黄荆子乙酸乙酯提取物对人鼻咽癌CNE-1细胞株增殖的影响

发布时间：2018-02-03 14:15

本文关键词： 黄荆子乙酸乙酯提取物 CNE-1细胞增殖抑制细胞周期　出处：《南华大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的初步探讨黄荆子乙酸乙酯提取物（Evn-50）对体外培养的人鼻咽癌CNE-1细胞株增殖的影响，探讨黄荆子乙酸乙酯提取物在鼻咽癌药物治疗中的应用价值，为其临床应用提供初步的理论依据。方法以人鼻咽癌细胞株CNE-1为研究对象，，将不同溶度（0、0.625、1.25、2.5、5、10ug/ml）黄荆子乙酸乙酯提取物作用CNE-1细胞后，用CCK-8法观察不同溶度（0、0.625、1.25、2.5、5、10ug/ml）黄荆子乙酸乙酯提取物作用不同时间（24h、48h和72h）后CNE-1细胞的增殖变化，并计数药物作用的IC50值，用于后续实验；将所选择药物浓度作用CNE-1细胞不同时间（24h、48h和72h）后，用流式细胞仪检测法测定黄荆子乙酸乙酯提取物作用不同时间后细胞的增殖和周期情况；采用RT-PCR和Western blotting检测黄荆子乙酸乙酯提取物作用不同时间后，CNE-1细胞内细胞周期相关蛋白p27和CDK2的表达情况。结果 1. CCK-8比色法结果显示：黄荆子乙酸乙酯提取物对CNE-1细胞增殖有显著的抑制作用，并且呈现出时间和剂量依赖性；其IC50值为2.5ug/ml。 2.流式细胞检测法结果显示：用2.5ug/ml黄荆子乙酸乙酯提取物处理CNE-1细胞不同时间后，CNE-1细胞的增殖受到明显抑制；G0/G1细胞比例明显增加，而S/G2/M期细胞明显减少，呈时间依赖性。 3. RT-PCR结果显示：用2.5ug/ml黄荆子乙酸乙酯提取物处理CNE-1细胞不同时间后，CNE-1细胞中G1-S期抑制蛋白p27mRNA表达增高，而G1-S期促进蛋白CDK2mRNA表达降低，呈时间依赖性。 4. Western blotting结果显示：用2.5ug/ml黄荆子乙酸乙酯提取物处理CNE-1细胞不同时间后，CNE-1细胞中G1-S期抑制蛋白p27蛋白表达增高，而G1-S期促进蛋白CDK2蛋白表达降低，呈时间依赖性。结论 1.黄荆子乙酸乙酯提取物（Evn-50）具有明显抑制CNE-1细胞增殖的作用，其抑制作用呈时间-剂量依赖性。 2.黄荆子乙酸乙酯提取物抑制CNE-1细胞增殖可能与通过阻断细胞周期（G1-S期）进展有关。 3.黄荆子乙酸乙酯提取物抑制细胞增殖可能与其上调G1-S期抑制蛋白p27和下调G1-S期促进蛋白CDK2表达有关。
[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.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R285.5;R739.63

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