靶向核仁素干扰、端粒损伤对胶质母细胞瘤生长抑制及其机制的研究

发布时间：2018-04-03 21:56

本文选题：核仁素　切入点：端粒　出处：《吉林大学》2017年博士论文

【摘要】：脑胶质瘤(glioma)是神经系统一种常见的原发肿瘤,其特点为恶性程度高,预后差,手术治疗存在复发风险性,对化疗治疗具有一定耐药性。研究表明,胶质瘤细胞存在端粒酶和核仁素(nucleolin,NCL)的过表达现象,并且成为了胶质瘤各种恶性特征和治疗抵抗的基础。核仁素普遍存在于真核细胞中,调控核糖体聚集、DNA代谢和RNA表达等生物活动。核仁素调控m RNA的转录的特性是控制细胞生物活动的主要作用。核仁素由707个氨基酸残基组成,主要含有三个主要的功能结构域,主要为羧基端、氨基端和中心区。氨基酸调控c DNA转录,核糖体前体包装,中心区含有4个较为保守的、序列一致型RNA结合域(RNA-Binding Domain,RBD),羧基端富含甘氨酸残基,可以使RNA更容易接近中心区的RNA结合域结构。核仁素的RBD具有特异性结合一系列m RNA的能力。包括Bcl-2,p53等调控细胞凋亡的蛋白。AS1411是一种富含26个单位的核酸配适体,目前被用于临床II期实验进行肿瘤治疗实验。其具有富有鸟嘌呤(G)结构的序列,具有易于吸收且不被核酶降解等优点,因此具有治疗肿瘤的潜能。端粒(telomere)位于染色体末端,端粒酶可保护端粒酶的降解因而具有保护染色体的作用。目前有报道证实AS1411可以竞争性结合核仁素的RBD,因此具有可以干扰核仁素调节蛋白的能力。三氧化二砷(As2O3)是一种古老的药物,俗称砒霜,我国自古就有应用砒霜等含砷的中药来治疗多种顽固疾病如哮喘等等,近年来被逐渐应用于肿瘤的治疗。As2O3可以抑制端粒酶从而产生对细胞的生物学效应。目前在胶质瘤中靶向干扰核仁素和端粒酶抑制对细胞生长抑制目前少有报道。但由于二者在恶性胶质瘤细胞中的高表达现象,因此我们选择分别利用AS1411干扰核仁素功能,As2O3抑制端粒酶活性,进而探索这两种药物通过作用两个关键蛋白而起到的胶质瘤细胞杀伤作用。最后,由于核仁素也可以调控端粒酶的表达,因此我们进一步探讨AS1411对核仁素的干扰进而造成端粒酶抑制等现象。在本文中,我们使用胶质瘤细胞系,胶质瘤组织,正常脑组织,星形胶质细胞以及胶质瘤种植小鼠作为实验对象,主要应用MTT,端粒酶活性TRAP assay,免疫印迹(western blot),免疫荧光,染色质免疫沉淀(Ch IP),si RNA基因沉默,Real time-RT-PCR,流式细胞术,衰老染色以及端粒荧光原位杂交分析等技术探究AS1411对核仁素的干扰,As2O3对端粒酶的抑制,进而造成相应的细胞凋亡,细胞周期阻滞,细胞衰老以及这些现象发生的主要机制。我们的主要实验结果如下:(1)AS1411可以通过与核仁素的RBD区域竞争性结合,上调p53以及下调Bcl-2和Akt1的表达。AS1411通过干扰核仁素上调p53和下调Bcl-2导致细胞的早期凋亡,AS1411通过下调Akt1抑制了胶质瘤细胞的侵袭和转移。(2)核仁素在胶质瘤组织和胶质瘤细胞中都过表达。AS1411通过干扰核仁素造成胶质瘤细胞的生长抑制,同时AS1411作用的体内实验显著减小了肿瘤的体积和延长了胶质瘤种植小鼠的生存期。(3)As2O3在胶质瘤细胞中产生了活性氧,因而造成了端粒酶的核质转移以及端粒酶磷酸化,进而造成了端粒酶活性抑制。(4)端粒酶的活性抑制造成的发生在染色体端粒部分的DNA损伤,并造成了ATM和ATR依赖途径的损伤应答。(5)As2O3诱导了胶质瘤细胞染色体末端融合,进一步研究发现,As2O3对脑胶质瘤端粒的3’悬端长度造成变化,出现融合缩短。(6)发生在端粒结构的DNA损伤又发了细胞凋亡,G2/M周期阻滞和细胞衰老现象的发生。(7)核仁素RBD的干扰导致了端粒酶活性的抑制,进而造成了细胞衰老等现象。结论:本文率先证实了在胶质瘤细胞中,AS1411靶向干扰核仁素可以造成p53的上调,Bcl-2和Akt1下调。利用As2O3抑制端粒酶活性可以造成ATM依赖的DNA损伤,进而引起胶质瘤细胞生长抑制,以及细胞凋亡,细胞损伤,周期阻滞,侵袭和迁移能力的抑制等等。
[Abstract]:Glioma (glioma) is a neurological primary tumor is common, characterized by high malignancy and poor prognosis, surgical treatment has the risk of recurrence, has a certain resistance to chemotherapy in the treatment of glioma cells. The study shows that the existence of telomerase and nucleolin (nucleolin, NCL) over expression of the phenomenon, and become the resistance of various malignant glioma characteristics and therapy. Nucleolin exists widely in eukaryotic cells, regulation of ribosome aggregation, DNA metabolism and the expression of RNA and other biological activities. The characteristics of transcriptional regulation of M nucleolin RNA is the main control cell biological activities. Nucleolin is composed of 707 amino acid residues, the main there are three major functional domains, mainly for the carboxyl terminal amino terminal, and the central region. The amino acid regulation of C DNA transcription, ribosomal precursor packaging, central area contains 4 of the more conservative sequence type RNA binding domain (RNA-B Inding Domain, RBD), carboxyl terminal glycine rich residues, can make the RNA more accessible to the central area of the RNA binding domain structure. Nucleolin RBD has a specific binding ability. A series of M RNA including Bcl-2, p53, cell apoptosis protein.AS1411 is a rich 26 single bit with nucleic acid aptamers, currently being used in phase II clinical trials in tumor treatment experiments. It has rich guanine (G) structure of the sequence is easy to absorb and not by ribozyme degradation. Thus, it has the potential for tumor treatment. Telomere (telomere) located on chromosome end, which can protect the degradation of telomerase telomerase have a protective effect on chromosome. There are reports indicating that AS1411 can compete with nucleolin RBD, so it can interfere with nucleolin regulated protein ability. Arsenic trioxide (As2O3) is a kind of ancient medicine, commonly known as arsenic, China There are ancient Chinese medicine containing arsenic arsenic to treat many kinds of stubborn diseases such as asthma and so on, in recent years has been gradually applied in the treatment of tumor.As2O3 can inhibit telomerase to produce biological effects on cells. At present in glioma targeting nucleolin and telomerase inhibition on cell growth inhibition was rarely reported. But because of the two in malignant glioma cells in high expression, so we choose respectively by AS1411 interference of nucleolin As2O3 function, inhibition of telomerase activity of glioma cells, and to explore the role of the two drugs through two key proteins and the killing effect. Finally, the expression of nucleolin can also regulate telomerase, therefore we further explore the AS1411 of nucleolin interference caused by telomerase inhibition phenomenon. In this paper, we use the glioma cell lines and glioma tissues, normal Brain tissue, astrocytes and glioma implant mice as experimental objects, the main application of MTT, the telomerase activity in TRAP assay, immunoblotting (Western blot), immunofluorescence, chromatin immunoprecipitation (Ch IP), Si RNA Real time-RT-PCR, gene silencing, flow cytometry, aging telomere staining and fluorescence in situ hybridization analysis these techniques explore AS1411 on nucleolin interference, inhibition of As2O3 on telomerase, resulting in cell apoptosis related, cell cycle arrest, cell senescence and the main mechanism of these phenomena. Our main results are as follows: (1) AS1411 can be combined by the RBD regional competition and nucleolin, upregulation of p53 and the expression of.AS1411 and down-regulation of Bcl-2 by Akt1 interference of nucleolin upregulation of p53 and downregulation of Bcl-2 apoptosis, AS1411 inhibits glioma cell invasion and metastasis through down-regulation of Akt1. (2) nuclear The kernel in glioma tissues and glioma cells in.AS1411 overexpression by interfering with nucleolin caused by glioma cell growth inhibition, and in vivo effect of AS1411 significantly reduced the tumor volume and prolong the survival time of the mice planting glioma. (3) As2O3 produced active oxygen in glioma cells. Thus telomerase and telomerase nuclear transfer phosphorylation, thus resulting in the inhibition of telomerase activity. (4) the activity of telomerase inhibition caused by DNA damage occurs in the part of the telomere, and caused ATM damage response and ATR dependent pathways. (5) As2O3 induced glioma cell chromosome end fusion, further study found that As2O3 on glioma telomere 3 'end length changes caused by suspension, the integration is shortened. (6) occurred in the DNA damage of telomere was cell cycle arrest and cell apoptosis, G2/M. Old phenomenon. (7) interference nucleolin RBD leads to inhibition of telomerase activity, thus resulting in the phenomenon of cell aging. Conclusions: This paper first confirmed in glioma cells, AS1411 targeting nucleolin can cause the upregulation of p53, Bcl-2 and Akt1. Downregulation of As2O3 can inhibit telomerase activity caused by ATM dependent DNA damage, thereby causing glioma cell growth inhibition, cell apoptosis and cell cycle arrest, injury, invasion and migration inhibition and so on.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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