【作者】 黄明敏；

【导师】 李万程；

【作者基本信息】 湖南师范大学 ， 发育生物学， 2005， 硕士

【摘要】 丝裂原活化蛋白激酶(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) 比较和检测了金鱼单倍体胚胎与二倍体胚胎的MAPK的表达差异。在尾芽时期,不同倍性的胚胎内的ERK蛋白总量存在着明显差别。单倍体胚胎的ERK表达水平要高于二倍体胚胎内的ERK表达。在色素期,ERK在正常自交二倍体胚胎中的表达水平上升,与单倍体胚胎之间的ERK表达差异程度不如尾芽期明显。而在眼睛不正常的单倍体胚胎中完全缺少了JNK的表达和活性。 (3) 将RAF、MEK、ERK的显性突变体基因通过显微注射转染至金鱼的受精卵,分析了当MAPK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DNMRAF,DNMMEK,DNMERK使金鱼胚胎的内源性RAF、MEK、ERK的活性被抑制。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 (ERK 1/2) are the most abundant in all of the six tissues examined. The expression of c-jun N-terminal kinase 1 (JNKl) 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) Both ERKs and JNKs are differentially expressed in haploid and diploid embryos of gold fish. At the tail-bud stage, the amounts of ERK proteins display distinct difference in haploid and diploid embryos, with the haploid showing much higher levels of ERK1/2 proteins than the diploid. However, at the pigment stage, the amount of ERK1/2 proteins increase substantially in diploid embryos, close to that of ERK1/2 found in haploid embryos. The expression patterns of JNK1/2 are very striking between hyploid and diploid embryos. While JNK1 is similarly expressed in the normal hyploid and diploid embryos, it is completely absent in the abnormal haploid embryo. For JNK2, it is only expressed in the diploid embryos of the tail-bud stage. Amazingly, the更多还原