【作者】 向斌；

【导师】 蒋宇钢；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 目的星形细胞胶质瘤是指以星形胶质细胞所组成的肿瘤,是最常见的神经上皮性肿瘤,约占胶质瘤的80%,且多数切除后有复发可能。目前,针对肿瘤细胞增殖、凋亡以及转移等方面的研究十分普遍,临床上普遍使用的抗肿瘤药物大都通过抑制肿瘤细胞的增殖和促进肿瘤细胞的凋亡来发挥作用的。自噬现象是指细胞自身消化和降解细胞器和蛋白质,重新利用氨基酸、核酸等小分子物质,以满足细胞新陈代谢的需要,以及更新某些细胞器的过程,也被称为Ⅱ型细胞死亡程序。自噬不仅可以维持细胞正常的生长、发育和分化,还可以保持细胞内的稳态,研究发现自噬具有特殊的调控通路,该调控通路的异常与肿瘤的发生有着直接的关系。继利用细胞凋亡之后,诱导自噬性细胞死亡已经成为抑制肿瘤增殖的新途径。同时自噬不仅可以启动细胞主动性的Ⅱ型细胞死亡程序,并可以通过平衡细胞内的合成与分解代谢,继而稳定细胞内环境,保护细胞防止周围环境带来的损害,维持细胞的存活。因此,利用药物诱导肿瘤细胞发生自噬并非总是引起细胞死亡,而是可能出现两种不同的结果。雷帕霉素作为mTOR的抑制剂,可诱导自噬发生,而且雷帕霉素在诱导细胞自噬发生的同时,也会增加细胞的凋亡。自噬与凋亡是两种不同的细胞死亡过程,PI3K/Akt/mTOR信号通路调控着肿瘤的生长和发展,且在多种瘤组织中过度激活表达,mTOR是P13K/Akt通路的下游分子,是控制包括胶质瘤在内的细胞生长、增殖及周期调节的中心环节。本研究试图探明雷帕霉素在抑制mTOR活性同时,是否反馈调节PI3K/Akt信号通路的蛋白表达,从而从细胞信号途径揭示雷帕霉素抑制U251增殖作用的分子机理。同时通过设置不同的雷帕霉素作用时间,观察其抑癌效应是否与作用时间有关,揭示作用时间与PI3K/Akt/mTOR信号传导通路蛋白表达的相关性。是否同时存在细胞凋亡率和细胞周期的改变,以探明雷帕霉素抑制U251细胞增殖能力(抗癌效应)的机理。方法1.用100nM的雷帕霉素处理人星形胶质瘤U251细胞分别在0、6、12、24h检测磷酸化的PI3K/AKT/mTOR蛋白分子的表达水平。2.IF检测LC3B的表达水平以评价自噬发生的水平。3.MTT法检测100nM雷帕霉素在随着时间的延长对U251细胞增殖能力的影响,OD570nm检测吸光度值,通过OD570nm吸光度值计算细胞存活率。4.通过FCM检测100nM雷帕霉素在随着时间的延长对U251细胞凋亡水平的影响。5.通过收集不同时间点的100nM雷帕霉素处理过的U251细胞,进行流式细胞术检测。结果1.用western blotting分析100nM的雷帕霉素处理人星形胶质瘤U251细胞分别在0、6、12、24h检测磷酸化的PI3K/AKT/mTOR蛋白分子的表达水平。随着处理时间的延长,PI3K的表达水平未有显著的升高降低趋势,AKT/mTOR的表达水平随着处理时间的延长均显著降低。2.免疫荧光发现随着处理时间的延长,LC3B的表达灰度值是不断升高的。3. MTT检测发现随着处理时间的延长,细胞的存活率呈下降的趋势。4.用流式细胞术检测发现,随着时间的延长,细胞的凋亡率呈上升的趋势。5.用流式细胞术检测发现,随着时间的延长,细胞的早起凋亡率呈上升的趋势。结论1.雷帕霉素可以有效抑制星形细胞胶质瘤U251细胞的增殖,使细胞停滞于G1期。2.雷帕霉素促进了U251细胞的自噬。3.雷帕霉素诱导了细胞凋亡,其机制涉及抑制了PI3K/Akt/mTOR信号通路。更多还原

【Abstract】 Objective Astrocytoma, which is composed of astrocytes, is the most common neuroepithelial tumor, and takes up80%of gliomas. The majority of astrocytoma may recur after the removal. At present, the study of tumor cell proliferation, apoptosis and metastasis is very common. In clinical, the commonly used anticancer drugs play a role through inhibition of tumor cell proliferation as well as apoptosis. Autophagy, also known as type Ⅱ cell death, is kind of cell self-digestion, degradation of organelles and proteins, re-use of small molecules such as amino acids, nucleic acids, in order to meet the needs of the cell metabolism, and to update some organelles. Autophagy is able to maintain normal cell growth, development and differentiation, to maintain the steady state intracellular. Studies have proved that there exists a special regulatory pathway involving in autophagy and it has a direct relationship with tumors. After the use of apoptosis, induced autophagic cell death has become a new way to inhibit tumor proliferation. At the same time, autophagy can not only initiate type Ⅱ cell death program, but also balance intracellular synthesis and catabolism, thereby stabilizing the intracellular environment, protecting cells against damage from the surrounding environment, and maintaining cell survival. Therefore, the use of drugs to induce tumor cell autophagy does not always caused cell death, but may leads to two different results. Rapamycin, as an inhibitor of mTOR, can induce the cell autophagy, and at the same time increase the cell apoptosis. Autophagy and apoptosis are two different cell death processes. PI3K/Akt/mTOR signaling pathway involves in tumor growth and development, which is excessively activated in a variety of tumors.mTOR, as a downstream molecule of the PI3K/Akt pathway, plays a central role in controlling the growth, proliferation and cycle of cells including astrocytoma. This study attempts to explore the feedback regulation of PI3K/Akt signaling pathway by rapamycin when inhibiting the activity of mTOR, in order to reveal the molecular mechanism of rapamycin in inhibiting U251proliferation from the aspect of signaling pathways.At the same time, this study attempts to revealthe relationship between the time and the PI3K/Akt/mTOR signal pathway by setting different action times of rapamycin and observing its tumor suppressor effect. We also wonder whether the cell apoptosis and the change of cell cycle exist at the same time, in order to reveal its mechanism of inhibition of U251cell proliferation (anti-cancer effects).Method100nM rapamycin was used to treat human astrocytoma U251cells, and the protein expressions of PI3K/AKT/mTOR were detected at0,6,12,24h respectively. IF was used to detect the LC3B expression in order to evaluate autophagy.MTT was performed to analyze in the proliferation capacity of U251cells treated by100nM rapamycin with time, and OD570nm absorbance value was used to determine the rate of cell survival. FCM was used to analyze the apoptosis levels of U251cells treated by100nM rapamycin at different time points.Result Western blotting was used to detect the expression levels of PI3K/AKT/mTOR protein in astrocytoma U251cells treated by100nM rapamycin at0,6,12,24h respectively. With the treatment time, the expression level of PI3K has no significant increase whilethe expression levels of AKT and mTOR weresignificantly decreased.Immunofluorescence result showed that with the treatment time, the expression of LC3B was increased gradually. MTT assay demonstrated that with the treatment time, the survival rate of U251cells showed a downward trend. Flow cytometry showed that with the treatment time, the apoptosis rate and the early apoptosis rate of U251cells showed an upward trend.ConclusionRapamycin can inhibit the proliferation of astrocytoma U251cellsand arrest them at G1phase.Rapamycin can promote the cell autophagy of U251cells.Rapamycin can induce cell apoptosis of U251cells, and its mechanism involves the inhibition of PI3K/Akt/mTOR signaling pathway.更多还原