mP19对细胞骨架和增殖的影响及其相互作用分子筛选

发布时间：2018-03-09 00:40

  本文选题：mP19　切入点：MARVELD1　出处：《哈尔滨工业大学》2010年硕士论文　论文类型：学位论文

【摘要】： 本课题组前期实验证实MARVELD1是一个在多种肿瘤细胞中呈现低表达的候选抑癌基因,其功能研究有待进一步深入。同源比对分析发现来自小鼠的mP19与人源MARVELD1氨基酸序列同源性达88%,且结构域高度保守。因此,本课题通过生化和细胞生物学技术探讨mP19的基本功能,旨在阐明mP19和MARVELD1的功能异同,为后续建立小鼠模型提供一定参考。本课题中通过生物信息学方法,我们推断mP19及其种属同源分子仅在脊椎动物中表达且在高等哺乳类中呈现高度保守性。mP19具有四个潜在的蛋白激酶C(PKC)磷酸化位点(Thr15, Ser19, Thr51, Thr120),而人源MARVELD1仅存在一个(Thr51),我们采用免疫沉淀技术及Western Blot方法检测证实mP19存在可被磷酸化的苏氨酸,而Thr51因其在人鼠序列中的高度保守性被认为是mP19的PKC磷酸化关键苏氨酸。我们采用免疫荧光技术,在共聚焦显微镜下观察到mP19呈现周期依赖性细胞定位:间期时定位于细胞核及细胞核周,分裂期时与纺锤体微管及胞质分裂器有明显共定位。佛波酯醇(PMA)激活PKC能促进mP19的核转位。通过GST-pull down和免疫共沉淀技术,我们证实mP19与α-tubulin存在相互作用,且秋水仙素能促进mP19向细胞膜转位。我们利用相差显微镜、共聚焦显微镜及扫描电镜从不同角度观察到过表达mP19抑制细胞微丝骨架组装、促进细胞表面超微结构和丝状伪足的形成。此外,我们证实在NIH3T3细胞中过表达mP19能明显抑制细胞增殖、造成细胞G1期阻滞、抑制细胞迁移运动。
[Abstract]:Our previous experiments confirmed that MARVELD1 is a low expression in many tumor cells candidate tumor suppressor gene, its function needs further study. Homology analysis found that mouse mP19 and human MARVELD1 amino acid sequence homology of 88%, and the domain is highly conserved. Therefore, the basic function of this subject through study of Biochemistry and cell biology, technology of mP19, mP19 and MARVELD1 to elucidate the functional similarities and differences, to provide a reference for the subsequent establishment of mouse model. Through bioinformatics in this topic studies, we conclude that mP19 and a homologous molecule expressed only in vertebrates and in mammals in highly conserved.MP19 has had four potential protein kinase C (PKC) phosphorylation sites (Thr15, Ser19, Thr51, Thr120), and there is only one source of MARVELD1 (Thr51), we used immunoprecipitation and Western Blot assay confirmed the existence of mP19 can be phosphorylated threonine, and Thr51 because of its highly conserved sequence in the rat was found to be in mP19 PKC phosphorylation of key threonine. We used immunofluorescence technique, mP19 were observed by confocal microscopy showed cell cycle dependent location: located in the nucleus and the interphase nuclear week, mitotic spindle microtubules and cytokinesis and has obvious colocalization. Phorbol ester alcohol (PMA) activation of PKC can promote the nuclear translocation of mP19. By GST-pull down and immunoprecipitation, we demonstrate that mP19 interacts with the alpha -tubulin, and colchicine can promote mP19 to cell membrane translocation. We use difference microscopy, confocal microscopy and scanning electron microscopy from different angles observed that overexpression of mP19 inhibits the actin cytoskeleton assembly, promote the formation of cell surface ultrastructure and filopodia. In addition, we confirm that overexpression of mP19 in NIH3T3 cells can significantly inhibit cell proliferation, cause G1 phase arrest and inhibit cell migration.

【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346;Q25

【参考文献】

相关期刊论文 前1条

1 王山;肖晟;赫杰;韩放;李钰;;核蛋白MARVELD1与核质转运受体蛋白Importin β1相互作用的鉴定[J];中国生物化学与分子生物学报;2009年08期

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