侧脑室注射Leptin对大鼠下丘脑前阿黑皮素mRNA表达的影响

给体重 180～ 2 0 0 g的雌性 SD大鼠侧脑室中注射重组瘦素 (L eptin) 1μg,采用 RT- PCR测定了其下丘脑前阿黑皮素 (POMC) m RNA和 L eptin受体 (OB- R) m RNA的表达水平。结果表明 ,注射重组 L eptin后 ,OB- R基因和 POMC基因表达量均上升 ,与生理盐水注射组、空白对照组相比差异显著 (P<0 .0 5 ,P<0 .0 1)。由此认为 ,L eptin在大鼠下丘脑 POMC的调控功能中起着重要作用     

Channel catfish, Ictalurus punctatus, leukocytes secrete immunoreactive adrenal corticotropin hormone (ACTH)

The interrelationships between the neuroendocrine and immune systems are becoming more understood, at least in mammalian systems. The most characterized of these relationships is that of hormonal signaling within the hypothalamo-pituitary-adrenal (HPA) axis. CNS-perceived signals stimulate the release of corticotropin releasing hormone (CRH) which in turn stimulates the release of pituitary corticotropin (ACTH) and ultimately the release of adrenal-cortex-derived corticosteroids. We demonstrate that channel catfish peripheral blood mononuclear cells, a channel catfish B-cell line (1G8) and a T-cell line (28S.1), constitutively and in response to CRF, secrete a molecule that is reactive with a mammalian RIA for ACTH (irACTH). The T-cell line was the most responsive to CRH and may provide a valuable model for understanding the interrelationships between the neuroendocrine and immune systems in lower vertebrates. Lymphoid derived ACTH, or ACTH-like products, in fish, as well as higher vertebrates, may represent a paracrine or autocrine control on lymphocyte function and immune regulation.     

GnRH and gpcr: laser-captured single cell gene profiling

We have developed a novel single cell real-time quantitative PCR technique, which incorporates harvesting marker-identified single cells using laser-capture. Here, for the first time in a vertebrate species, using this innovative single cell gene profiling technique, we report the presence of G-protein coupled receptors in individual gonadotropin-releasing hormone (GnRH) neurons and endocrine cells of the pituitary of the tilapia Oreochromis niloticus. The differential expression of multiple combinations of three GnRH receptor types (R1, R2 and R3) in individual gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity. The differential use of GnRH receptors in corticotropes, melanotropes and thyrotropes during gonadal maturation and reproductive behaviors suggests new roles for these hormones. Further, we provide evidence of the structure of a novel nonmammalian G-protein coupled receptor (GPR54) for kisspeptins, encoded by Kiss-1 gene, which is highly conserved during evolution and expressed in GnRH1, GnRH2 and GnRH3 neurons. We hypothesize GPR54 stimulates GnRH secretion and is crucial for pubertal maturation. We speculate, the use of this method will allow the identification and quantification of known and unknown genes in single cells, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells in vertebrates species.     

刀鲚POMC基因的cDNA克隆及其应激应答

为了研究刀鲚(Coilia nasus)阿黑皮素原(proopiomelanocortin,POMC)基因的应激应答及调控,克隆了刀鲚POMC基因的c DNA全长。刀鲚POMC基因的c DNA全长2030 bp,编码区699 bp,编码232个氨基酸。二级结构预测显示,刀鲚POMC包括信号肽(signal peptide)、N末端区(N-terminal region,NPP)、促肾上腺皮质激素结构域(adrenocorticotropic hormone,ACTH)、β-脂解素(β-LPH)、α-促黑素(α-MSH)、促肾上腺皮质激素样垂体中叶多肽(CLIP)、γ-脂解素(γ-LPH)、β-促黑素(β-MSH)和β-内咖肽(β-EP)结构域。运用荧光定量的方法检测了该基因在刀鲚不同组织中的表达分布。结果显示,POMC在健康刀鲚的脑高表达,鳃、肾、精巢相对高表达,肝、脾、肠、头肾、肌肉和卵巢中微量表达。运输胁迫后,POMC基因的表达量显著下降(P0.05);10‰ NaCl组4 h和6 h POMC基因的表达量显著上调(P0.05),6 h的表达量恢复至应激前。POMC基因在脂肪代谢及应激调控中发挥重要的功能,本研究结果可为该基因在刀鲚后续育种及应激调控工作提供重要基础。     

Correlation analysis of hypothalamic mRNA levels of appetite regulatory neuropeptides and several metabolic parameters in 28‐day‐old layer chickens

Various lines of evidence suggest that appetite‐related neuropeptides in the hypothalamus are regulated by adiposity signals such as leptin and insulin in mammals. In the present study, we examined age‐dependent changes in the weight of abdominal fat and hypothalamic mRNA levels of neuropeptide Y (NPY, an orexigenic neuropeptide) and proopiomelanocortin (POMC, a precursor of anorexigenic neuropeptides) in growing chickens at 7, 14, 21 and 28 days of age. Hypothalamic NPY mRNA levels were significantly (P < 0.05) decreased after 14 days of age, whereas hypothalamic POMC mRNA levels were significantly (P < 0.05) increased at 28 days of age. The percentage of abdominal fat was significantly increased after 14 days of age in chickens. We next examined the correlation of hypothalamic NPY and POMC mRNA levels and several parameters at 28 days of age. There were no significant correlations between hypothalamic mRNA levels of NPY or POMC and the percentage of abdominal fat. These findings suggest that the gene expressions of NPY and POMC do not depend on adiposity in chickens, at least in 28‐day‐old layer chickens.     

Central insulin suppresses feeding behavior via melanocortins in chicks

Growing evidence suggests that insulin interacts with both orexigenic and anorexigenic peptides in the brain for the control of feeding behavior in mammals. However, the action of central insulin in chicks has not yet been identified. In the present study, we investigated the effects of central injection of insulin on feeding behavior in chicks. Intracerebroventricular (ICV) administration of insulin, at doses that do not influence peripheral glucose levels, significantly inhibited food intake in chicks. Central injection of insulin in chicks significantly increased expression of pro-opiomelanocortin (POMC) mRNA, and decreased that of neuropeptide Y (NPY) mRNA. Finally, co-injection of the melanocortin antagonist (SHU9119 or HS014) prevented the reduction in food intake caused by ICV administration of insulin. These data suggest that insulin functions in chicks as an appetite-suppressive peptide in the central nervous system, and that the central melanocortin system mediates this anorexic effect of insulin, as in mammals.     

4个不同地理种群中华鳖POMC基因全长cDNA克隆及其序列分析

为探讨中华鳖不同地理种群的POMC基因的多态性,建立区分中华鳖不同地理种群的分子遗传标记,采用RT-PCR的方法扩增得到4个不同地理种群(太湖鳖种群、沙鳖种群、台湾鳖种群和黄河鳖种群)中华鳖的POMC基因,对该基因进行克隆及序列测定,并对所测序列进行比较,分析其SNP位点,并对所得的SNP位点进行验证。结果显示:4个不同地理种群中华鳖所扩增的POMC基因的核苷酸序列均为786bp;以megalign软件比较分析4个不同地理种群中华鳖的POMC基因,发现该序列中共有15个多态性核苷酸位点,经过验证,第129位核苷酸的变异为台湾鳖种群不同于其他种群的变异,第441位核苷酸变异为太湖鳖种群不同于其他种群的变异,第168和396位核苷酸为黄河鳖种群不同于其他种群的变异,第531和618位核苷酸为沙鳖种群不同于其他种群的变异。结果表明,中华鳖的POMC基因存在多态性,不同地理种群中华鳖POMC基因均有各自特异的核苷酸序列,可以作为区分中华鳖的不同地理种群的分子遗传标记。     

RFRP-3对大鼠采食行为和生长性能的影响

为了探讨RFRP-3对大鼠采食行为以及生长性能的影响,选取体重相近的清洁级SD大鼠30只,随机分为3组,分别为对照组[腹腔注射生理盐水200 μL/(次·只)]、RFRP-3低剂量组[腹腔注射1 μg/100 μL RFRP-3,200 μL/(次·只)]和高剂量组[腹腔注射10 μg/100 μL RFRP-3,200 μL/(次·只)],每组10只,雌、雄各半,每天注射2次,连续腹腔注射14 d。观察和记录大鼠的采食量、采食行为和体重变化,计算料肉比及Lee指数,分离血清并检测血脂相关指标,并对下丘脑食欲相关因子的表达水平进行检测。结果表明,与对照组相比,仅在光照环境中腹腔注射高剂量RFRP-3会导致大鼠采食量极显著(P<0.01)增加;采食行为方面主要表现为采食频率极显著(P<0.01)增高和饱腹率明显下降;生长性能方面主要表现为体重增长极显著(P<0.01)加快以及料肉比显著(P<0.05)降低;在肥胖指标中,Lee指数显著(P<0.05)增加,血清中甘油三酯和总胆固醇的含量极显著(P<0.01)升高;下丘脑NPY mRNA表达水平显著(P<0.05)上升,而POMC mRNA表达水平显著(P<0.05)下降。试验表明,腹腔注射RFRP-3可促进大鼠的采食行为并提高其生长性能,且这种生理功能的发挥可能是通过调节下丘脑食欲相关因子的变化完成的。     

POMC在不同毛色绵羊皮肤中的表达与定位

【目的】对不同毛色绵羊皮肤中的阿黑皮素原（proopiomelanocortin,POMC）进行定位和表达差异分析,确定POMC与毛色形成的关系,进一步丰富有关哺乳动物毛色形成机制的理论知识。【方法】选取年龄相近的雄性黑色和白色成年绵羊各3只,用取皮器分别在臀部采集皮肤组织3块,对其中一块制作石蜡切片后采用免疫组织化学法确定POMC在不同绵羊皮肤中的定位,另两块置于液氮中冻存后分别提取皮肤总RNA和总蛋白,总RNA经反转录成cDNA后,经NCBI分析比对后设计POMC特异性PCR引物,采用QRT-PCR方法检测POMC基因在mRNA水平的表达差异;总蛋白经SDS-PAGE凝胶电泳后,采用Western blotting方法加入POMC多克隆抗体检测POMC在蛋白水平的表达差异,得出的数据用SPSS19.0软件进行差异显著性分析,探讨POMC对毛色形成机制的调控作用。【结果】免疫组织化学结果显示POMC在黑色和白色绵羊皮肤中均有表达,且表达部位都位于毛囊根鞘周围和表皮的皮肤角质形成层细胞内;QRT-PCR结果显示POMC在黑色绵羊皮肤中mRNA的相对表达量为2.5004±0.2577^**,而在白色绵羊皮肤中POMC的mRNA的相对表达量为1.0032±0.0350,黑色绵羊皮肤中POMC的mRNA的相对表达量为白色绵羊皮肤的2.5倍,二者表达差异极显著（P＜0.01）;Western boltting结果显示黑色绵羊皮肤POMC蛋白水平相对表达量为1.5253±0.0426^*,而白色皮肤中POMC蛋白水平相对表达量为1.0005±0.0180,黑色皮肤POMC蛋白表达量是白色皮肤的1.55倍（P＜0.05）,两者差异显著。【结论】试验表明POMC可在黑色和白色绵羊皮肤中正常表达,且其表达位置一致,但POMC在不同毛色绵羊皮肤组织中mRNA和蛋白的表达水平存在一定的差异,表明POMC的表达量变化会在一定程度上影响毛色的生成。