RFC调控神经嵴发育与尼克氨酸杀伤胶质瘤细胞机制的研究
文献类型：学位论文
| 作者 | 李杰晶 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 毛炳宇 |
| 关键词 | 叶酸 叶酸转运蛋白 神经嵴 组蛋白甲基化 表观遗传修饰 |
| 其他题名 | Regulation of neural crest development by reduced folate carrier in Xenopus and apoptosis of glioma cell induced by nicotinic acid |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 叶酸是B族维生素家族成员，是细胞中合成核酸的重要前体物质。叶酸的另一个重要功能是参与S-腺苷甲硫氨酸（SAM）的合成，而SAM是体内甲基化反应的甲基供体。在胚胎早期发育过程中叶酸缺乏可以导致严重的出生缺陷，包括神经嵴发育相关畸形（neurocristopathic malformations）和神经管缺陷等。其中神经嵴发育相关畸形是由于神经嵴细胞的诱导、迁移、分化或增殖缺陷所导致的，主要包括唇腭裂、白化病、巨结肠、心脏流出道狭窄等。在胚胎早期持续补充叶酸可有效预防神经嵴发育相关畸形的发生，但其作用机制却并不清楚。 大部分脊椎动物不能合成叶酸，只能从环境中吸收。叶酸及其衍生物是亲水性分子，主要通过细胞膜上的通道转运入胞。在脊椎动物体内叶酸及其衍生物进入体内主要依赖叶酸受体（folate receptors，FR）和还原性叶酸转运蛋白（Reduced folate carrier，RFC）。RFC是一个12次跨膜蛋白，是活性叶酸5-甲基四氢叶酸（5-MTHF）进入细胞的主要通道。我们克隆了非洲爪蛙的还原性叶酸转运蛋白（XRFC），发现它在爪蛙神经嵴发育中具有重要作用并对其作用机制进行了初步研究。 XRFC在诱导和迁移时期的神经嵴细胞中高表达，用特异性Morpholino阻断XRFC翻译后，胚胎出现颅面软骨缺陷、黑色素减少、心包积液等神经嵴发育缺陷相关表型，而共注射XRFC mRNA可以明显挽救这些缺陷，表明XRFC可能与爪蛙神经嵴的发育相关。进一步研究发现XRFC可特异性调节神经嵴相关基因Zic1、Slug、FoxD3和Twist1等的表达，而且这种调节作用依赖于XRFC的5-甲基四氢叶酸转运功能。共注射5-甲基四氢叶酸或SAM都可以挽救XRFC敲低所导致的缺陷，表明XRFC可能通过调节叶酸代谢而参与神经嵴发育的调节。在爪蛙动物帽诱导实验中，RFC或5-甲基四氢叶酸可调节组蛋白H3的甲基化水平。此外，我们还初步研究了爪蛙组蛋白乙酰转移酶延伸因子3（Elp3）在神经嵴发育中的功能。XElp3在神经嵴中高表达，通过功能阻断实验发现XElp3参与调节神经嵴迁移，而这一过程依赖于XElp3的组蛋白乙酰化功能。上述结果表明表观遗传学修饰在神经嵴诱导与迁移调控中具有重要作用，而叶酸等营养因子可 通过表观遗传学机制参与调节神经嵴的发育和相关疾病的发生。 脑胶质瘤是儿童中最频发的脑肿瘤，我们发现一定浓度的尼克氨酸（Nicotinic acid，NA）能打破脑胶质瘤细胞内钙的平衡，致使胶质瘤细胞内钙持续升高，并引起细胞内质网（ER）和细胞质之间钙分布改变，同时激活Caspase依赖的凋亡途径和内质网应激(ER stress)反应，从而有效诱导脑胶质瘤细胞的凋亡。在同样条件下，尼克氨酸却不引起神经元内钙发生类似变化，因而不能杀伤神经元。同时，我们还观察到一定浓度的尼克氨酸可以上调脑胶质瘤TRPM7钙离子通道的表达，使得Myosin IIA发生磷酸化解聚，抑制细胞迁移前缘结构（leading edge）的形成，从而抑制脑胶质瘤的迁移能力。我们的研究提示尼克氨酸可通过靶向于胶质瘤细胞的钙系统而诱导其凋亡，从而有望成为有效的胶质瘤治疗药物。 |
| 英文摘要 | Defective neural crest development leads to a broad spectrum of congenital malformations, collectively called neurocristopathies, which includes defects in pigmentation, and abnormal craniofacial and heart development. Folic acid has been shown to have protective effect against neurocristopathic malformations and has been used as a powerful medicine for pregnant women for more than 20 years. However, the underlying mechanism remains largely unknown. Folate, a member of the vitamin B family, functions in a series of vital biological processes, including the synthesis of DNA and amino acid, cell cycle control, and one carbon unit cycle which is important for DNA and protein methylation. As a water-soluble B class of vitamin, the uptake of folate by cells is mediated by specific carriers or receptors, including folic acid receptors (FRs) and reduced folic acid carrier (RFC).We showed here that in Xenopus RFC (XRFC) is specifically expressed in neural crest tissues. Knockdown of XRFC resulted in severe neurocristopathies, as well as loss of neural crest marker gene expressions. On the other hand, over-expression of RFC or injection of 5-methyltetrahydrofolate expanded the neural crest territories. In animal cap assays, knockdown of RFC dramatically reduced the mono- and trimethyl-Histone3-K4 levels and co-injection of the lysine methyltransferase hMLL1 largely rescued the XRFC morpholino phenotype. These results suggested that the RFC mediated folate metabolic pathway likely potentiates neural crest gene expression through epigenetic modifications and manifested the role of nutrients in gene transcriptional regulation through epigenetic modification. We also found that elongation protein 3 (Elp3) is involved in Xenopus neural crest development. Elp3 contains a SAM radical domain and a HAT (histone acetyltransferase) domain and has DNA demethylation and histone acetylization activities.We found that Xenopus Elp3 is highly expressed in migrating neural crest.Overexpression and knockdown of XElp3 affect neural crest induction and migration and both the SAM domain and the HAT domain are required for its activity.Immune-staining experiment indicates that Elp3 is required for proper H3/H4 acetylization in Xenopus embryos. Glioma is the most common primary brain tumor.We found that nicotinic acid (NA) specifically disrupted the intracellular calcium balance in glioma but not neuronal cells.In glioma cells,NA induced cell apoptosis through caspase dependent and independent pathways(ER stress). We also showed that in glioma cells, NA distributed the leading edge formation through the up-regulation of trpm7, which phosphorylates Myosin IIA causing disassembly of the leading edge. Further work in rat model indicated that NA was also able to inhibit glioma growth in vivo and might be a new candidate for clinical glioma therapy. |
| 语种 | 中文 |
| 公开日期 | 2013-04-22 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7364]  |
| 专题 | 昆明动物研究所_发育生物学 |
| 推荐引用方式 GB/T 7714 | 李杰晶. RFC调控神经嵴发育与尼克氨酸杀伤胶质瘤细胞机制的研究[D]. 北京. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：昆明动物研究所
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