【作者】 郑凯迪；

【导师】 罗凌飞； 李云；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 IGFs系统对发育、生长和生殖都起着重要的调控作用。胰岛素样生长因子酸不稳定亚基(insulin-like growth factor acid-labile subunit, IGFALS)是IGFs系统中的重要一员,可以与IGFs、IGFBP3/5形成三聚体,延长IGFs半衰期,从而参与对IGFs的调控。IGFs系统中的配体和受体以及结合蛋白都受到高度重视,得到了深入而广泛的研究,但迄今为止,对IGFALS的研究仅限于哺乳动物出生后生长过程。为阐明IGFALS的功能,我们用斑马鱼作为研究模型,联合使用PCR、RT-PCR、荧光定量RT-PCR、RACE、western blot、原位杂交、显微注射、过表达、生物软件分析等技术和方法对IGFALS进行研究,研究结果如下。斑马鱼IGFALS基因位于第3号染色体上,DNA序列长为3694bp,包括2个外显子(长度分别为58bp和3466bp)和1.个内含子(170bp)。开放阅读框位于第二外显子中,长度为1845bp,编码20种614个氨基酸残基,其中亮氨酸含量达到20.5%,推测分子量为69127.8Da,等电点为7.65。N-端的前19个氨基酸为信号肽,成熟肽中含有19个亮氨酸富集重复基序(LRR),每个LRR包括24个氨基酸残基,可以用xLxxLxLxxNxLxxLxxxxFxxLx表示,L、N、F分别主要代表Leu、Asn、Phe,各LRR中保守氨基酸的保守程度存在差异。IGFALS氨基酸序列中包含11个半胱氨酸残基和5个天冬酰胺糖基结合位点,与哺乳动物分布位点相比,其中10个Cys分布相同,2个糖基结合位点分布相同。蛋白二级结构包括α-螺旋、延伸链、β-转角、随机卷曲,其中α-螺旋最为丰富(占50.98%),β-转角在LRR的分布位置稳定。IGFALS三级结构形似马蹄,C-端和N-端均向内折,彼此靠近,19个LRR分布不均匀,负电荷主要分布在C-端和N-端,正电荷主要分布在LRR表面。斑马鱼IGFALS氨基酸序列与其它动物差异性很大,与麻雀差异最小(43.9%),与海鞘的差异最大(85.8%),和哺乳动物的差异为48.0-50.8%。在IGFALS系统发育树上,斑马鱼和爪幨亲缘关系最近。克隆得到了GC含量较低的斑马鱼igfals5’端侧翼序列2910bp。和哺乳动物不同,序列中具有TATA-box、CAAT-box和GC-box元件,并且均为两个,这暗示斑马鱼igfals可能能够转录两种mRNA。在该序列的215个区域出现了54种转录因子结合位点,GH的应答元件为GAS2,无GAS1。将不同长度5’侧翼序列连接到pGL3 basic和pEGFP enhancer载体中成功构建了重组质粒P1-P7和EP1-7,发现斑马鱼igfals5’侧翼序列中在-372至-812区域内有启动子活性,在-1623至-2041区域存在能够使启动子转录活性急剧增强的元件。注射EP1后,从19hpf开始可以在斑马鱼胚胎中观察到EGFP表达,而且均表达在体表细胞上。斑马鱼igfals mRNA表达在成鱼多种组织器官,在雌雄鱼的肝胰脏中表达最多,肠道、脾脏、肾脏、肌肉、鳃中表达较强,眼睛、脑和心脏中表达微弱,但在雌雄性腺中的表达存在差异。雌雄斑马鱼肝胰脏igfals的表达水平在一个繁殖周期(7d)内的变化趋势相同,交配后0d显著降低,交配后1d急剧升高,从第2天开始没有显著差异。雌雄鱼肝脏igfbp5 mRNA变化趋势相似,igfs mRNA和igfbp3 mRNA变化在雌雄鱼之间则几乎相反,不过同一性别各种igfs mRNA的变化相似,其它基因的变化趋势则差异很大。在一天内,光照条件变化对处于产卵周期中的斑马鱼雌鱼IGFs系统基因的表达存在显著影响,但对非产卵周期的斑马鱼影响不显著。在6hpf斑马鱼胚胎中可以检测到微弱的igfals mRNA,之后表达量上升,24hpf-48hpf表达量变化不明显,5dpf时表达量大幅度增加。利用pCS2+MT质粒成功构建了IGFALS的融合表达载体pALSMT,过表达IGFALS之后,斑马鱼胚胎出现了如下表型：眼部轮廓模糊,脑部体积减小,不能形成正常脊索,两排体节之间的宽度增加,单个体节的宽度减小,脊背神经组织扭曲,身体背部加宽,体节模糊,体节数减少,体长显著变短,但是对尾芽、近轴中胚层和后脑菱脑节的发育没有显著影响。IGFALS过表达明显增加了igf2a、igf2b、igfbp3、gh、ghr的mRNA水平,但对igf1ra、igf1rb、igfbp5、gh的表达没有显著影响,igf3的表达水平在10hpf显著降低,在24hpf显著升高。过表达IGFALS之后,JAK2的磷酸化水平显著升高,AKT的磷酸化程度没有显著性变化,表明IGFALS(?)(?)可能通过JAK2信号通路影响胚胎发育。在一个产卵周期内,卵巢igfals的表达水平在产卵后3-5d显著升高。igfals在卵巢的滤泡细胞中表达。igfals能够显著促进斑马鱼卵母细胞的成熟。繁殖周期内卵巢igfs和igfbp3/5的表达变化趋势和igfals明显不同,各种igfs mRNA的变化趋势也存在差异。雄性信息素对igf2b和igfbp3的表达有负调控作用。处于产卵周期中的斑马鱼卵巢igfals的表达受光周期影响。DES、DHP、hCG能够显著促进滤泡细胞igfals的表达,E2则对卵巢igfals的表达没有显著影响。igfals和DES、hCG、DHP共作用对卵母细胞成熟的促进,在一定剂量范围内具有协同效应。饥饿显著增加斑马鱼肝脏igfals和igfbp3的表达,不同饥饿时间对表达量的影响存在差异,恢复投喂后表达量先升高后急剧下降至正常水平,但是对igf1表达水平的影响则不显著。腹腔注射GH对斑马鱼肝脏igfals、igf1、igfbp3表达水平的影响相似,先显著抑制表达水平,之后和对照组没有显著差异。腹腔注射E2后肝胰脏中igfals和igfbp3 mRNA水平的变化趋势相同,均为先显著低于对照组,后显著高于对照组,最后恢复到正常水平,但对igf1 mRNA水平的影响不同。斑马鱼IGFALS无论是在基因结构、碱基组成、推导氨基酸的组成以及蛋白质空间构象,还是在启动子序列、转录因子结合位点都和哺乳动物差异较大,另外在GH等刺激作用下,斑马鱼igfals基因表现出和哺乳动物不同的变化趋势,表明igfals基因在斑马鱼中的功能可能和哺乳动物存在差异。在斑马鱼igfals启动子序列中存在54种215个转录因子结合位点,有很多潜在的调控因子,并且在很多情况下,igfals和三聚体其它2个基因的表达水平完全不同,表明igfals基因在斑马鱼发育和生命活动中的功能可能远比目前了解的要复杂。因此通过对斑马鱼igfals的研究,希望能够为揭示IGFALS在低等脊椎动物生长和发育中的功能,为探讨IGFALS在调控脊椎动物生长发育过程中的机制,以及为更加全面和深入了解IGF系统在生物体内的功能提供重要的资料。更多还原

【Abstract】 IGFs play important roles in regulating development, growth and reproduction. Insulin-like growth factor acid-labile subunit (IGFALS), one of the important members of IGF system, forms ternary complex with IGFs and IGFBP3/5 and significantly increases the half-lives of both IGFs and IGFBP3/5 in serum and maintains a circulation pool of these molecules. Until now, almost all reports about IGFALS has been limited to mammals. Little is learned about IGFALS in non-mammal vetebrates. In order to elucidate the role of IGFALS in lower vertebrates, we employed molecular developmental biological techniques, such as PCR, RT-PCR, Q-PCR, RACE, western blot, in situ hybridization, microinjection and overexpression to explore developmental and physiological actions of IGFALS in zebrafish.Zebrafish igfals gene was located in chromosome 3 with a whole sequence of 3694bp, including two exons (58bp and 3466bp) and one intron (170bp). The open reading frame, which coded 614 amino acids (20 kinds) was limited in the second exon with a sequence of 1845bp. The leucine was the richest amino acid in the sequence with a ratio of 20.5%. First 19 amino acids in the N-terminal were signal peptides. There were 19 leucine rich repeats (LRR) in mature IGFASL peptide and every LRR contained 24 amino acids. The LRR was showed as xLxxLxLxxNxLxxLxxxxFxxLx (L, N, F represent leucine, asparagine and phenylalanine respectively in most time). IGFALS contains 11 cysteine residues and 5 N-linked glycosylation sites. The secondary structure was formed by extension chain,β-turn, random coil and a-helix, and the latter (50.98%) was the richest type. The tertiary structure of IGFALS was just like a horse hoof. The electrostatic potential was negative in C-terminal and N-terminal and positive in the surface of LRR. The difference in amino acids of IGFALS between zebrafish and other animals was 43.9-85.8%. In phylogenetic tree analysis with amino acids of IGFALS, zebrafish was close to Xenopus laveis.A 2910bp 5’flanking sequence of igfals was cloned from zebrafish. It contained double TATA-boxes, CAAT-boxes and GC-boxes. Two hundred and fifteen potential sites identified in the 5’flanking sequence can be binded by 54 transcription factors. GAS2 instead of GAS1 was the responding element to GH in zebrafish igfals. Transcription activities of zebrafish igfals promoter were significantly improved when sequence between-2041 to-2910 were added to vector in zebrafish embryoes and HepG2 cells. EGFP was first observed in the surface cells in 19hpf zebrafish embryos, which were injected with pEGFP enhancer vector including igfals promoter.In adult zebrafish, igfals mRNA expressed in many tissues and mainly expressed in liver tissue, modestly expressed in intestine, spleen, kidney, muscle and gill, and weakly expressed in eyes, brain and heart. Interestingly, the level of igfals was significant different between male and female gonad tissue. The expression patterns of igfals and igfbp5 between male and female zebrafish were similar in a reproduction cycle. However the expression patterns of igfs and igfbp3 was quite different between male and female. Expressions in ovary were significantly affected by photoperiod in female in spawning, but photoperiod had no effects on these genes expressions of female out of spawning cycle. Weak igfals mRNA could be detected in 6hpf embryoes, and then igfals mRNA gradually increased, but not significantly different between 24 hpf and 4dpf embryos. The level of igfals mRNA was obviously increased in 5dpf embryoes.Overexpression of igfals caused defects in midline, notochord and somite development of zebrafish embryos. Expressions of igf2a、igf2b、igfbp3、gh and ghr mRNA were significantly increased by overexpression of igfals, but expressions of igf1ra、igf1rb、igfbp5 and gh mRNA were unaffected. Western blot analysis indicated a marked increase in the level of phosphorylated JAK2 in igfals overexpression zebrafish embryoes, but the level of phosphorylated Akt was not significantly changed.In a spawning cycle, the igfals mRNA levels from 3-5d female zebrafish after ovulation were higher than other days in ovary.igfals mRNA was exclusively detected in the follicle cells. Overexpression of igfals could significantly increased the number of GVBD follicles and igfals potently promotes maturation of follicles. The expression patterns of igfs and igfbps were quite different from igfals in ovary. Level of igfals mRNA of female was not influenced by sex pheromone from male zebrafish, but igf2b and igfbp3 mRNA levels were negatively regulated by sex pheromone from male zebrafish. In addition, photoperiod could also affect expression of igfals in zebrafish ovaries. DES, DHP, hCG significantly contributed to increase ovary igfals mRNA expression with a dose-dependent manner, but no obviously impact was observed in ovary treated with E2 on igfals mRNA expression. Injection igfals vector and then treatment with DES, hCG and DHP can obviously enhanced maturation of follicle cells.Fasting strikingly increased expression of igfals and igfbp3 in zebrafish liver, but have no significant effects on igf1. After refeeding 3 weeks, levels of igfals and igfbp3 mRNA were reduced to control level. Intraperitoneal injection of GH to adult zebrafish influenced expression of igfals, igfl and igfbp3 in a similar manner. Their expressions were decreased at first few days, but increased to the level of control gradually. After intraperitoneal injection of E2, igfals and igfbp3 mRNA expression were similiarly changed. Levels of liver igfals and igfbp3 mRNA were significantly lower than that of control at early days, and then significantly higher than that of control. At last, their expressions went to normal levels. However, E2 influenced igfl mRNA expression in a different manner.Not only in gene and amino acid sequence, but in response to hormones, zebrafish igfals is quite different from mammals, which suggested that the function of igfals in zebrafish may be different from mammals. IGFALS could play more complicated roles than our predictation in the development, growth and reproduction.更多还原