肌肉异位表达pGHRH的两种质粒对大鼠的促生长作用

构建了两种猪生长激素释放激素(GHRH)的肌肉特异表达质粒ppG-A53f-H6-GHRH(ppG-H6)及ppG-A53f-H4-GHRH(ppG-H4).通过对大鼠进行肌肉注射,并于注射后分别记录0,3,7,11,14,17,20,24,27,31,34,38以及第41天的体重变化结果,同时分析了第20、27、34和第41天的血清生长激素(GH)浓度,以研究促生长表达质粒在动物体内的促生长作用.通过研究表明,外源的GHRH表达质粒在肌肉中获得了表达,并使动物体内的GH水平维持在一个相对稳定的较高水平上,导致的直观效果表现为:通过肌肉注射表达质粒ppG-H6对大鼠的生长有促进效果,在第34天的净增重显著于对照组((200.57±3.99)gvs(185.85±9.45)g,P<0.05).实验结果预示,利用注射肌肉特异异位表达GHRH蛋白可能促进动物生长.     

共表达生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)对动物生长的影响

生长激素释放激素(GHRH)与垂体腺苷酸环化酶(PACAP)在序列及功能方面均相似,且同为PACAP/胰高血糖素超家族成员．研究了这二者对生长激素释放的刺激作用,以及对动物生长的影响．构建了3个表达载体,pIRES1- GHRH-PACAP(P-G-P),pIRES1-GHRH(P-G) 及 pIRES1-PACAP(P-P),并转染到CHO细胞中,进行RT-PCR,Dot-ELISA以及Westen-blot检测．此外,给大鼠注射细胞上清表达产物,检测其生物学活性．注射8 h后,注射表达P-G-P上清的大鼠血清中IGF-Ⅰ浓度显著高于其他组(P < 0.05)．用PLGA微球包裹各种质粒,并注射到家兔后肢胫前肌．观察家兔生长情况,并于注射后0,15,30,45天时分别采集家兔血液,检测血液中IGF-Ⅰ浓度．结果显示,三质粒注射组动物体重变化及血液中IGF-Ⅰ浓度均高于对照组．注射后30天时,P-G-P组增重较对照组提高81% (P < 0.01),P-G组比对照组提高15%(P > 0.05),P-P组比对照组高7%(P > 0.05)．另一方面,P-G-P组动物血液中IGF-Ⅰ含量比分别比P-G、P-P及对照组提高16.68% (P > 0.05),17.14%(P > 0.05),50.46%(P < 0.05)．以上结果揭示：给动物注射PLGA微球包裹的共表达GHRH与PACAP质粒,可以增强动物体内生长激素(GH)的分泌,并促进动物生长．通过上述研究发现,肌肉注射PACAP表达质粒可以促进家兔的生长,PACAP和GHRH 共表达可以起到协同作用．这可能为动物的促生长研究提供新的方法．     

生长激素/胰岛素样生长因子1与阿尔茨海默病

生长激素／胰岛素样生长因子-1（GH／IGF-1）轴的合成、分泌、调节及生物学活性与阿尔茨海默病（AD）有密切关系。生长激素（GH）的合成和分泌受生长激素释放激素（GHRH）正向调节。GH／IGF-1轴活性下降导致一系列生理功能变化。GH／IGF-1缺乏可引起衰老及神经退行性变（AD）而导致认知功能的下降,相应激素的补给可以抑制或逆转这种认知障碍。越来越多的证据表明：GH／IGF-1参与AD型痴呆病理过程,对AD有很好的治疗应用前景。本文就生长激素／胰岛素样生长因子1在AD发病中的机理和药理学研究做一综述。     

达氏鲟生长激素基因cDNA克隆、表达及免疫荧光定位研究

为研究达氏鲟(Acipenser dabryanus)生长激素(Growth Hormone, GH)基因的功能, 合成了达氏鲟垂体SMART cDNA, 克隆得到GH全长cDNA序列。达氏鲟GH全长cDNA序列为1008 bp, 由52 bp的5'端非编码区(Untranslated region, UTR)、编码214个氨基酸的645 bp开放阅读框(Open reading frame, ORF)和311 bp的3'UTR构成。运用GH氨基酸序列构建进化树分析发现, 达氏鲟与两栖类、爬行类和哺乳类的一致性要高于真骨鱼类。实时荧光定量PCR结果表明, 达氏鲟GH mRNA主要在垂体和下丘脑中表达, 且垂体中GH的表达量约为下丘脑的110倍; Western-blot研究结果与qRT-PCR一致, 仅在垂体和下丘脑中检测到生长激素蛋白, 且垂体中GH的表达量远高于下丘脑。免疫荧光定位结果显示, GH主要定位于垂体中部, 下丘脑中也有少量荧光信号; 苏木精-伊红组织切片染色研究表明, GH主要是由嗜酸性的生长激素分泌细胞分泌。研究为深入研究脊椎动物生长激素基因的进化和人工养殖达氏鲟的生长调控提供了基础。       

SRIF及CSH对斜带石斑鱼脑垂体生长激素合成和分泌的调控

斜带石斑鱼 (Epinepheluscoioides)属于雌性先成熟、具有性转变的雌雄同体鱼类。生长激素释放抑制因子 (SRIF)是鱼类生长激素 (GH)分泌的主要抑制性调节剂 ,半胱胺 (CSH)可抑制SRIF的作用。本文采用静态孵育系统 ,应用RPA及RIA研究SRIF及CSH对斜带石斑鱼GHmRNA表达及GH分泌的调节。结果显示 ,SRIF能以剂量依存方式抑制斜带石斑鱼脑垂体释放GH ,时间越长作用越强。但SRIF作用 2 4h对GHmR NA水平的影响不显著 ,表明SRIF是斜带石斑鱼GH释放的抑制性调节剂 ,对GHmRNA的表达没有明显影响。较低剂量的CSH (10 -4- 10 -2 mol/L)使斜带石斑鱼的GH释放量增加 ,较高剂量 (10 -1mol/L)的CSH引起的GH增加趋势减缓 ,这种现象可能与较高剂量的CSH不仅抑制下丘脑SRIF的释放 ,同时影响GHRH的释放 ,使得GH的分泌量增幅下降有关 ;无论是较高剂量还是较低剂量的CSH都不能使GHmRNA的水平增加 ,表明CSH只能引起GH的释放量增加 ,不影响GH的合成。GnRH与CSH共同作用引起的GH释放量明显高于CSH单独作用的效应 ,其主要原因是由于GnRH促进GHmRNA的表达所致     

生长激素和生长激素受体的多样性

生长激素及其受体对动物生长发育起着重要的作用。转录过程选择性剪接和存在多种降解途径可能是GH或GHR产生多样性的原因。随着GH结构形态的改变,其功能也在发生变化。GH基因的多样性对鸡的抗病选择性反应与产蛋性能有相关,GH和GHR基因的多样性会影响奶牛的产奶生产性能。GHR的分子多样性可能导致动物生长发育模式的变异,例如动物的矮小病。     

生长激素促分泌素受体(GHS-R)的发现及影响

生长激素的分泌不仅受到GHRH、SRIF的调节 ,还受到GHS R及其配体GHS的作用。本文综述了GHS R的发现背景、结构特点以及生理功能 ,讨论了GHS R促GH分泌的分子机制及其对多种组织器官可能影响 ,同时还探讨了除Ia和Ib亚型外还存在其它GHS R亚型的可能性。     

鱼类生长激素生物活性和定量免疫测定技术的研究进展

生长激素(growth hormone, GH)是调节鱼类生长、发育和代谢的一种重要激素.为了弄清鱼体内GH水平与鱼体生长的关系, 以及研究外源GH基因在受体鱼体内的表达部位、表达效率和表达调控等问题, 首先必须建立鱼类GH灵敏、特异的微量检测技术.此外, 在进行鱼类GH分离纯化中, 很重要的一点就是鉴定所获得的GH制品是否具有生物活性, 同样也需要建立鱼类GH灵敏、特异的生物活性检测技术.     

6种重要经济鱼类生长激素完整cDNA的克隆和序列分析

通过RT-PCR、3′RACE、5′-RACE方法,从6种重要经济鱼类——大眼鳜(Siniperca kneri)、石斑鱼(Epinephelus coioides)、黄鳝(Monopterus albus)、鲶鱼(Silurus asotus)、泥鳅(Misgurnus anguillicaudatus)和方正银鲫(Carassius auratus gibelio Bloch,Fang Zheng crucian carp)中克隆了生长激素(Growth Hormone,GH)的完整cDNA序列(除石斑鱼序列外,其他生长激素序列均系第一次克隆),并详细分析了其序列特征。测序结果显示,克隆的6种GH cDNA长度依次为953bp、1023bp、825bp、1082bp、1154bp和1180bp,它们均包含一个长度为600个左右核苷酸的完整阅读框,分别编码一个200个左右氨基酸的蛋白：大眼鳜、石斑鱼和黄鳝GH为204个氨基酸,鲶鱼GH为200个氨基酸,泥鳅和方正银鲫GH为210个氨基酸。这6种蛋白序列与其他已知的鱼类GH序列都有较高的同源性,特别是与相同目的鱼类序列相比。通过序列比对,在这些蛋白序列内鉴定了许多保守的氨基酸残基,其中的大多数聚集而成5个保守域。基于这6种鱼类序列的编码区和其他鱼类的GH编码序列进行分子系统学分析,结果(MP和NJ树)与根据形态特征构建的系统发育树基本一致,特别是在硬骨鱼类较大分类阶元(目间、目以上)的系统发育研究方面比较一致,尽管仍存在一定差异,说明生长激素基因的编码区应该在硬骨鱼类系统发育研究领域得到更多的重视。     

生长激素释放激素和人血清白蛋白融合蛋白的克隆表达

目的:通过与人血清白蛋白(HSA)融合,延长生长激素释放激素(GHRH)在体内的半衰期。方法:根据毕赤酵母偏爱密码子重新设计GHRH的核酸序列,并通过化学合成和重叠PCR法将GHRH的N端与HSA的C端通过一个11肽的接头连接,获得GHRH和HSA融合的全长基因序列。构建pPIC9-HSA-GHRH表达载体,电击转化毕赤酵母GS115感受态细胞,通过表型筛选和诱导表达实验得到蛋白表达工程菌,对表达产物进行分离纯化和生物学活性分析。结果:克隆了HSA-GHRH融合基因,构建了pPIC9-HSA-GHRH融合表达载体;电击转化后通过表型筛选和诱导表达实验得到蛋白表达工程菌;经分离纯化后,对表达产物的生物学活性分析显示其在体内有促进生长的作用。结论:与人血清白蛋白的融合有效地提高了GHRH的表达水平,并延长了GHRH的半衰期。     

PLGA Microsphere-Mediated Growth Hormone Release Hormone Expression Induces Intergenerational Growth

To improve animal growth, growth hormone-releasing hormone (GHRH) expression vectors that maintain constant GHRH expression can be directly injected into muscles. To deliver the GHRH expression vectors, biodegradable microspheres have been used as a sustained release system. Although administering GHRH through microspheres is a common practice, the intergenerational effects of this delivery system are unknown. To investigate the intergenerational effects of polylactic-co-glycolic acid (PLGA) encapsulated plasmid-mediated GHRH supplements, pCMV-Rep-GHRH microspheres were injected into pregnant mice. Growth and expression of GHRH were measured in the offspring. RT-PCR and immunohistochemistry reveal GHRH expression 3–21 days post-injection. The proportion of GH-positive cells in the GHRH treated offspring was 48.2% higher than in the control group (P < 0.01). The GHRH treated offspring were 6.15% (P < 0.05) larger than the control offspring. At day 49 post-injection, IGF-I serum levels were significantly higher in the treatment group than in the control group. This study confirms that intramuscular expression of GHRH mediated by PLGA microspheres significantly enhances intergenerational growth.     

Growth hormone responses to growth hormone-releasing hormone (1-29)-NH2 and a D-Ala2 analog in normal men

Synthetic analogs of growth hormone-releasing hormone, GHRH(1-29)-NH2 and D-Ala2 GHRH(1-29)-NH2 were administered as a bolus intravenous injection to five normal men in a dose range of 0.015 to 0.5 micrograms/kg body weight. Vehicle only was administered in a control study. Peak responses to GHRH analogs occurred at 15 or 30 min. An increase in the integrated plasma growth hormone (GH) response was observed at each dose. The dose-response curve of GHRH(1-29)-NH2 indicated that it has a similar molar potency to GHRH(1-40) and GHRH(1-44). The potency of D-Ala2 GHRH(1-29)-NH2 was approximately twice that of GHRH(1-29)-NH2. Neither analog affected blood levels of PRL, TSH, LH, FSH, ACTH, insulin, glucagon, glucose, cortisol, free thyroxine, and free triiodothyronine. No side effects were noted other than transient flushing with the highest dose administered. The findings demonstrate GHRH(1-29)-NH2 and its D-Ala2 analog are potent stimulators of GH release and have potential application in clinical medicine.     

Chronic orchidectomy does not influence the sensitivity of the pituitary somatotropes to varying doses of GHRH administered intravenously to the adult male rhesus monkey

In the present study, the pituitary growth hormone (GH) response to graded doses of GH-releasing hormone (GHRH) was determined in intact (n = 3) and chronically orchidectomized (n = 3) adult rhesus monkeys (Mucaca mulatta). GHRH in doses of 0, 6.25, 12.5 and 25 microg/kg BW was infused through a teflon cannula implanted in the saphenous vein. Blood samples were collected 60 min before and 90 min after the injection of the neurohormone at 15 min intervals. All bleedings were carried out under ketamine hydrochloride anesthesia. The plasma levels of GH were determined by using AutoDELFIA time-resolved flouroimmunoassay, whereas plasma levels of testosterone and estradiol were determined using specific radioimmunoassay systems. The GH responses to GHRH were not significantly different between intact and chronically orchidectomized monkeys at any of the dose levels tested (p > 0.05). The administration of GHRH resulted in a significant (p < 0.05) stimulation of GH secretion at all the doses tested and in both the groups studied. In both intact and orchidectomized animals, the greatest response was observed at 6.25 microg/kg and no further increase was noted with the higher doses of GHRH. In conclusion, the present study suggests that chronic orchidectomy does not influence the sensitivity of the pituitary somatotropes to GHRH stimulation implying that the responsiveness of the pituitary somatotropes to GHRH is independent of testicular steroid modulation.     

Anatomy of the Hypophysiotropic Somatostatinergic and Growth Hormone-releasing Hormone System Minireview

The central control of growth hormone (GH) secretion from the pituitary gland is ultimately achieved by the interaction between two hypothalamic neurohormones, somatostatin which inhibits and growth hormone-releasing hormone (GHRH) which stimulates GH release. The regulation of the somatostatin and GHRH release from the hypothalamus is regulated by a range of other neuropeptides, neurotransmitters, neurohormones. In this mini review we attempt to provide a short summary covering the anatomy and chemical characteristics of the various cell populations regulating GH secretion as a tribute to Miklós Palkovits who pioneered the field of functional neuroanatomy of hypothalamic networks.Special Issue Dedicated to Miklós Palkovits.     

睡眠中间断低氧对大鼠下丘脑-垂体-肾上腺轴和生长激素的影响

目的:探讨睡眠中间断低氧对大鼠下丘脑-垂体-肾上腺轴和生长激素水平的影响.方法:大鼠分别给予吸入空气,持续低氧和间断低氧气体,在1 d,3 d,7 d和30 d后测定下丘脑促肾上腺皮质激素释放激素(CRH)和生长激素释放激素(GHRH)mRNA水平,并测定30d后血浆CRH,GHRH,促肾上腺皮质激素(ACTH)和皮质酮水平,分析其间的变化关系.结果:与对照组比较,在低氧后1 d,3 d,7 d后大鼠下丘脑CRH mRNA升高,GHRH mRNA降低,在30 d后,间断低氧组下丘脑CRH mRNA升高,GHRH mRNA降低,而持续低氧组则接近正常.间断低氧30 d后,血浆CRH、ACTH,皮质酮均升高,GHRH降低,而生长激素没有明显变化.结论:大鼠睡眠中慢性间断低氧可以引起下丘脑-垂体-肾上腺轴激素水平升高,反馈调节紊乱,可引起GHRH分泌抑制.     

Growth hormone and ghrelin secretion in severely obese women before and after bariatric surgery

Objective: The objective was to evaluate ghrelin and growth hormone (GH) interactions and responses to a growth hormone‐releasing hormone (GHRH)/arginine test in severe obesity before and after surgically‐induced weight loss. Research Methods and Procedures: Our study population included 11 severely obese women 39 ± 12 years of age, with a mean BMI of 48.6 ± 2.4 kg/m², re‐studied in a phase of stabilized body weight, with a BMI of 33.4 ± 1.2 kg/m², 18 months after having successfully undergone biliopancreatic diversion (BPD). A GHRH/arginine test was performed before and 18 months after BPD to evaluate ghrelin and GH interactions. Active ghrelin, measured by radioimmunoassay (RIA), and GH, measured by chemiluminescence assay, were assayed before and after the GHRH/arginine test. Results: Fasting serum GH levels and GH area under the curve (AUC) significantly increased from 0.2 ± 0.05 ng/mL to 1 ± 0.3 ng/mL (p < 0.05) and from 514.76 ± 98.7 ng/mL for 120 minutes to 1957.3 ± 665.1 ng/mL for 120 minutes after bariatric surgery (p < 0.05), respectively. Although no significant change in fasting ghrelin levels was observed (573 ± 77.9 before BPD vs. 574.1 ± 32.7 after BPD), ghrelin AUC significantly increased from ?3253.9 ± 2180.9 pg/mL for 120 minutes to 1142.3 ± 916.4 pg/mL for 120 minutes after BPD (p < 0.05). Fasting serum insulin‐like growth factor (IGF)‐1 concentration did not change significantly (133.6 ± 9.9 ng/mL before vs. 153.3 ± 25.2 ng/mL after BPD). Discussion: Our study demonstrates that the mechanisms involved in ghrelin and GH secretion after the secretagogue stimulus (GHRH/arginine) are consistent with patterns observed in other populations.     

MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk

Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.