转录共激活因子TAZ在胶质瘤细胞增殖中的作用及其机制研究

发布时间：2018-04-03 17:07

本文选题：胶质瘤　切入点：转录共激活因子　出处：《西南大学》2017年硕士论文

【摘要】：神经胶质瘤是中枢神经系统最常见的和致命原发性肿瘤,约占中枢神经系统恶性肿瘤的80%。根据生长速度和对邻近脑组织的浸润程度,胶质瘤可分为低级别(世界卫生组织(WHO)I级和II级),以及高级别(WHO III级和IV级)[3]。即使经过目前最有效的治疗方法如手术、放疗、联合化疗等治疗后,高级别胶质瘤病人的中位生存期仍不容乐观(14.6个月)。这可能是由于胶质瘤的具体特性,如高度浸润性和缺乏明确的边界等引起的。此外,血脑屏障(BBB)的物理和化学约束也是胶质瘤综合治疗的主要障碍。因此,研究恶性胶质瘤形成的分子机制,寻找恶性胶质瘤新的药物靶点是迫切需要的。转录共激活因子TAZ是哺乳动物Hippo通路下游最主要的效应因子之一,包含400个氨基酸,其结构由一个TEAD结合区域(TB),一个WW区域,一个卷曲螺旋结构,一个转录激活区域,以及C端的一个PDZ结合模序组成。TAZ参与其下游靶因子的转录共激活过程,调控细胞的增殖、凋亡以及迁移等多种生理过程。近年来研究发现,在胃癌、非小细胞肺癌、乳腺癌及神经母细胞瘤等多种恶性肿瘤中TAZ的表达均高于正常癌旁组织。肿瘤细胞的恶性增殖往往和细胞周期进程的改变密切相关,EGFR信号通路在此过程中发挥着至关重要的作用。EGFR属于ErbB受体家族,在哺乳动物上皮细胞、成纤维细胞、胶质细胞、角质细胞等细胞表面均有广泛分布,EGFR及其下游信号通路对细胞的生长、增殖和分化等过程至关重要。EGFR一经磷酸化激活,其下游的PI3K/AKT和Ras-ERK通路随即被激活,进而加速细胞周期进程。研究发现,TAZ能够通过促进EGFR配体双调蛋白(AREG)的表达,进而促进乳腺癌细胞MCF10A不依赖于生长因子(EGF)增殖。但是,TAZ对于胶质瘤细胞的增殖的影响的研究还尚未有报道。本文旨在研究转录共激活因子TAZ对胶质瘤细胞增殖的影响及其可能的分子机制,通过实验取得如下结果:1.TAZ对胶质瘤的影响及表达分析我们通过查询数据库及基因芯片检测TAZ与胶质瘤病人预后的关系,及TAZ在不同级别的胶质瘤中的表达情况,结果表明TAZ的表达情况与胶质瘤病人的预后密切相关,胶质瘤级别越高,TAZ的表达量越高。利用Western blot技术检测TAZ在多种不同胶质瘤细胞系中的表达情况,结果显示TAZ在U251、LN229、U87、A172、U118及胶质瘤组织中均有较高的表达。2.TAZ对恶性胶质瘤细胞增殖及肿瘤形成能力的影响采用慢病毒介导的shRNA干扰技术和稳定质粒表达技术在U87和LN229两种胶质瘤细胞系中对TAZ蛋白进行下调和上调,利用MTT、Brdu技术检测两种细胞系的增殖情况,建立荷瘤小鼠模型和利用Soft Agar实验检测TAZ在体内及体外成瘤及自我更新能力的影响。结果显示细胞增殖实验结果表明,细胞的增殖率与TAZ的表达量呈正相关,TAZ下调后细胞的增殖受到抑制,在体内及体外成瘤及自我更新能力也受到相应的抑制,TAZ上调的结果恰好与此相反。3.TAZ对胶质瘤细胞细胞周期的调控利用流式细胞仪检测TAZ对细胞周期的调控,采用Western blot技术检测相应蛋白的表达量结果显示TAZ下调的情况下细胞周期阻滞在G1期,相关周期蛋白CDK2和cyclin E2表达伴随其下调,TAZ上调的结果恰好与此相反。4.TAZ调控恶性胶质瘤细胞增殖的机制研究采用Western blot技术检测EGFR/AKT/ERK信号通路的变化情况,最后利用EGFR信号通路抑制剂阻断由TAZ促进的细胞增殖检测胶质瘤细胞的增殖情况变化,结果显示下调TAZ后相关的EGFR信号通路受到相应的抑制,TAZ上调的结果恰好与此相反,EGFR信号通路抑制剂Eriotinib可以阻断由TAZ过表达引起的相关反应,从而初步确定了TAZ是通过EGFR信号通路调控胶质瘤细胞的增殖与迁移的。
[Abstract]:Glioma is the most common central nervous system and fatal primary tumors, accounting for malignant tumors of central nervous system 80%. according to the growth rate and degree of infiltration of adjacent brain tissue and glioma can be divided into low level (the WHO (WHO) I and II), and WHO (high level III and IV [3].) even after the most effective treatment such as surgery, radiotherapy, chemotherapy and other treatment, the median survival time of patients with high grade gliomas is still not optimistic (14.6 months). This may be due to the specific characteristics of glioma, such as high degree of infiltration and the lack of clear boundaries as a result of the. In addition, the blood brain barrier (BBB) a major obstacle to the physical and chemical constraints are also comprehensive treatment of gliomas. Therefore, the research of molecular mechanism of malignant glioma formation of malignant glioma, looking for new drug targets is urgently needed. The transcriptional co activator TA Z is one of the most important effect factor downstream of the mammalian Hippo pathway, contains 400 amino acids, which is composed of a TEAD binding domain (TB), a WW region, a coiled coil structure, a transcriptional activation domain, transcription of a PDZ and C end binding motifs of.TAZ and its downstream target. The total factor activation, regulation of cell proliferation, migration and apoptosis of a variety of physiological processes. Recent studies have found that in gastric cancer, non-small cell lung cancer, breast cancer and neuroblastoma and other malignant tumors in TAZ were higher than that in normal adjacent tissues. The change is closely related to the malignant proliferation of tumor cells and often the process of cell cycle, EGFR signaling pathway in this process plays a crucial role in the.EGFR belongs to the family of ErbB receptors in mammalian epithelial cells, fibroblasts, glial cells, keratinocyte cell surface has wide The distribution, growth of EGFR and its downstream signaling pathway on cell proliferation and differentiation, and other critical.EGFR by phosphorylation, PI3K/AKT and its downstream Ras-ERK pathway was activated, thus speeding up the process of cell cycle. The study found that TAZ can promote EGFR ligands amphiregulin (AREG) expression, and promote MCF10A breast cancer cell growth factor independent proliferation (EGF). However, the effect of TAZ on glioma cell proliferation has not yet been reported. This paper aims to study the effect of CO transcriptional factor TAZ on the proliferation of glioma cells and its possible molecular mechanism. The results are as follows: through the experiment and the effect of 1.TAZ on the expression of glioma analysis we through the relationship between database query and gene chip detection of TAZ and the prognosis of patients with glioma, and expression of TAZ in gliomas of different grades of the results show that the TAZ The expression and prognosis of glioma patients is closely related to the higher level of glioma, the expression of TAZ was higher. The expression of blot using Western technology to detect TAZ in different glioma cell lines, the results showed that TAZ in U251, LN229, U87, A172, were down regulated and up-regulated, TAZ protein U87 and LN229 in two glioma cell lines affect the expression of U118 and glioma tissues showed higher expression of.2.TAZ forming ability on the proliferation of human glioma cells and tumor by lentivirus mediated shRNA interference and stable plasmid by MTT, the proliferation of Brdu technology for detection of two kinds of cell lines, the establishment of model tumor bearing mice and effects of tumor formation and self-renewal in vivo and in vitro by Soft Agar assay. The results showed that TAZ cell proliferation experiment results showed that the expression of TAZ and cell proliferation rate was positively related to the down-regulation of TAZ After the cell proliferation was inhibited in vitro and in vivo tumorigenicity and self-renewal capacity by corresponding suppression, upregulation of TAZ results are just the opposite regulation of.3.TAZ glioma cell cycle by flow cytometry TAZ the regulation of the cell cycle, the expression of the corresponding protein detection results showed that the amount of TAZ down under the condition of cell cycle arrest in G1 phase by Western blot, cyclin CDK2 and cyclin related with the down-regulation of E2 expression and mechanism of TAZ up regulation results are just the opposite of.4.TAZ regulating the proliferation of malignant glioma cell research using Western blot technology to detect the changes of EGFR/AKT/ERK signaling pathway, the EGFR pathway inhibitor blocked by TAZ promoted cell proliferation assay proliferation of glioma cells, results showed that the EGFR signaling pathway related to down-regulation of TAZ by the corresponding suppression In conclusion, the results of TAZ up regulation are just the opposite. EGFR signaling pathway inhibitor Eriotinib can block the related reactions induced by over expression of TAZ, thus initially determined that TAZ regulates the proliferation and migration of glioma cells through EGFR signaling pathway.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.4

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