靶向端粒G-四链体配体诱导胶质瘤端粒紊乱和端粒酶移位的机制研究

发布时间：2018-05-03 21:41

本文选题：胶质瘤 + 端粒　；参考：《吉林大学》2016年博士论文

【摘要】：恶性胶质瘤是最常见的人类神经系统原发性恶性肿瘤,同时也是造成儿童和青少年“肿瘤相关死亡”的第二大恶性肿瘤。胶质母细胞瘤是恶性胶质瘤中相对较常见的一种病理类型,而且还是恶性度最高,发展速度最快,侵袭性最强的恶性胶质瘤。目前的研究表明胶质母细胞瘤对于术后放化疗的敏感性也是最弱的。胶质母细胞瘤是预后极差的一种恶性胶质瘤,统计显示,诊断为胶质母细胞瘤的患者,经过手术治疗和联合放化疗治疗后的平均生存期不足18个月。人类细胞的端粒结构位于染色体末端,由重复的TTAGGG序列构成,作用是帮助细胞分辨正常的染色体末端和双链DNA损伤。人类端粒的3′悬端是由一个富G序列单链DNA构成。3′悬端的DNA序列有两种形态,当DNA序列打开时,能够与端粒酶结合,进行端粒DNA的延长反应。而当单链DNA关闭时,形成了所谓的T-loop结构和D-loop结构,主要是为了保持端粒末端的完整性。如果能够采用措施使端粒末端维持一种打开状态,那么就会导致端粒结构的紊乱,表现为染色体末端融合、后期桥和端粒末端降解,最后导致细胞走向凋亡和衰老。恶性肿瘤细胞最显著的标志是长度永远不缩短的端粒DNA,从而使细胞拥有无限增殖的能力。研究证明大约85%的人恶性肿瘤细胞中表达处于激活状态的端粒酶来维持端粒长度。这些研究为制定靶向端粒酶结构的抗肿瘤治疗方案的可行性提供了依据。端粒酶抑制剂能够与端粒3'悬端单链DNA有效结合形成稳定的G-quadruplex四链体结构,抑制端粒酶催化的端粒合成反应和帽状结构的形成。这种有效抑制端粒延长的作用使得端粒酶抑制剂成为很有潜力的化疗药物选择。虽然端粒酶抑制剂是一种很有潜力的抗癌治疗药物,但是传统的端粒酶抑制剂的弊端在于,达到能够诱导细胞走向凋亡和衰老的端粒DNA缩短所需要的时间过长。而且,有研究显示,部分端粒DNA配体作用于恶性肿瘤细胞后,产生的端粒DNA缩短效果会诱导恶性肿瘤细胞激活体内的端粒DNA长度延长机制,进而维持恶性肿瘤细胞内的端粒长度。这也是阻碍端粒酶抑制剂成为临床用药的重要因素。BRACO-19是结合端粒形成G-quadruplex结构最有效的端粒DNA配体之一,研究显示它具有显著的抑制端粒酶活性和抗肿瘤作用。然而,G-quadruplex配体分子作用于端粒结构的研究仍处于临床前阶段,因此,确定其潜在反应机制仍是主要的挑战性课题。在本论文中,我们主要应用TRAP assay,免疫荧光,western blotting,染色质免疫沉淀(Ch IP)以及端粒荧光原位杂交分析BRACO-19作用后产生的端粒酶的活性抑制,端粒结构紊乱及细胞增殖的抑制。本论文的主要观点如下:1、我们发现BRACO-19能显著抑制端粒酶活性,选择性抑制胶质瘤细胞增殖,对于普通胶质细胞的抑制作用不明显。BRACO-19可以明显的诱导人胶质母细胞瘤细胞内端粒酶出现核质转运效应。进一步研究发现BRACO-19诱导胶质瘤细胞出现的增值抑制与细胞内出现的DNA损伤反应有密切的联系。而且,我们证实了进一步证实BRACO-19诱导的DNA损伤反应发生在端粒上;2、BRACO-19诱导了TRF2和POT1(端粒结合蛋白)从端粒上解离,这一结论间接证明了胶质瘤细胞内G-quadruplex形成及T-loop结构解散。进一步研究发现,BRACO-19作用后的胶质瘤细胞出现了明显的端粒DNA 3′-悬端的缩短;3、BRACO-19诱导胶质瘤细胞出现了细胞周期阻滞,细胞凋亡和衰老,这可能是端粒结构紊乱的结果,而且p53和p21蛋白的上调与这些生物学效应有密切的联系;4、BRACO-19作用胶质瘤细胞后出现了端粒融合和后期桥形成等染色体异常现象,这是端粒结构紊乱的结果。通过端粒-末端脱氧核糖核酸转移酶(Td T)的共定位分析证实了BRACO-19作用后胶质瘤细胞出现的端粒去保护状态。本文率先证明了BRACO-19结合端粒从而抑制端粒酶活性对于脑肿瘤细胞的生长抑制作用,单独用药或者联合其他治疗方法,比如放射性治疗,可以为脑胶质母细胞瘤以至其他种类的恶性肿瘤的治疗提供新的依据。
[Abstract]:Malignant glioma is the most common primary malignant tumor of the human nervous system. It is also the second major malignant tumor causing "tumor related death" in children and adolescents. Glioblastoma is a relatively common pathological type of malignant glioma, and it is also the most malignant, fast and aggressive evil. The present study shows that the sensitivity of glioblastoma to postoperative chemoradiotherapy is also the weakest. Glioblastoma is a malignant glioma with poor prognosis. Statistics show that the average survival time of patients diagnosed as glioblastoma is less than 18 months after surgical treatment and combined radiotherapy. Human cells The telomere structure is located at the end of the chromosome and consists of a repeat TTAGGG sequence that helps the cells to distinguish between the normal chromosome ends and the double strand DNA damage. The 3 'end of the human telomere is a DNA sequence consisting of a.3' suspended from a single strand DNA rich in G sequence. When the DNA sequence is opened, it can be combined with telomerase to carry out telomere D. When the single strand DNA is closed, the so-called T-loop structure and D-loop structure are formed, mainly to maintain the integrity of the telomere end. If the end of the telomere end is maintained, the telomere structure disorder, the fusion of the end of the chromosome, the end of the terminal bridge and the end of the telomere will be reduced. The most significant sign of malignant tumor cells is that the telomere DNA, which is never shortened in length, makes the cells have the ability to proliferate indefinitely. Studies have shown that about 85% of human malignant tumor cells express telomerase in active state to maintain telomere length. These studies are designed to formulate target telomere. The enzyme structure provides a basis for the feasibility of antitumor therapy. Telomerase inhibitors can effectively bind to the telomere 3'suspension single strand DNA to form a stable G-quadruplex four chain body structure, inhibit telomerase catalyzed telomere synthesis and cap formation. This effective inhibition of telomere prolongation makes telomerase inhibitors Although telomerase inhibitors are a potential anticancer drug, the disadvantage of traditional telomerase inhibitors is that it takes a long time to achieve the telomere DNA shortening that can induce cell apoptosis and aging. Furthermore, some studies have shown that some telomere DNA ligands act on evil. After the tumor cells, the telomere DNA shortening can induce the telomere DNA length extension mechanism in the malignant tumor cells, and then maintain the telomere length in the malignant tumor cells. This is also an important factor hindering telomerase inhibitors from the clinical use of.BRACO-19 is the most effective end of the binding telomere to form the G-quadruplex structure. One of the grain DNA ligands, research shows that it has a significant inhibition of telomerase activity and anti-tumor effect. However, the study of G-quadruplex ligand molecules on telomere structure is still in the preclinical stage. Therefore, it is still a major challenge to determine its potential reaction mechanism. In this paper, we mainly use TRAP assay, immunofluorescence, w Estern blotting, chromatin immunoprecipitation (Ch IP) and telomere fluorescence in situ hybridization analysis of the activity inhibition of telomerase, disorder of telomere structure and inhibition of cell proliferation. The main points of this paper are as follows: 1, we found that BRACO-19 can significantly inhibit telomerase activity and selectively inhibit the proliferation of glioma cells. The inhibitory effect of common glial cells is not obvious.BRACO-19 can obviously induce the nuclear transport effect of telomerase in human glioblastoma cells. Further studies have found that the proliferation inhibition of BRACO-19 induced glioma cells is closely related to the DNA damage reaction in cells. Furthermore, we have confirmed the further evidence. The actual BRACO-19 induced DNA damage reaction occurred on the telomere; 2, BRACO-19 induced the dissociation of TRF2 and POT1 (telomere binding protein) from the telomere, which indirectly demonstrated the formation of G-quadruplex in glioma cells and the dissolution of the T-loop structure. Further studies showed that the telomere DNA 3 '- a distinct telomere of the glioma cells after BRACO-19 was used. 3, BRACO-19 induced cell cycle arrest, apoptosis and senescence in glioma cells, which may be the result of the disorder of telomere structure, and the up regulation of p53 and p21 proteins is closely related to these biological effects; and 4, after the action of BRACO-19 glioma cells, there are telomere fusion and late bridge formation of chromosomes. Common phenomenon is the result of telomere structure disorder. Through co localization analysis of telomere terminal deoxyribonucleic acid transferase (Td T), the telomere deactivation state of glioma cells after BRACO-19 action is confirmed. This article is the first to prove that BRACO-19 binding telomeres can inhibit the growth inhibition of telomerase activity to brain tumor cells. Alone or in combination with other treatments, such as radiation therapy, can provide a new basis for the treatment of glioblastoma and other types of malignant tumors.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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