詞： 黃荊子乙酸乙酯提取物 CNE-1細(xì)胞 增殖抑制 細(xì)胞周期　出處：《南華大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的初步探討黃荊子乙酸乙酯提取物（Evn-50）對(duì)體外培養(yǎng)的人鼻咽癌CNE-1細(xì)胞株增殖的影響，探討黃荊子乙酸乙酯提取物在鼻咽癌藥物治療中的應(yīng)用價(jià)值，為其臨床應(yīng)用提供初步的理論依據(jù)。 方法以人鼻咽癌細(xì)胞株CNE-1為研究對(duì)象，，將不同溶度（0、0.625、1.25、2.5、5、10ug/ml）黃荊子乙酸乙酯提取物作用CNE-1細(xì)胞后，用CCK-8法觀(guān)察不同溶度（0、0.625、1.25、2.5、5、10ug/ml）黃荊子乙酸乙酯提取物作用不同時(shí)間（24h、48h和72h）后CNE-1細(xì)胞的增殖變化，并計(jì)數(shù)藥物作用的IC50值，用于后續(xù)實(shí)驗(yàn)；將所選擇藥物濃度作用CNE-1細(xì)胞不同時(shí)間（24h、48h和72h）后，用流式細(xì)胞儀檢測(cè)法測(cè)定黃荊子乙酸乙酯提取物作用不同時(shí)間后細(xì)胞的增殖和周期情況；采用RT-PCR和Western blotting檢測(cè)黃荊子乙酸乙酯提取物作用不同時(shí)間后，CNE-1細(xì)胞內(nèi)細(xì)胞周期相關(guān)蛋白p27和CDK2的表達(dá)情況。 結(jié)果 1. CCK-8比色法結(jié)果顯示：黃荊子乙酸乙酯提取物對(duì)CNE-1細(xì)胞增殖有顯著的抑制作用，并且呈現(xiàn)出時(shí)間和劑量依賴(lài)性；其IC50值為2.5ug/ml。 2.流式細(xì)胞檢測(cè)法結(jié)果顯示：用2.5ug/ml黃荊子乙酸乙酯提取物處理CNE-1細(xì)胞不同時(shí)間后，CNE-1細(xì)胞的增殖受到明顯抑制；G0/G1細(xì)胞比例明顯增加，而S/G2/M期細(xì)胞明顯減少，呈時(shí)間依賴(lài)性。 3. RT-PCR結(jié)果顯示：用2.5ug/ml黃荊子乙酸乙酯提取物處理CNE-1細(xì)胞不同時(shí)間后，CNE-1細(xì)胞中G1-S期抑制蛋白p27mRNA表達(dá)增高，而G1-S期促進(jìn)蛋白CDK2mRNA表達(dá)降低，呈時(shí)間依賴(lài)性。 4. Western blotting結(jié)果顯示：用2.5ug/ml黃荊子乙酸乙酯提取物處理CNE-1細(xì)胞不同時(shí)間后，CNE-1細(xì)胞中G1-S期抑制蛋白p27蛋白表達(dá)增高，而G1-S期促進(jìn)蛋白CDK2蛋白表達(dá)降低，呈時(shí)間依賴(lài)性。 結(jié)論 1.黃荊子乙酸乙酯提取物（Evn-50）具有明顯抑制CNE-1細(xì)胞增殖的作用，其抑制作用呈時(shí)間-劑量依賴(lài)性。 2.黃荊子乙酸乙酯提取物抑制CNE-1細(xì)胞增殖可能與通過(guò)阻斷細(xì)胞周期（G1-S期）進(jìn)展有關(guān)。 3.黃荊子乙酸乙酯提取物抑制細(xì)胞增殖可能與其上調(diào)G1-S期抑制蛋白p27和下調(diào)G1-S期促進(jìn)蛋白CDK2表達(dá)有關(guān)。
[Abstract]:Objective to investigate the ethyl acetate extract of Vitex negundo seed (Evn-50) on the proliferation of cultured human nasopharyngeal carcinoma cell line CNE-1, to investigate the application value of the ethyl acetate extract of Vitex negundo seed in nasopharyngeal carcinoma in drug therapy, provide the theoretical basis for its clinical application.
Methods human nasopharyngeal carcinoma cell line CNE-1 as the research object, the different solubility of Huang Jingzi (0,0.625,1.25,2.5,5,10ug/ml) of ethyl acetate extract of CNE-1 cells, observe the different solubility by the method of CCK-8 (0,0.625,1.25,2.5,5,10ug/ml) Huang Jingzi effect at the same time the ethyl acetate extract (24h, 48h and 72h) proliferation of CNE-1 cells, and counting the number of drugs IC50 for subsequent experiments, the selection of different time; drug concentration of CNE-1 cells (24h, 48h, and 72h) were detected by flow cytometry method for the determination of extract of different time after cell cycle and proliferation of Vitex negundo seed ethyl acetate; using RT-PCR and Western blotting detection of ethyl acetate extract of Vitex negundo seed after different time the expression of CNE-1, cell cycle related proteins p27 and CDK2.
Result
The results of 1. CCK-8 colorimetry showed that Huang Jingzi ethyl acetate extract had a significant inhibitory effect on the proliferation of CNE-1 cells, and showed a time and dose dependent manner, and the IC50 value was 2.5ug/ml..
2. the results of flow cytometry assay showed that the ethyl acetate extract of Vitex negundo seed by 2.5ug/ml in CNE-1 cells treated with different time, the proliferation of CNE-1 cells was inhibited; the proportion of G0/G1 cells was significantly increased, while cells in S/G2/M phase decreased, in a time dependent manner.
3. RT-PCR results showed that 2.5ug/ml of ethyl acetate extract of Vitex negundo seed treatment of CNE-1 cells at different time after CNE-1 cells in G1-S phase inhibition of protein expression of p27mRNA, G1-S and promote the expression of CDK2mRNA protein decreased in a time-dependent manner.
4. Western blotting results showed that 2.5ug/ml of ethyl acetate extract of Vitex negundo seed treatment of CNE-1 cells at different time after CNE-1 cells in G1-S phase inhibition of protein expression of p27 protein, G1-S protein and promote the expression of CDK2 protein decreased in a time-dependent manner.
conclusion
1. ethyl acetate extract of Vitex negundo seed (Evn-50) can inhibit the proliferation of CNE-1 cells, the inhibitory effect was time and dose dependent.
2. ethyl acetate extract of Vitex negundo seed could inhibit the proliferation of CNE-1 cells by blocking cell cycle progression (G1-S).
3. inhibition of ethyl acetate extract of Vitex negundo seed inhibitory protein p27 and down-regulation of G1-S promote the expression of CDK2 protein on cell proliferation may be related to the up regulation of G1-S.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R285.5;R739.63

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