论文标题:DADS对人鼻咽癌细胞系CNE2周期阻滞及组蛋白乙酰化的研究
Arrest Effect of Diallyl Disulfide on Cell Cycle in Human Nasopharyngeal Carcinoma Cell Line CNE2
论文作者
论文导师 苏琦,论文学位 硕士,论文专业 病理学与病理生理学
论文单位 南华大学,点击次数 60,论文页数 49页File Size1525K
2007-05-01论文网 http://www.lw23.com/lunwen_444083842/
diallyl disulfide;; nasopharyngeal carcinoma;; CNE2 cell line;; histone acetylation
目的:在前期工作的检测上,进一步探讨二烯丙基二硫(diallyl disulfide, DADS)对人鼻咽癌CNE2细胞周期的阻滞作用,并研究组蛋白乙酰化状态及其调控p21WAF1蛋白表达改变的情况。 方法:MTT实验、细胞计数法观察不同浓度和作用时间DADS对CNE2细胞的生长抑制作用;HE染色观察不同浓度和作用时间DADS对体外培养的CNE2细胞形态的影响;流式细胞术分析不同浓度和作用时间DADS对CNE2细胞的周期分布;免疫印迹分析不同浓度DADS诱导CNE2细胞组蛋白H3和H4乙酰化及周期相关蛋白P21WAF1表达变化。 结果:MTT比色法检测显示,DADS能明显抑制CNE2细胞增殖,呈浓度和时间依赖性,90μmol/L,140μmol/L,240μmol/L和400μmol/L DADS处理48h后,对CNE2细胞增殖的抑制率分别为3.33%、12.90%、28.38%和56.90%,与对照组相比,差异有显著性意义(P<0.05)。细胞计数结果表明,常规培养的CNE2细胞群体倍增时间为24.51h,当DADS的浓度由90μmol/L增加到400μmol/L时,其细胞群体倍增时间由27.68h延长到93.10h,与对照组比较,差异有显著性意义(P<0.05)。细胞形态学观察显示:90μmol/L,140μmol/L,240μmol/L和400μmol/L DADS处理细胞48后,与对照组细胞相比,异型性明显减少,核浆比例明显减小。流式细胞仪分析结果提示:DADS呈浓度依赖性将CNE2细胞阻滞在G0/G1期。Western blot分析,不同浓度的DADS处理CNE2细胞48h后,组蛋白H3乙酰化的表达均较对照组有增加,伴有药物浓度依赖性,90、140、240和400μmol/L处理分别比NS对照组增加20%、32%、42%和30%,差异有显著性(P<0.05);与SB对照组比较时,除240μmol/L DADS处理组外,其余组均有差异显著(P<0.05)。同期实验发现,组蛋白H4乙酰化程度较对照组差异无显著性;p21WAF1蛋白表达量随着DADS浓度升高而上升。 结论: 1. DADS对CNE2细胞有抑制增殖作用,呈剂量依赖效应。 2. DADS对CNE2细胞的抑制增殖的机制,与组蛋白H3乙酰化水平提高, P21WAF表达上调,将细胞阻滞在G0/G1期有关。 3. DADS可能是一种潜在的组蛋白去乙酰化酶抑制剂。
Objective: To investigate the arrest effect of diallyl disulfide (DADS) in the cell cycle of human nasopharyngeal carcinoma CNE2 cell line and its molecular mechanism. Methods:The growth inhibition effect of DADS on CNE2 cell line was measured by MTT assay and cell counting. Light microscope was employed to observe the morphlogical change after DADS treatment. Phase distribution of cell cycle was analyzed by flow cytometry. Expression of H3, H4 and P21WAF1 were determined by western blotting analysis. Results: The MTT assay showed that DADS inhibited growth of CNE2 cells significantly and exhibited a dose dependent modal. Adding 90μmol/L,140μmol/L,240μmol/L and 400μmol/L DADS for 48 hours suppressed CNE2 growth by 3.33%、12.90%、28.38%、56.90%, respectively. Cell counting showed that average doubling time retarded from 24.51h in normal cultured CNE2 cells to 93.10h in 400μmol/L DADS experimental CNE2 cells (P<0.05). Light microscope examination indicated that heteromorphism and karyoplasmic ratio were decreased when treated with 90μmol/L,140μmol/L,240μmol/L and 400μmol/L DADS for 48 hours in a dose and time dependent. Flow cytometry analysis revealed that treating CNE2 cells with increasing quantities of DADS increased the percentage of cells in the G0/G1 phase. By western blot, the acetylation level of histone H3 in CNE2 cell line was elevated after treated by DADS, and the maximum effect was higher 42% than untreated cells at 48h. At the same time,the expression of p21WAF1 also increased in a dose dependent. But the acetylation level of histone H4 did not changed. Conclusion: 1. DADS had a role in inhibiting the proliferation of CNE2 cell line in a dose dependent. 2. The antiproliferation property of diallyl disulfide (DADS) in cultured human nasopharyngeal carcinoma CNE2 cell line relates to its ability to arrest G0/G1 through over-expression of P21WAF1. 3. DADS might be a potential deacetylation drug.
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