自噬相关蛋白4EBP1与肿瘤发生相关性的初步研究

发布时间：2018-02-14 17:14

本文关键词： 脑瘤 4EBP1 RNAi 细胞增殖　出处：《山东大学》2016年硕士论文　论文类型：学位论文

【摘要】：mTOR (the mammalian target of rapamycin)信号通路作为细胞内最重要的信号信号转导途径之一,在调节细胞的分裂、增殖等方面至关重要。其中,蛋白4EBP1和eIF4E是该信号转导通路中的重要组成部分,己被证明在多种肿瘤细胞中表达,可作为多种肿瘤发生的分子标志以及肿瘤治疗的靶标。有关4EBP1和eIF4E两种蛋白在中国人常见的脑肿瘤中表达的研究比较少。因此,我们首先探讨了4EBP1和eIF4E在脑膜瘤、星形细胞瘤、胆脂瘤和少突胶质细胞瘤组织中的表达及其差异性。研究结果显示,四种脑部肿瘤组织中均有4EBP1和eIF4E mRNA和蛋白的表达,同时也检测到自噬标志蛋白LC-3和LAMP-2A的高表达。免疫组化结果表明,与正常脑组织相比,4EBP1和eIF4E两种蛋白在四种脑部肿瘤组织中的表达量明显增高,结合其他文献和本实验结果可以看出,4EBP1、eIF4E也可作为脑部肿瘤发生的分子标志之一。利用Hep-2细胞为实验材料,采用RNAi方法敲低Hep-2细胞中4EBP1蛋白的表达量,然后检测了4EBP1敲低对细胞增殖、细胞周期进程等的影响,结果显示,在转染有效4EBP1 siRNA敲低4EBP1的表达后,细胞内p-4EBP1的含量也同步性地减少；转染有效4EBP1 siRNA 48h-72h后,Hep-2细胞的增殖被明显抑制,主要表现为DNA复制活性降低,S期以及S期以后的G2、M期的细胞数目减少；推测敲低细胞内4EBP1表达引起p-4EBP1含量的同步性减少,导致了细胞内游离的eIF4E降低,从而抑制了细胞增殖和细胞周期进程。利用敲低Hep-2细胞中4EBP1蛋白表达量联合5-氟尿嘧啶(5-FU)处理的方法,研究了4EBP1下调与低剂量的5-FU对细胞增殖以及细胞周期改变是否有协同作用。初步的研究结果显示,与单独敲低或单独5-FU处理相比,两者联合实验可使S期细胞数占总细胞数的比例有更显著的增加,说明二者联用加重了细胞的S期阻滞,为将来继续探讨5-FU与自噬抑制相结合,通过抑制自噬使肿瘤细胞增强对抗肿瘤药物的敏感性、开拓抗肿瘤研究新思路提供理论和实验基础。
[Abstract]:As one of the most important signal transduction pathways in cells, mTOR / the mammalian target of rapamycin signaling pathway plays an important role in regulating cell division and proliferation, among which protein 4EBP1 and eIF4E are important components of the signal transduction pathway. It has been proved that it can be expressed in many kinds of tumor cells and can be used as a molecular marker for many kinds of tumorigenesis and as a target for tumor treatment. There are few studies on the expression of 4EBP1 and eIF4E in common brain tumors in Chinese. We first investigated the expression and difference of 4EBP1 and eIF4E in meningioma, astrocytoma, cholesteatoma and oligodendrocytoma. Meanwhile, the high expression of autophagy marker protein LC-3 and LAMP-2A was also detected. The results of immunohistochemistry showed that the expression of eIF4E and 4EBP1 proteins were significantly higher in four kinds of brain tumors than in normal brain tissues. In combination with other literatures and the results of this study, we can see that 4EBP1eIF4E can also be used as one of the molecular markers of brain tumorigenesis. Using Hep-2 cells as experimental materials, the expression of 4EBP1 protein in Hep-2 cells was reduced by RNAi method. Then the effects of 4EBP1 knockout on cell proliferation and cell cycle progression were examined. The results showed that after transfection of 4EBP1 siRNA knockout 4EBP1, the content of p-4EBP1 decreased synchronously. The proliferation of Hep-2 cells was significantly inhibited after transfection of 4EBP1 siRNA 48h-72 h, mainly manifested by the decrease of DNA replication activity in S phase and the number of cells in G2M phase after S phase, suggesting that 4EBP1 expression in knockout cells caused the simultaneous decrease of p-4EBP1 content. It resulted in the decrease of intracellular free eIF4E, which inhibited cell proliferation and cell cycle progression. The method of low expression of 4EBP1 protein combined with 5-FU of 5-fluorouracil was used to reduce the expression of 4EBP1 protein in Hep-2 cells. The effects of 4EBP1 down-regulation and low-dose 5-FU on cell proliferation and cell cycle change were studied. The combination of the two methods can increase the proportion of S phase cells to the total number of cells significantly, which indicates that the combination of the two methods has aggravated the S phase arrest of the cells and further explored the combination of 5-FU and autophagy inhibition in the future. Inhibition of autophagy enhances the sensitivity of tumor cells to antitumor drugs and provides a theoretical and experimental basis for the development of a new approach to antitumor research.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.2

【相似文献】