【作者】 黄明敏；

【导师】 李万程；

【作者基本信息】 湖南师范大学 ， 细胞生物学， 2008， 博士

【摘要】 丝裂原激活蛋白激酶(mitogen-activated protein kinases,MAPKs)是细胞内的至关重要的蛋白激酶。研究证实,MAPK信号转导通路存在于大多数细胞内,将细胞外刺激信号转导至细胞及其核内,并引起细胞生物学反应(如细胞增殖、分化、转化及凋亡等)的过程中具有至关重要的作用。MAPK可分为三个主要的亚群,即细胞外信号调节激酶(ERK),应激活化蛋白激酶(JNK)和p38MAPK。本文应用Western Blot分析、免疫沉淀、激酶活性分析、显微注射等技术研究和探讨了MAPK对金鱼发育的影响和调控作用。所得实验结果如下:(1)MAPK三种主要成员在金鱼的晶体上皮、晶体纤维、视网膜、大脑、心脏以及肌肉六种组织中的表达和活性存在着组织特异性。ERK的表达和活性水平最高,在金鱼的六种组织中都存在着强烈的表达和活性水平;JNK1只在晶体纤维、视网膜、大脑和心脏中表达,在晶体上皮中的表达极其微弱;JNK2在视网膜、心脏、大脑以及肌肉组织中均有一定程度的表达;但在晶体上皮,JNK2只有微弱的表达;p38在金鱼的视网膜、大脑、心脏以及肌肉中存在表达,而在晶状体的上皮和纤维中没有表达,在金鱼六种组织中不存在p38的磷酸化活性。(2)克隆了金鱼的ERK2基因编码区序列全长。金鱼erk2基因编码区共有1110个碱基。翻译成369个氨基酸。金鱼erk2基因核苷酸序列在斑马鱼、鼠、沟鼠、人、狗、鸡、牛、爪蟾同源性依次降低。金鱼erk2核苷酸与斑马鱼同源性最高,与爪蟾同源性最低。而金鱼ERK2蛋白同源性与斑马鱼最高,其次是爪蟾,与鼠类的同源性最低,但同源度仍然高达89.9%。这为进一步研究更深入的探索ERK蛋白在金鱼发育中的调控机制和以及此过程所涉及的调控因子具有极其重要的意义。(3)对ERK2基因和JNK1基因在金鱼的9种组织和10个发育时期mRNA水平的表达进行了分析。实验结果显示,ERK和JNK在金鱼早期胚胎发育各个时期都有很高水平的表达,呈现出典型的时间表达模式。同时,ERK、JNK在金鱼成体的多种组织中分布也很广泛,在各种不同组织间具有组织表达差异性。二者均在卵巢组织中表达最高。本实验的结果表明ERK、JNK是金鱼体内重要的蛋白激酶。(4)将ERK的显性突变体基因通过显微注射转染至金鱼的受精卵,分析ERK信号通路被抑制后对金鱼发育的影响。通过激酶活性分析证实注射的DNM-ERK使金鱼胚胎的内源性ERK的活性被抑制。对注射DNM-ERK的金鱼胚胎的连续的显微观察发现,抑制ERK信号通路将导致金鱼多种器官的形成出现障碍,尤其是外胚层来源的眼睛以及中胚层来源的肌肉体节。本实验揭示了ERK在脊椎动物的早期胚胎发育的至关重要的主导作用。(5)将JNK的显性突变体基因通过显微注射转染至金鱼的受精卵,探索JNK信号通路被抑制后对金鱼发育的影响。实验结果显示JNK在金鱼发育过程中必不可少,抑制JNK信号通路将导致金鱼多种器官的形成出现障碍,特别是眼睛、神经管以及肌肉。本实验揭示了JNK在脊椎动物的早期胚胎发育的至关重要的主导作用。首次运用功能缺失的方法确定了JNK在脊椎动物眼睛发育过程中的重要作用。更多还原

【Abstract】 Mitogen-activated protein kinases (MAPKs), a family of special protein kinases, exist in most cells and play significant roles in signal transduction from extra-cellular circumstances to nuclei, which mediate various cellular processes such as proliferation, differentiation, transformation and apoptosis. Through Western blot analysis、immunoprecipitation-linked kinase assays、and microinjection of the dominant negative mutant RAF,MEK and ERK, the expression patterns of MAPKs and the function of the ERK signaling pathway on development of gold fish have been investigated and the results are summarized below:(1)、The three types of MAPKs are differentially expressed in lens epithelium, lens fiber, retina, heart, brain and muscle of gold fish with distinct tissue specificity. Among three types of MAPKs, the expression and activities of the extracellular signal-regulated kinases (ERK1/2) are the most abundant in all of the six tissues examined. The expression of c-jun N-terminal kinase 1 (JNK1) is only observed in lens fiber, retina and brain tissues. In contrast, expression of JNK2 is detected in retina, heart, brain and muscle with minor expression in lens epithelium. The expression of p38 is detected in retina, heart, brain and muscle. However, its activity is absent in all the tissues of gold fish examined.(2)、The whole length open reading frame (ORF) of gold fish ERK2 gene has been cloned. It is 1100 bp long and encodes a protein of 369 amino acid residues. A decreased homology can be obtained of gold fish ERK2 gene to those of zebrafish, mouse, rat, human, dog, chicken, bovine and Xenopus laevis, among which zebrafish is the highest while Xenopus laevis the lowest. However, at the amino acid level, the highest homology is of gold fish ERK2 to zebrafish, and to Xenopus laevis the second high, while the lowest to mouse or rat, despite an 89.9% high score can still be drawn to both of these rodents. These data greatly contribute to further exploring the regulatory mechanism of ERK and the understanding of involved transcription factors in the development process of gold fish.(3)、It has been analyzed of the two major MAP kinases of ERK2 and JNK1 in nine different gold fish tissues in ten gold fish developing stages at the mRNA level. Experimental results show that in different early embryonic developing stages of gold fish, the two genes of ERK and JNK are highly expressed, which demonstrates a time-dependent pattern. Meanwhile, ERK and JNK are widely expressed in different tissues of adult gold fish, and an apparent tissue-specific difference can be observed among all these tissues, among which ovary expression is the highest. All these results indicate that ERK and JNK are two important protein kinases in gold fish.(4)、Microinjection of dominant negative mutant ERK( DNM-ERK) into gold fish zygotes apparently blocks the ERK signaling pathway of the injected embryos as reflected by the attenuated or abolished ERK activity at different stages of development. When the ERK signaling pathway is blocked by DNM-ERK, the development of gold fish is severely interrupted as shown by the appearance of abnormal phenotypes of stunted embryos and abnormal phenotypes which include abnormal shape of ocular lens, asymmetric pigment, reduced somite number, curled tails, reduced heartbeat, unconspicuous movement of muscles and high lethality rate, etc.These results indicate ERKs play an impotant role in vertebrate embryo development.(5)、The DNMJNK gene has been microinjected into zygote of gold fish to explore its affection on development after blocking the JNK signaling pathway. Experimental results display an absolutely necessary role of JNK in gold fish development, for the inhibition of JNK signaling pathway can obstruct the formations of many gold fish organs, especially eye, nervous tube and muscle. The present experiment demonstrates that JNK plays an important leading role in the early embryonic developing period of vertebrate, and this firstly confirms that JNK serves a significant function in vertebrate eye development by the method of functional absence.更多还原