吴茱萸碱对人胶质瘤U251细胞增殖及凋亡影响的研究

发布时间：2018-05-31 14:23

本文选题：吴茱萸碱 + 胶质瘤　；参考：《苏州大学》2014年硕士论文

【摘要】：胶质瘤是中枢神经系统中最常见的颅内肿瘤，并且有着很高的致死率。与此同时，它有较高的侵袭性且不易于治疗。对胶质瘤的各种临床治疗仅仅只能延长患者几个月的生存期，预后极其不佳。因此，发掘一个新的抗癌药物对于胶质瘤的治疗至关重要。传统中药用于治疗肿瘤已有很长一段时间，，大量研究表明许多中药的提取物或者他们的混合物在体内外都有抗癌作用。吴茱萸碱是从吴茱萸的中提取的主要生物碱，它具有抑制肿瘤生长和转移等多种生物学特性。然而吴茱萸碱对胶质瘤的作用及其作用机制均不明确。目的研究吴茱萸碱（Evodiamine)对人胶质瘤U251细胞增殖和凋亡的影响及其可能的作用机制。方法体外培养人胶质瘤U251细胞，并将其分为空白对照组及25、50、100μg.mL-1吴茱萸碱4组。应用MTT法检测吴茱萸碱在24、48和72h对U251细胞的增殖抑制作用；Hoechst33258荧光染色法检测吴茱萸碱诱导胶质瘤U251细胞凋亡；采用AnnexinV-FITC/PI双染法检测各组24h细胞早期凋亡率；Western blot法分析凋亡相关蛋白的变化。结果与空白对照组同期比较，25、50、100μg.mL-1吴茱萸碱组生长抑制率在24、48、72h均增加，随着吴茱萸碱浓度增加和作用时间延长，细胞增殖抑制越明显，其抑制作用呈现剂量依赖性和时间依赖性，差异有统计学意义(P＜0.05)。Hoechst33258荧光染色显示吴茱萸碱作用24h后U251细胞出现典型的细胞凋亡特征，各处理组均可见凋亡小体。U251细胞凋亡小体数量随着吴茱萸碱浓度的增加而逐渐增多，且碎裂的颗粒逐渐变小。随着吴茱萸碱浓度增加和作用时间延长，细胞的凋亡率逐渐升高。与空白对照组自发早期凋亡率3.12%比较，25、50、100μg.mL-1吴茱萸碱组早期凋亡率分别为8.65%、19.47%及28.97%，差异均有统计学意义(P＜0.05)。Westernblot实验显示，与空白对照组同期比较，25、50、100μg.mL-1吴茱萸碱上调了Fas、FADD、Caspase-8及Caspase-3蛋白表达，Bcl-2蛋白表达明显下降，Bax蛋白表达明显上升，差异均有统计学意义(P＜0.05)。上述指标均呈时间和剂量依赖性。结论吴茱萸碱对U251细胞具有明显的抑制细胞增殖和促进细胞凋亡的作用，其机制可能与上调Fas途径和下调Bcl-2/Bax有关。
[Abstract]:Glioma is the most common intracranial tumor in the central nervous system and has a high mortality. At the same time, it is highly invasive and difficult to treat. The clinical treatment of glioma can only prolong the survival of patients for a few months, and the prognosis is extremely poor. Therefore, the discovery of a new anti-cancer drug is essential for the treatment of gliomas. Traditional Chinese medicine has been used to treat cancer for a long time. A lot of studies have shown that many extracts of traditional Chinese medicine or their mixture have anticancer effect in vivo and in vitro. Rutaecarpine is the main alkaloid extracted from Evodia rutaecarpa. It has many biological characteristics such as inhibiting tumor growth and metastasis. However, the effect of rutaecarpine on glioma and its mechanism are not clear. Aim to study the effect of Evodiamine on proliferation and apoptosis of human glioma U251 cells and its possible mechanism. Methods Human glioma U251 cells were cultured in vitro and divided into control group and 2550100 渭 g.mL-1 rutaecarpine group. The inhibitory effect of evodiamine on U251 cells was detected by MTT method. Hoechst33258 fluorescence staining was used to detect the apoptosis of U251 glioma cells induced by evodiamine. The changes of apoptosis-related proteins were detected by AnnexinV-FITC/PI double staining and Western blot assay. Results compared with the control group, the growth inhibition rate of Evodia officinalis 2550100 渭 g.mL-1 group increased at 24: 48 h. With the increase of Evodia rutaecarpine concentration and the prolongation of the action time, the cell proliferation inhibition was more obvious, and the inhibitory effect was in a dose-and time-dependent manner. The difference was statistically significant (P < 0.05).Hoechst33258). The typical apoptotic characteristics of U251 cells were observed after Evodipine treatment for 24 hours. The number of apoptotic corpuscles in U251 cells increased with the increase of Evodia rutaecarine concentration in all treatment groups. And the broken particles gradually become smaller. With the increase of Evodia officinalis concentration and the prolongation of the action time, the apoptosis rate increased gradually. Compared with the control group, the spontaneous early apoptosis rate was 3.12%. The early apoptotic rate was 8.65%, 19.47% and 28.97% respectively in the 25 ~ 50100 渭 g.mL-1 rutaecarpine group. The difference was statistically significant (P < 0.05).Westernblot). Compared with the control group, Evodia rutaecarcinae 2550100 渭 g.mL-1 upregulated the expression of Caspase-8 and Caspase-3 protein of Fas-FADDN, and the expression of Bax protein increased significantly (P < 0.05). All the above indexes were in a time and dose dependent manner. Conclusion rutaecarpine can inhibit cell proliferation and promote cell apoptosis in U251 cells, and its mechanism may be related to up-regulation of Fas pathway and down-regulation of Bcl-2/Bax.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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