拟穴青蟹GnRH-R的免疫识别

促性腺激素释放激素(GnRH)及其受体(GnRH-R)对生殖具有重要的调控作用.迄今无脊椎动物GnRH及GnRH-R的研究尚少.本研究采用兔抗人GnRH-R、兔抗果蝇GnRH-R和兔抗海鞘GnRH-R的抗体,应用免疫印迹和免疫共沉淀技术对拟穴青蟹(Scyllaparamamosain)GnRH-R进行了免疫识别,所得结果如下:1)经免疫印迹检测,拟穴青蟹脑、胸神经团、视神经节和精巢中共有的免疫阳性条带分子质量为45~55 ku.脑和胸神经团中另有一条36 ku条带;在精巢中,兔抗人GnRH-R和兔抗果蝇GnRH-R抗体只有一条28 ku条带,而兔抗海鞘GnRH-R抗体显示有28,36和30 ku的条带.2)利用免疫共沉淀技术,分离出与GnRH-R抗体相结合的两种物质,分子质量分别为38.1和54.0 ku,分别与哺乳动物和非哺乳动物GnRH-R的分子质量接近.该发现提示拟穴青蟹体内存在GnRH-R,为深入了解甲壳动物生殖调控机理提供了理论依据.     

雌激素受体在锯缘青蟹视神经节的免疫识别

利用免疫细胞化学SABC法对锯缘青蟹(Scylla serrata)视神经节雌激素受体(ER)进行定位研究.结果表明:视神经节的视外髓、视内髓和视端髓存在ER免疫阳性细胞.视神经节存在的ER免疫阳性细胞可以为雌激素提供结合位点.本研究为雌激素参与视神经节神经内分泌调节作用提供了形态学依据.     

拟穴青蟹β-肌动蛋白基因的克隆与分析

β-肌动蛋白广泛存在于真核生物中,在维持细胞结构、细胞运动和细胞分裂等生理活动中发挥着重要作用.运用RACE技术克隆了拟穴青蟹(Scylla paramamosain)β-肌动蛋白基因,并用RT-PCR方法检测该基因在成体各组织中的表达情况.拟穴青蟹β-肌动蛋白cDNA全长1 337 bp,5′端非编码区为67 bp,3′端非编码区为139 bp,开放阅读框1 131 bp编码376个氨基酸.拟穴青蟹β-肌动蛋白与其他节肢动物β-肌动蛋白氨基酸序列的相似性高达98％～99％.系统进化树显示拟穴青蟹β-肌动蛋白基因的分子进化地位与其生物学分类地位一致.半定量RT-PCR分析结果表明,β-肌动蛋白基因在拟穴青蟹视神经节、脑神经节、胸神经节、性腺、鳃、心、胃、肌肉、肝胰腺共9个组织器官中的表达基本一致,具有良好的稳定性.     

拟穴青蟹细胞核DNA含量的测定

以拟穴青蟹(Scylla paramamosain)的血淋巴细胞为材料,刀额新对虾(Metapenaeus ensis)的血淋巴细胞为参照系,利用倍性分析仪结合外标定法测定其细胞核DNA含量.拟穴青蟹的血淋巴的细胞核DNA含量是2.677 pg/2c.此外,本文作者还发现拟穴青蟹血淋巴与肌肉组织细胞核DNA含量存在差异.报道了我国拟穴青蟹细胞核DNA含量,并且提供了拟穴青蟹的细胞遗传学特征,为蟹类系统重建与演化过程分析提供新资料.     

锯缘青蟹视神经节FSH和LH的免疫识别

甲壳动物的视神经节含有性腺抑制激素(Gonad inhibiting hormone, GIH),长期以来视神经节被理解为生殖抑制效应.本研究应用脊椎动物卵泡刺激素(Fllicle stimulating hormone,FSH)抗体和黄体生成素(Luteinizing hormone,LH)抗体,从锯缘青蟹(Scylla serrata)视神经节的视外髓、视内髓和视端髓检测到FSH和LH免疫细胞化学阳性反应.锯缘青蟹视神经节中的FSH和LH免疫阳性物质,可能参与了生殖神经内分泌调节.     

雄性三疣梭子蟹神经器官GnRH受体的免疫组织化学研究

采用MaxVisionTM免疫组织化学方法,以兔抗人GnRH受体的多克隆抗体,对雄性三疣梭子蟹(Portunus trituberculatus)的神经器官(视神经节、脑神经节和胸神经团)进行免疫组织化学定位.结果显示:GnRH受体的免疫阳性物质在雄性三疣梭子蟹视神经节、脑神经节和胸神经团的多个部位都有较为广泛的存在,定位在神经细胞的细胞质和神经髓质中,不同神经器官GnRH受体的免疫阳性强度有一定的差异.三疣梭子蟹神经器官存在GnRH受体的免疫阳性物质,为GnRH参与其神经调节作用提供了形态学依据.     

人工感染呼肠孤病毒对拟穴青蟹部分免疫因子的影响

采用注射和浸泡的方式人工感染拟穴青蟹呼肠孤病毒,研究了其对拟穴青蟹血细胞密度以及血清中酚氧化酶(PO)、超氧化物歧化酶(SOD)和碱性磷酸酶(AKP)活力的影响.结果表明:注射(第Ⅱ组)和浸泡(第Ⅳ组)方式均能感染健康青蟹,病发死亡时间为7～9 d,死亡率达100%,血细胞平均密度在试验36～72 h间迅速上升并达最高值,其血细胞密度最高值分别为1.4008×107cells/mL与1.8243×107cells/mL;感染病毒的青蟹PO活性均大致呈现下降的趋势,其中,第Ⅳ组感染12 h青蟹PO活性相对对照组显著升高(P<0.05),其值为(6.90±1.54),达实验所测PO活性最高值;各实验组血清SOD活性呈无规律的变化;AKP活性感染组与对照组表现不同.表明测定血细胞密度以及PO和AKP活性可用来辅助诊断青蟹疾病,而SOD活性变化不能很好地表征青蟹受病毒感染的状况.     

中国东南沿海青蟹属不同种类的mtDNA COI基因序列分析及其系统发育

对采自中国东南沿海青蟹属的4种青蟹锯缘青蟹Scylla serrata(Forskal,1775),紫螯青蟹S.tranquebarica(Fabricius,1798),榄绿青蟹S.olivacea(Herbst,1796),拟穴青蟹S.paramamosain(Estampador,1949)的线粒体COI基因片段进行了测定,分析研究了其种间遗传差异及系统发育关系.研究结果显示:4种青蟹在COI基因片段上存在差异,种间序列差异远大于种内差异.青蟹属在我国的优势种与GenBank上的S.paramamosain的遗传距离为0.001 3,与S.serrata的遗传距离为0.121 3.序列特征、遗传距离和系统进化等分析结果表明中国青蟹属的优势种为拟穴青蟹S.paramamosain.     

拟穴青蟹ANT2基因的克隆与低温胁迫表达分析

腺苷酸转移酶(ANT)是线粒体中最丰富的蛋白质之一,其主要作用是介导ADP/ATP在细胞质和线粒体基质之间的运输.为探讨该基因在拟穴青蟹(Scylla paramamosain)低温适应中的作用,采用反转录PCR(RT-PCR)、cDNA末端快速扩增技术(RACE)等技术,从拟穴青蟹中获得了ANT2的cDNA全长序列,并运用实时荧光定量PCR(qPCR)技术检测了ANT2在不同组织和不同温度下的表达谱.该序列全长1 348bp,开放阅读框(ORF)为930bp,编码309个氨基酸残基.同源分析显示,该蛋白具有3个保守的线粒体跨膜功能结构域,形成能量分子传导转运通道,催化细胞质中ADP和线粒体内ATP的跨膜交换.qPCR结果表明ANT2基因在拟穴青蟹多个组织中均有表达,且表达量不同,表明ANT2基因具有组织表达特异性.第1期仔蟹在10,15,20,25℃不同温度条件下,在1,3,12,24,36h,10和15℃组表达量显著低于20和25℃组(p0.05);且在1,3,6,12,24h,10℃组的表达量显著低于15℃组表达量(p0.05).15℃组在48h内,ANT2呈现高低起伏的表达模式.拟穴青蟹第1期仔蟹ANT2基因表达变化,提示该基因与能量代谢功能相关,可能参与拟穴青蟹低温胁迫应答.     

亚硝酸盐急性胁迫对拟穴青蟹相关免疫指标的影响

【目的】探讨亚硝酸盐急性胁迫对拟穴青蟹(Scylla paramamosain)机体免疫力的影响,为拟穴青蟹养殖模式调整与产业化升级提供基础资料。【方法】测定拟穴青蟹在不同浓度亚硝酸盐(0 mg/L、20 mg/L、60 mg/L、100mg/L、140mg/L)胁迫下其组织中碱性磷酸酶(AKP)活力、过氧化氢酶(CAT)活力、丙二醛(MDA)含量在受胁迫48h内的动态变化。【结果】亚硝酸盐胁迫浓度达60mg/L以上时,青蟹肝胰腺及肌肉中AKP活力变化显著,肌肉中AKP活力随时间增加呈先升高后降低趋势,各组织中CAT活力变化也呈先升高后降低趋势,表现出一定的"毒兴奋效应",肝胰腺及鳃中MDA含量则随胁迫时间与胁迫浓度变化呈递增趋势。【结论】亚硝酸盐胁迫可通过破坏组织中非特异性免疫酶类体系运转,加剧组织中脂质过氧化方式导致机体免疫力下降。在拟穴青蟹人工养殖中,将亚硝酸盐浓度控制在20mg/L以下,有助于青蟹养殖模式调整与产业化升级。     

Peptidergic transmitters in synaptic boutons of sympathetic ganglia

In sympathetic ganglia of the bullfrog, a slow synaptic potential lasting for minutes--the late slow excitatory postsynaptic potential (e.p.s.p.)--was discovered. This slow response, unlike other previously known synaptic potentials in the autonomic nervous system, is not mediated by acetylcholine or monoamines. Similar non-cholinergic, non-adrenergic slow synaptic potentials have since been found in several other vertebrate autonomic ganglia. We found that the late slow e.p.s.p. is probably mediated by a peptide that is identical to, or closely resembles, mammalian luteinizing hormone releasing hormone (LHRH), because (1) when applied directly to sympathetic neurones, LHRH and its agonists elicit a slow depolarization, associated with similar changes in membrane conductance and excitability as those occurring during the late slow e.p.s.p. Furthermore, both peptide-induced and nerve-evoked responses are blocked by antagonists of LHRH; and (2) radioimmunoassays indicate that a chain of sympathetic ganglia contains 100-800 pg of a LHRH-like peptide. Its distribution among spinal nerves, the great reduction of this substance following denervation, and its release from ganglia following isotonic KCl treatment or nerve stimulation suggest that the LHRH-like material is contained in preganglionic nerve fibres. Here we report that immunohistochemical staining of sympathetic ganglia shows that LHRH-like immunoreactivity is indeed present in synaptic boutons. We also show that the two types of ganglion cells (B cells and C cells) receive strikingly different patterns of peptidergic innervation.     

正常与性早熟河蟹视神经节蛋白的二维电泳初步分析

利用双向电泳技术比较正常和性早熟河蟹视神经节蛋白的表达差异,旨在探索蛋白质组学技术在甲壳动物中尤其是河蟹的性早熟研究当中的应用.结果发现,明显差异的蛋白点共24个,为以后通过蛋白质组学手段筛选出真正影响性早熟发生的蛋白质或酶奠定了基础.     

GAP-43在锦鲤荒漠沙蜥和雉鸡视网膜内分布的免疫组化研究

GAP-43具有多种功能,主要与神经元轴突的生长、再生、神经递质的释放及膜泡的吞噬有关.本研究用免疫组织化学的方法观察了正常成年锦鲤、荒漠沙蜥和雉鸡视网膜内GAP-43分布.结果显示GAP-43主要分布在内网层,另外,在内核层、外网层、光感受器细胞层因动物不同也呈现不同的分布特点,而在节细胞层中3种动物均未发现GAP-43阳性染色.在锦鲤视网膜中GAP-43主要分布在内网层和内核层的无长突细胞;在荒漠沙蜥视网膜中GAP-43主要分布在内网层和外网层,在雉鸡视网膜中GAP-43主要分布在内网层、外网层、外核层和光感受器外节,其中在雉鸡视网膜外核层和光感受器外节中发现阳性分布是在脊椎动物此层发现GAP-43的首次报道.     

Retrograde axonal transport of target tissue-derived macromolecules

Neurones depend on contact with their target tissues for survival and subsequent development. The protein, nerve growth factor (NGF), can be selectively taken up by sympathetic nerve terminals and reaches the neuronal perikaryon by a process of retrograde intra-axonal transport, suggesting that its role in vivo is to act as a target tissue-derived trophic factor. The development of the neurones of the chick ciliary ganglion requires the presence of structures derived from the optic cup. Several studies in vitro have shown that media conditioned by non-neuronal cells contain factors that result in the survival of neurones from ciliary ganglia. In particular, chick embryo iris, ciliary body and choroid contained large amounts of these factors indicating the presence of a target tissue-derived trophic factor for the cholinergic ciliary ganglion. This study demonstrates that neurones of the ciliary ganglion accumulate, by retrograde intra-axonal transport, proteins synthesized and released by optic tissues in culture.     

金鱼视觉系统内中、高分子量神经丝免疫组织化学定位

RT97是一种特异性识别中、高分子量神经丝蛋白抗原簇的抗体.采用组织化学技术对金鱼(Carassiusauratus)视觉系统内RT97识别神经丝的表达分布进行了观察.结果表明,RT97在金鱼视网膜、视神经和视顶盖中均有阳性反应:①在视网膜内视纤维层、内网层、内核层的水平细胞、光感受器的外节有RT97阳性反应;②在视乳突部和视神经有少量生长纤维为RT97阳性;③在视顶盖中RT97阳性反应集中在顶盖的边缘层(SM),同时观察到有RT97阳性纤维从中央纵突区(TL)通过顶盖腹面延伸至顶盖边缘区.实验结果提示,RT97不仅可作为金鱼视觉系统视觉生长纤维的一种标记蛋白,同时在视顶盖由RT97识别的蛋白也可能是一种吸引视觉纤维生长的靶蛋白.     

Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit

Retinal ganglion cells are the projection neurons that link the retina to the brain. Peptide immunoreactive cells in the ganglion cell layer (GCL) of the mammalian retina have been noted but their identity has not been determined. We now report that, in the rabbit, 25-35% of all retinal ganglion cells contain substance P-like (SP) immunoreactivity. They were identified by either retrograde transport of fluorescent tracers injected into the superior colliculus, or by retrograde degeneration after optic nerve section. SP immunoreactive cells are present in all parts of the retina and have medium to large cell bodies with dendrites that ramify extensively in the proximal inner plexiform layer. Their axons terminate in the dorsal lateral geniculate nucleus, superior colliculus and accessory optic nuclei, and these terminals disappear completely after contralateral optic nerve section and/or eye enucleation. In the dorsal lateral geniculate nucleus large, beaded, immunoreactive axons and varicosities make up a narrow plexus just below the optic tract, where they define a new geniculate lamina. The varicosities make multiple synaptic contacts with dendrites of dorsal lateral geniculate nucleus projection neurons and presumptive interneurons in complex glomerular neuropil. This is direct evidence that some mammalian retinal ganglion cells contain substance P-like peptides and strongly suggests that, in the rabbit, substance P (or related tachykinins) may be a transmitter or modulator in a specific population or populations of retinal ganglion cells.     

The neostriatal mosaic: compartmentalization of corticostriatal input and striatonigral output systems

The striatum (caudate-putamen) of the basal ganglia in the mammalian forebrain is a mosaic of two interdigitating, neurochemically distinct compartments. One type, the 'patch' compartment, is identified by patches of dense opiate receptor binding, and is enriched in enkephalin- and substance P-like immunoreactivity. The other compartment, the 'matrix', has a high acetylcholinesterase activity, and is shown here to have a dense plexus of fibres displaying somatostatin-like immunoreactivity. The present study demonstrates the two compartments have distinct connections, using a method that concurrently reveals striatal input, output and neurochemical systems in the rat. Patches receive inputs from the prelimbic cortex (a medial frontal cortical area with direct 'limbic' inputs from the amygdala and hippocampus); they also project to the substantia nigra pars compacta (the source of the nigrostriatal dopaminergic system). Conversely, the matrix receives inputs from sensory and motor cortical areas; here it is shown to project to the substantia nigra pars reticulata (the source of the non-dopaminergic nigrothalamic and nigrotectal system). Also, an intrinsic striatal somatostatin-immunoreactive system is described that may provide a link between the two compartments. The striatal patch and matrix compartments thus appear to be functionally distinct and interactive parallel input-output processing channels.     

榆紫叶甲 Ambrostoma quadriim-pressum脑及咽下神经节GABA阳性神经元分布模式的构建

采用树脂石蜡(CP)组织包埋切片技术,结合免疫组化链霉菌抗生物素蛋白-过氧化物酶(SP)双染法,初步构建了榆紫叶甲脑及咽下神经节GABA阳性神经元的分布模式.结果显示,榆紫叶甲脑结构比较特殊:视叶不发达,但具内髓板;嗅叶不发达;蕈形体冠高度退化,而根叶相对发达;中央复合体结构明显;后脑经由围咽神经束与咽下神经结相连,咽下神经结非常发达.榆紫叶甲的所有髓区几乎都有GABA免疫阳性纤维分布.一侧脑半球及咽下神经节共存在约2 800个GABA阳性神经元,分布广泛,且趋向于集群分布.该研究表明榆紫叶甲的脑结构及GABA阳性神经元的分布模式与榆紫叶甲的生活习性、进化地位等密切相关.