【作者】 朱河水；

【导师】 王艳玲；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2008， 博士

【摘要】 为探讨妊娠、泌乳期小鼠下丘脑、垂体、外周内分泌腺中催乳素释放肽(PrRP)及其受体(PrRP-R) mRNA的表达规律,首先取小鼠下丘脑,提取RNA,进行RT-PCR。PCR产物与pMD-19T连接后转化E. coli DH5a,检测阳性克隆并测序,克隆出小鼠PrRP基因。利用DNAstar软件对小鼠PrRP基因进行序列分析,并和大鼠、人、牛、绵羊PrRP核苷酸、氨基酸序列进行比较。然后选用36只雌性小鼠,随机分为6组(n=6)。分别在妊娠6,12,18d和泌乳6,12,18 d处死小鼠,取下丘脑、垂体、甲状腺、肾上腺、卵巢组织,提取RNA。以GAPDH作为参照,利用半定量RT-PCR方法测定PrRP、PrRP-R mRNA在下丘脑、垂体、外周内分泌腺中相对表达水平。同时采取血样,制得血浆,通过放射免疫法(RIA)测定血浆孕酮(P)、雌二醇(E2)和催乳素(PRL)水平。运用SPSS13.0软件对组织PrRP、PrRP-R mRNA水平和血浆P、E2、PRL水平进行双侧相关分析,探究下丘脑、垂体、外周内分泌腺PrRP、PrRP-R mRNA与血浆P、E2、PRL的相关关系。基因克隆结果表明,小鼠PrRP基因阅读框(ORF)全长276bp,编码92个氨基酸。核苷酸同源性分析表明小鼠PrRP与大鼠、绵羊、牛、人PrRP核苷酸同源性分别为88.5%、74.8%、73.3%、78.2%。氨基酸同源性分析表明小鼠PrRP与大鼠、绵羊、牛、人PrRP氨基酸序列同源性分别为87.1%、71.6%、70.5%、71.6%。小鼠PrRP-31与大鼠、绵羊、牛、人PrRP-31氨基酸序列同源性分别为100.0%、93.8%、93.8%、84.4%；PrRP-20氨基酸同源性分别为100.0%、95.0%、95.0%、90.0%。几种种属PrRP氨基酸的核心序列同为RGIRPVGRFGRRRA(45-58),此段氨基酸序列为哺乳类PrRP的保守序列和特征序列,可进一步修饰为RF肽。PrRP mRNA表达规律研究表明：妊娠、泌乳期下丘脑、垂体、甲状腺、肾上腺、卵巢中都有PrRP mRNA的表达。其中表达水平最高的组织为卵巢,其次为下丘脑。妊娠期甲状腺、卵巢PrRP mRNA表达水平和下丘脑表达水平呈显著正相关关系,肾上腺PrRP mRNA表达水平和垂体表达水平呈显著正相关关系,提示下丘脑PrRP可能通过影响甲状腺、卵巢PrRP,而垂体PrRP通过影响肾上腺PrRP参与妊娠的调控。泌乳期PrRP mRNA表达方面,甲状腺、肾上腺、卵巢和垂体,垂体和下丘脑都呈正相关关系,结果提示泌乳期机体可能通过下丘脑、垂体正向调控甲状腺、肾上腺、卵巢PrRP mRNA的表达,参与机体泌乳的相关调控。妊娠、泌乳期下丘脑、垂体、外周内分泌腺中PrRP mRNA表达水平的变化提示PrRP可能通过下丘脑、垂体、外周内分泌腺参与妊娠、泌乳的调控。PrRP-R mRNA表达规律研究发现：妊娠、泌乳期PrRP-R mRNA在下丘脑、垂体、甲状腺、肾上腺、卵巢中都有表达。其中妊娠期甲状腺、下丘脑表达水平显著著高于垂体表达水平。妊娠期下丘脑PrRP-R与垂体PrRP显著正相关,与卵巢PrRP显著负相关,提示垂体PrRP可通过作用下丘脑调节相关生理活动,而卵巢PrRP可能通过作用于非下丘脑器官参与机体调节。泌乳期下丘脑PrRP-R与甲状腺PrRP显著正相关,而甲状腺PrRP-R与下丘脑PrRP显著负相关,提示在甲状腺PrRP可反馈作用于下丘脑,而下丘脑PrRP可能通过作用于垂体,而不是直接作用于甲状腺进行作用；垂体PrRP-R与卵巢PrRP显著正相关,提示泌乳期卵巢PrRP可能通过作用于垂体调节相关泌乳活动。血浆激素分析结果表明：妊娠期小鼠血浆P水平与垂体PrRP mRNA表达量显著负相关,提示妊娠期血浆高水平的P可能通过抑制垂体PrRP的表达,进而影响下丘脑,调节相关妊娠活动。泌乳期小鼠血浆E2水平与卵巢PrRP mRNA表达量之间显著负相关,提示卵巢PrRP可能直接抑制卵巢的分泌活动,或者通过作用于垂体间接调节卵巢分泌活动,进而影响泌乳相关生理活动。妊娠、泌乳期内血浆PRL与下丘脑、垂体、卵巢等组织中PrRP及其受体表达量均无显著相关关系,提示血浆PRL水平可能不受组织中的PrRP表达的影响。更多还原

【Abstract】 ABSTRACT To investigate PrRP and PrRP-R mRNA expression in hypothalamus, pituitary and periphery glands during pregnancy and lactation, Firstly, total RNA was extracted from mouse hypothalamus and amplified by RT-PCR. The PCR products were purified and ligated into the pMD-19T vector, then transformed into competent cells of E. coli DH5a.The positive cloning was sequenced to gain mouse prolactin-releasing peptide cDNA. The nucleotide and amino acid sequence of mouse PrRP were analyzed and blasted to those of rat, human, cattle, sheep by DNAstar software. Then 36 female mouses were divided randomly into six groups (n=6). hypothalamus, pituitary, thyroid, adrenal and ovary were sampled for mRNA extraction. Expression levels of PrRP and PrRP-R mRNA in these tissues were measured by simi-RT-PCR with GAPDH as control. At the same time, blood were sampled on 6,12 and 18 d of pregnancy and lactation respectively to determine plasma concentration of progestin(P), estrogen(E2) and prolactin(PRL) by RIA. Corelation between PrRP, PrRP-R mRNA and plasma P, E2 and PRL were analyzed by SPSS 13.0 software to investigate the relation among them.As cloning result showed mouse PrRP consisted of a 276 bp open reading frame encoding 92 amino acids. The nucleotide sequences alignment result indicated that mouse PrRP nucleotide for the coding region showed 88.5%,74.8%,73.3%,78.2% identity with that of rat, sheep,cattle, human respectively. The amino acid sequences alignment result showed that deduced amino acids from mouse PrRP had 87.1%, 71.6%,70.5%,71.6% identity with that of rat, sheep,cattle, human respectively. Further analysis showed that deduced amino acid sequence for mouse PrRP-31 had 100.0%、93.8%、93.8%、84.4% identity with that of rat, sheep,cattle, human respectively. Mouse PrRP-20 had 100.0%、95.0%、95.0%、90.0% identity with that of rat, sheep,cattle, human respectively. The key amino acid residues segments RGIRPVGRFGRRRA (45-58) of PrRP, which were conservative and characteristic to mammal PrRP and indispensible for further modification to RF peptide, were identical among these species.Study on PrRP mRNA indicated that:during pregnancy and lactation, hypothalamus, pituitary, thyroid, adrenal and ovary all showed PrRP mRNA expression. Ovary had highest level and hypothalamus followed. During pregnancy, adrenal PrRP mRNA had significant positive correlation with those of pituitary and thyroid, at the same time, ovary PrRP mRNA had significant positive correlation with those of hypothalamus.Results indicated that hypothalamus and pituitary PrRP may regulated pregnancy by modulation of thyroid, ovary and adrenal respectively.During lactation, correlation between hypothalamus PrRP mRNA and pituitary, pituitary PrRP mRNA and thyroid, adrenal, ovary, all showed positive relation, which indicated that target gland PrRP mRNA might be positive regulated by hypothalamus-pituitary during lactation. The data that tissue PrRP mRNA expression levels changed on different phases indicated that PrRP might take part in relative physiological regulation during pregnancy and lactation by action on hypothalamus, pituitary and periphery glands.Study on PrRP-R mRNA indicated that:PrRP-R mRNA expressed in hypothalamus, pituitary, thyroid, adrenal and ovary during pregnancy and lactation. During pregnancy, thyroid and hypothalamus had significant higher PrRP-R level than pituitary. Hypothalamus PrRP-R had significant positive correlation with pituitary PrRP and negative correlation with ovary PrRP, which suggested that pituitary PrRP might action on hypothalamus and ovary PrRP action on non-hypothalamus to participate in physiological regulation. During lactation, Hypothalamus PrRP-R had significant positive correlation with throid PrRP, however throid PrRP-R had negative correlation with hypothalamus PrRP, which suggested that, among hypothalamus, pituitary and throid, throid PrRP might feedback on hypothalamus and hypothalamus PrRP action on non-throid to participate in physiological regulation. Pituitary PrRP-R had significant positive correlation with ovary PrRP indicated that ovary PrRP might feedback on pituitary to modulate lactation.Analysis for plasma hormones showed plasma P had significant negative correlation with PrRP mRNA in pituitary during pregnancy, which suggested that higher plasma P might inhibit PrRP expression in pituitary, then by which to regulate pregnancy. During lactation, plasma E2 had significant negative correlation to PrRP mRNA in ovary, which indicated that ovary PrRP might directly or indirectly inhibit the secretion of E2 from ovary. Plasma PRL had no signifant correlation with PrRP, PrRP-R of hypothalamus, pituitary and periphery glands provided further evidence that tissue PrRP maybe did not regulated plasma PRL.更多还原