斜带石斑鱼淋巴器官个体发育的组织学

本文应用连续组织切片技术和组织学观察,对出膜后1～60天的斜带石斑鱼(Epinephelus coioides)各期仔鱼、稚鱼和幼鱼的淋巴器官组织进行了研究,描述了淋巴器官的个体发育过程和组织学结构特征。研究表明：实验水温为22．0～27．8℃时,孵化后第10天出现头肾原基。头肾原基由未分化的造血干细胞组成。随着鱼体的生长,头肾原基的造血干细胞很快分化成不同类型的细胞;头肾主要由网状内皮系统支持下的淋巴造血组织构成。第11天出现脾脏原基。脾脏原基由造血细胞组成,淋巴化速度相对较慢。脾脏在整个发育过程中,红细胞和类红细胞占优势,没有红髓和白髓之分。第13天出现胸腺原基。胸腺发育速度较快,是明显的淋巴器官。胸腺主要由胸腺细胞(淋巴细胞)和上皮细胞组成,外区和内区没有明显的界限,但很容易区分。胸腺外被单层的上皮细胞层与咽腔相隔,保持浅表的位置,并且在整个发育过程中,胸腺与头肾是独立分开的。免疫器官原基出现顺序是头肾、脾脏和胸腺;而免疫器官淋巴化的顺序是胸腺,头肾和脾脏。和其它硬骨鱼类一样,斜带石斑鱼在早期发育阶段,淋巴器官的发育较迟,出现相对滞后的现象[动物学报49(6)：819～828,2003]。     

体重对斜带石斑鱼能量收支的影响

鱼类能量学是研究能量在鱼体内转换的学科,其核心问题之一是能量收支各组分之间的定量关系及其各种因子(如温度[1—5]、盐度[6]、体重[7—11]、性别[12]、摄食水平[13—18]、饵料种类[19,20]等)的影响作用。欧美等发达国家对鱼类能量学研究起步较早,迄今已经初步建立了多种鱼类的能量收支模式[21,22];国内在该领域较系统的研究起始于90年代初[23—25],主要局限于淡水鱼类,近年来又对海水鱼类进行了大量研究[26]。斜带石斑鱼(Epinephelus coioides)俗称青斑,为暖水性中下层鱼类,是广东省海水网箱养殖的主要品种之一。斜带石斑鱼分布于西太平洋的硫球群岛、澳大利亚以及贝劳和菲济群岛的东部,常栖息于大陆沿岸和大岛屿,但在河口和离岸100m深的水域中也可发现[27]。通过本项研究将有助于揭示海洋暖水性中下层鱼类的能量学特征。1材料与方法1·1材料来源与驯养实验用斜带石斑鱼,采自广东省大亚湾水产试验中心。实验用斜带石斑鱼经淡水浸泡10min后,置于室内容积0·5t桶内驯养,待摄食和生长趋于正常后,开始实验。驯化时间为30d。实验于2006年10—11月在广东省大亚湾水产试验中心进行。1...     

斜带石斑鱼(♀)×鞍带石斑鱼(♂)受精细胞学的组织切片观察

人工诱导雌核发育二倍化的关键是掌握极体排出和卵裂的时机,掌握该规律最直接的方式即进行受精细胞学的研究。以鞍带石斑鱼(Epinephelus lanceolatus)为父本,斜带石斑鱼(Epinephelus coioides)为母本,通过人工授精获得受精卵,收集受精后不同时刻的卵子,经Smith′s液固定、石蜡包埋、切片、苏木精-伊红染色和显微组织观察。结果表明:受精后第30~60 s精子入卵;第3 min精子星光出现;第5 min卵子排出第二极体;第7~15 min雌雄原核相互靠近,并最终融合;第15 min以后合子核开始分裂。鞍带石斑鱼精子进入斜带石斑鱼卵子的时间与多数硬骨鱼类比较没有显著差别;但是,第二极体排出及第一次卵裂的时间较早。研究展示了斜带石斑鱼与鞍带石斑鱼杂交的受精细胞学的基本发育过程,并为后续的人工诱导石斑鱼雌核发育研究提供了理论依据。     

斜带石斑鱼胸腺的显微和超微结构

本文通过解剖及组织切片技术、光学显微镜、透射和扫描电子显微镜技术,对斜带石斑鱼(Epinephelus coioides)胸腺器官组织进行了观察研究。结果表明:斜带石斑鱼胸腺实质主要由胸腺细胞(淋巴细胞)和网状上皮细胞构成。鱼体从Ⅰ龄之后,其胸腺发生明显的变化,与幼鱼有所不同,主要是胸腺可明显区分为三个区域:胸腺外皮质区、内皮质区和髓质区。外皮质区主要由网状上皮细胞、黏液细胞、成纤维细胞和少量淋巴细胞构成,细胞排列疏松;内皮质区主要由密集的淋巴细胞和网状上皮细胞组成,以含有大量的淋巴细胞为特征;髓质区主要由淋巴细胞和较多的网状上皮细胞构成,总体特征是淋巴细胞数量比内皮质区的少,且细胞排列较疏松。外皮质区、内皮质区相当于高等脊椎动物的皮质;髓质区相当于高等脊椎动物的髓质。髓质区之下有结缔组织,在Ⅱ龄以上的成体出现胸腺小体(Hassall's corpuscles)或类似胸腺小体的结构,而且随着年龄的增加,胸腺外皮质区增厚,结缔组织增加,还表现在内皮质区和髓质区组织逐渐萎缩变薄,胸腺的细胞组成类型和淋巴细胞数量上有所变化等等。这些现象在Ⅱ龄鱼开始出现,即胸腺呈现退化迹象,在Ⅲ龄以上鱼体呈现明显的退化和萎缩。胸腺表面扫描电镜结果表明:其上皮细胞表面具有微嵴以及由微嵴组成的指纹状结构,有一些微孔分布。透射和断面扫描电镜的结果进一步表明:胸腺组织内的细胞成分复杂,除了淋巴细胞和网状上皮细胞外,还具有巨噬细胞、肥大细胞、肌样细胞、浆细胞、指状镶嵌细胞和纤维细胞等。     

斜带石斑鱼(♀)×鞍带石斑鱼(♂)杂交子代(青龙斑)消化系统的早期发育

本文研究斜带石斑鱼(Epinephelus coioides)(♀)×鞍带石斑鱼(E.lanceolatus)(♂)杂交子代(青龙斑)仔、稚、幼鱼的消化系统发育,描述了其消化器官发育过程和组织学结构特征,充实青龙斑生物学研究文库,为其发育生物学研究和苗种培育提供技术支撑。青龙斑苗种培育于2012年6~8月期间进行。水温为(30±1)℃,盐度为28±1。利用形态学和连续组织切片技术,对出膜后0~40日龄幼鱼的消化系统进行了观察和研究。消化系统发育可划分为内源性营养、混合营养和外源性营养3个阶段:0至3日龄为内源性营养阶段,初孵仔鱼消化管为一简单的直形管,卵黄囊大,椭圆形,口和肛门尚未与外界相通;口腔中出现鳃弓的雏形,3日龄仔鱼食道由2~3层的复层立方上皮细胞组成,形成较低的褶皱;胃与小肠和食道的分界明显,上皮由单层柱状细胞组成;肠道分化,肛门开通体外,开始摄食;肝细胞团和胰腺细胞团形成。4~5日龄为混合营养阶段,6日龄之后进入外源性营养阶段,卵黄囊已经完全被吸收,前、中、后肠和直肠区分明显,肠黏膜上皮中出现少量的杯状细胞,由肠腔面向深层依次可以分为黏膜层、黏膜下层和浆膜层,肌层不明显。至25日龄,消化系统的结构和功能已经较为完善。38日龄时,胃、幽门盲囊、肠以及直肠各段分界明显,黏膜褶皱高度为前肠中肠后肠;肌层厚度为后肠前肠中肠;消化道和消化腺组织结构与成鱼基本相同。青龙斑的消化系统发育和分化是与其生理功能的逐步完善同步的。     

斜带石斑鱼PACAP的原核表达及活性分析

自1989年从绵羊下丘脑提取物发现垂体腺苷酸环化酶激活多肽（Pituitary adenylate cyclase activating polypeptide,PACAP）以来（Miyata et al．,1989）,已证明它能促进垂体激素释放,同时还具有神经递质、神经调质和神经营养等作用,使对PACAP的研究成为十分活跃的领域。PACAP属于血管活性肠肽（VIP）-胰高血糖素-生长激素释放因子-分泌素家族（Campbell and Scanes,1992）成员,已鉴别出包含27和38个氨基酸两种类型。对原索动物（McRory et al．,1997）、两栖类（蛙）（Alexandre et al．,2000）、爬行类（蜥蜴）（Pohland Wank,1998）、鸟类（鸡）（McRory et al．,1997）,啮齿类（鼠）（Ghatei et al．,1993）等脊椎动物PACAP的研究多集中在结构与进化方面,对功能了解甚少。     

斜带石斑鱼黏膜免疫系统结构的研究

对斜带石斑鱼皮肤、眼角膜、鳃、前肠及后肠等黏膜相关免疫组织结构进行了研究。通过组织切片的H E染色及透射电镜的观察 ,描述了黏膜组织的显微结构、主要免疫相关细胞在黏膜组织中的分布情况及各免疫细胞的超微结构。结果表明黏膜组织中存在杯状细胞、淋巴细胞、巨嗜细胞、单核细胞、嗜曙红细胞、嗜中性粒细胞等免疫相关细胞 ,具有在黏膜局部独立完成免疫应答的细胞基础。另外 ,还观察到了皮肤表皮对异物的吞噬过程。本文还就黏膜免疫组织的非特异和特异性免疫应答 ,以及黏膜免疫是否独立于系统免疫等问题进行了讨论。     

斜带石斑鱼仔鱼变态过程中甲状腺的发育变化

斜带石斑鱼(Epinephelus coioides)属鲈形目,科,石斑鱼属,为暖水性中下层鱼类,是我国南方沿海重要的经济养殖鱼类。长期以来,许多海水鱼仔稚鱼培育阶段的一大难题是成活率低1。目前斜带石斑鱼的大规模苗种培育仍未很好解决,仔鱼经历第二背鳍棘从生长到吸收的变态过程时死亡率很高。       

斜带石斑鱼CD4基因克隆和组织表达分析

CD4 作为TCR的共受体可以提高TCR/抗原-MHC复合体的稳定性,辅助TCR识别抗原,并且参与T细胞活化.本研究从斜带石斑鱼Epinephelus coioides头肾中克隆得到全长2240 bp的CDM cDNA序列,该序列包含长1410 bp的ORF,编码469个氨基酸,预测蛋白分子包含一段信号肽,4个Ig样区...     

重组斜带石斑鱼生长激素及其抗血清在放射免疫测定中的应用

将斜带石斑鱼(Epinephelus coioides)生长激素成熟多肽cDNA序列克隆到质粒pRSET,与6x组氨酸等原核编码序列融合获得重组质粒pRGH6,转入大肠杆菌BL21(DE3),获得高效表达,表达量占细菌总蛋白的43％。免疫印迹证明表达产物为含斜带石斑鱼生长激素的融合蛋白,Ni^2 亲合层析柱纯化融合蛋白,以此为抗原免疫家兔制备特异性的抗血清。以纯化的重组生长激素和特异性的抗血清建立斜带石斑鱼生长激素的放射免疫测定法,该方法的灵敏度、特异性和重复性均达到测定血液生长激素的水平。研究了多巴胺的受体激动剂阿扑吗啡对静态孵育斜带石斑鱼脑垂体碎片释放生长激素的影响,结果表明,阿扑吗啡能以剂量依存方式促进斜带石斑鱼垂体释放生长激素。     

斜带石斑鱼阿片黑素促皮质素原全长cDNA克隆及序列分析

通过构建斜带石斑鱼垂体cDNA库和随机测序,克隆了其阿片黑素促皮质素原(Proopiomelanocortin,POMC)全长cDNA。斜带石斑鱼POMC全长cDNA为863bp(含Poly(A)),编码的POMC多肽前体为219aa。氨基酸序列比较分析表明：斜带石斑鱼POMC前体包含有促肾上腺皮质激素(Adrenocorticotropin,ACTH),α—促黑素(α—melanocyte stim—datinghormone,α—MSH),β—促黑素(β—MSH),γ—促脂素(γ-lipotrophic hormone,γ-LPH),β—内啡肽(β-endorphin)等,但缺失了γ—促黑素(γ-MSH)和大部分连接区。斜带石斑鱼POMC与哺乳动物POMC的同源性为39％—42％左右,与鸟类的同源性为42％左右,与两栖类的同源性为36％—41％,与其他鱼类POMC的同源性为38％—77％。斜带石斑鱼和罗非鱼的POMC的ACTH功能区都为40个氨基酸残基,而其他脊椎动物为39个氨基酸残基。     

六种植物对Pb的吸收与耐性研究

 为了选择和筛选重金属Pb的耐性与富集植物,在温室砂培盆栽条件下对铅锌尾矿区附近生长的6种植物(山野豌豆（Vicia amoena Fisch）、草木樨(Melilotus suavena Ledeb)、披碱草(Elymus dahuricus Turca)、酸模（Rumex acetosa）、紫苜蓿(Medicago sativa)和羽叶鬼针草(Bidens maximowicziana Oett))体内Pb的含量与分布、重金属Pb的迁移总量、根系的耐性指数做了研究;拟定了６种植物对Pb的耐性临     

侧脑室注射牛胰多肽对家兔糖耐量和血清胰岛素浓度的影响

在家兔侧脑室内微量注射牛胰多肽能增加其糖耐量及血清胰岛素浓度。这种作用与牛胰多肽注射剂量有明显依从关系,并被预先皮下注射阿托品所部分阻断。结果提示,中枢给予牛胰多肽可能部分通过迷走神经途径增加血清胰岛素的释放而增加糖耐量。     

Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells

The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ～20%. Consistent with this, analysis of GU in muscle cells from α1(-/-)/α2(-/-) AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ～35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.     

Metabolic Reprogramming of Macrophages: GLUCOSE TRANSPORTER 1 (GLUT1)-MEDIATED GLUCOSE METABOLISM DRIVES A PROINFLAMMATORY PHENOTYPE*

Glucose is a critical component in the proinflammatory response of macrophages (MΦs). However, the contribution of glucose transporters (GLUTs) and the mechanisms regulating subsequent glucose metabolism in the inflammatory response are not well understood. Because MΦs contribute to obesity-induced inflammation, it is important to understand how substrate metabolism may alter inflammatory function. We report that GLUT1 (SLC2A1) is the primary rate-limiting glucose transporter on proinflammatory-polarized MΦs. Furthermore, in high fat diet-fed rodents, MΦs in crown-like structures and inflammatory loci in adipose and liver, respectively, stain positively for GLUT1. We hypothesized that metabolic reprogramming via increased glucose availability could modulate the MΦ inflammatory response. To increase glucose uptake, we stably overexpressed the GLUT1 transporter in RAW264.7 MΦs (GLUT1-OE MΦs). Cellular bioenergetics analysis, metabolomics, and radiotracer studies demonstrated that GLUT1 overexpression resulted in elevated glucose uptake and metabolism, increased pentose phosphate pathway intermediates, with a complimentary reduction in cellular oxygen consumption rates. Gene expression and proteome profiling analysis revealed that GLUT1-OE MΦs demonstrated a hyperinflammatory state characterized by elevated secretion of inflammatory mediators and that this effect could be blunted by pharmacologic inhibition of glycolysis. Finally, reactive oxygen species production and evidence of oxidative stress were significantly enhanced in GLUT1-OE MΦs; antioxidant treatment blunted the expression of inflammatory mediators such as PAI-1 (plasminogen activator inhibitor 1), suggesting that glucose-mediated oxidative stress was driving the proinflammatory response. Our results indicate that increased utilization of glucose induced a ROS-driven proinflammatory phenotype in MΦs, which may play an integral role in the promotion of obesity-associated insulin resistance.     

The effects of ketamine anesthesia on glucose clearance in African green monkeys

Ketamine hydrochloride's effect on glucose clearance-insulin secretion during intravenous glucose tolerance testing was studied in five African green monkeys. Animals were tested with ketamine anesthesia and then had indwelling cannulas implanted and were retested both in the presence and absence of ketamine anesthesia. Serum glucose and insulin concentrations were determined. There were no significant differences in the glucose clearance rate (K value), basal glucose and insulin concentrations, maximum insulin concentration, and area under the insulin response curve, among the three different conditions.     

Glucose Metabolism and Regulation in Lactating Mink (Mustela Vison) - Effects of Low Dietary Protein Supply

Eighteen lactating mink raising litters of 6 to 7 kits were fed ad libitum from parturition on diets with 32% of ME derived from protein and decreasing fat:carbohydrate ratios [high fat:low carbohydrate (HFLC): 67:1, medium fat:medium carbohydrate (MFMC): 52:16, low fat:high carbohydrate (LFHC): 37:31]. Four weeks post partum the dams were fitted with a jugular vein catheter, and the experiment started with a 3 hours fasting period, after which the dams were fed 210kJ ME of the experimental diets. Blood samples were collected 10 and 5min before feeding and 30, 60, 90, 120, 150 and 180min postprandially. Two hours postprandially a single dose of 50µCi U-¹⁴C-labelled glucose was administered to each dam and blood samples were collected 5, 10, 20, 30, 45 and 60min after the tracer administration. Plasma concentrations of glucose and insulin 30 to 120min postprandially were higher in dams fed the LFHC diet, than in dams fed the HFLC diet, values for dams fed the MFMC diet being intermediate. Plasma glucagon concentrations were not significantly affected by dietary treatment. The glucagon:insulin ratios decreased postprandially in all dams, the response being significant in dams fed the LFHC diet. Plasma concentrations of urea were not significantly affected by dietary treatment. Plasma FFA concentrations tended to increase postprandially in dams fed the HFLC diet. Glucose turnover rates were approximately 4.0% permin in all dams, irrespective of dietary treatment. However, the daily glucose flux was lower in dams fed the HFLC diet than in dams fed the LFHC diet, and tended to be lower than in dams fed the MFMC diet. In conclusion, a dietary protein supply of 32% of ME simultaneously with a carbohydrate supply of 16% or 31% of ME had no adverse effects on glucose homeostasis or glucose metabolism in lactating mink.