水螅基盘的固着行为及机理的初步研究

采用形态学、组织化学及分子克隆方法对水螅基盘固着行为及相关细胞和分子机理进行了初步研究。研究结果表明,水螅固着时基盘吸附面接近圆形,仅吸附面的圆形周边区域与固着物表面直接接触、而吸附面中央区域不与固着物表面直接接触,并且吸附面周边区域外胚层细胞还沿着固着物表面形成伪足。水螅基盘粘液细胞分子标志物水螅基盘特异性过氧化物酶的表达模式表明粘液细胞在基盘吸附面上不是均匀分布,而仅分布在吸附面的周边区域即吸附面与固着物直接接触的部位。对水螅基盘特异性过氧化物酶氨基酸序列进行生物信息学分析发现该蛋白隐含actin交联因子fascin蛋白的保守结构域,而伪足形成的主要物质基础是细胞质中的actin蛋白分子交联集聚。基盘特异性过氧化物酶可能具有的fascin蛋白活性有助于基盘粘液细胞伪足的形成、从而增强基盘对固着物的吸附力。     

水螅营养积累对基盘组织更新进程的影响

目的观察大乳头水螅（Hydra magnipapillata ）基盘组织更新进程,探讨水螅营养积累对基盘组织更新进程的影响。方法设定水螅喂食频率梯度（代表不同的营养积累水平）,记录和观察喂食频率对水螅更新基盘组织进程的影响。通过ABTS细胞化学染色法检测水螅基盘分子标志物过氧化物酶的表达,观察水螅老基盘组织脱落后水螅体主体新生基盘组织的再生过程。结果喂食频率对水螅更新基盘组织进程有明显的影响。水螅基盘组织更新的标准过程如下;在一定的喂食频率下培养水螅,水螅体出芽区逐渐有芽体产生,随后在出芽区和基盘之间靠近芽体的位置出现缢痕,最后水螅体在缢痕处断裂为水螅体主体和老基盘组织两部分。缢痕断裂后对水螅体主体保持既定的喂食频率,其伤口能愈合但不能再生出新的基盘组织;对其降低喂食频率直至其伤口上方的芽体全部脱落后伤口处重新启动新生基盘组织的再生进程。另外,脱落的老基盘组织有两种不同的命运,即大部分老基盘组织不能发育成正常水螅体、最终解体;而小部分的老基盘组织能发育成正常的水螅体。结论水螅营养积累可能促进基盘组织更新进程,靠近断裂伤口处的芽体能抑制水螅体主体新生基盘的再生进程。     

水螅芽体发育与头部再生进程间的相互作用

目的观察具有单个芽体的大乳头水螅(Hydra magnipapillata)头部再生进程,探讨水螅头部结构的再生进程与芽体发育过程之间可能存在的相互作用。方法选取具有单个年幼芽体的水螅体,在水螅母体上紧贴芽体着生部位的上方进行切除手术,观察母体头部再生进程。通过ABTS细胞化学染色法检测水螅基盘分子标志物过氧化物酶的表达,观察水螅芽体基盘与母体间的结构联系。结果水螅母体伤口在手术后2h内愈合。随着再生时间的延长,出现两种不同命运的芽体发育方式。一种情况是水螅芽体基盘紧贴母体手术切口,芽体发育成熟后可正常脱离母体;在芽体脱离母体前母体头部再生进程被抑制,在芽体脱落后母体头部再生进程重新启动、且在其后48h内母体头部再生完成。另一种情况是水螅芽体基盘组织与母体手术切口不产生结构联系而向外突出生长,母体头部再生进程完全停止,芽体胃区与母体相连且芽体发育成熟后不脱离母体。结论靠近水螅母体手术伤口的年幼芽体能延迟或阻断母体头部的再生进程,而手术切口也可能干扰了发育成熟的芽体与母体脱离的正常机制。     

水螅异常极性体轴的形成及矫正进程的观察

目的:如何建立和维持体轴是一个基本的发育生物学问题,而淡水水螅是适合进行形态发生和个体发育调控机制研究的重要模式生物。本文观察了大乳头水螅异常极性体轴的形成及矫正进程,初步探讨水螅极性体轴的维持和调控机制。方法:先切取水螅的整个头部,再获得带二根触手的口区组织。通过ABTS细胞化学染色法检测水螅基盘分子标志物过氧化物酶的表达,判别水螅基盘组织(水螅足区的末端)是否形成。结果:从40块口区组织再生得到的水螅个体中有1例极性体轴发育异常的个体,其身体两端均发育成头区,且两端的头区均具有捕食能力。随后水螅其中一端头区的触手逐渐萎缩、退化,最终该端头区转化成具有吸附能力的基盘组织。结论:水螅组织的再生涉及极性体轴的重建,而一些特殊因素可能造成临时性的水螅极性体轴调控紊乱。本研究表明水螅具备自我矫正异常极性体轴的能力。另外,本研究结果显示水螅触手可以萎缩直至退化,该现象涉及的细胞学过程可能是非常复杂的,有可能涉及到触手细胞的凋亡转化过程,也可能是触手的高度分化细胞仍然具备去分化能力、去分化后再转移到身体其他地方,其具体机制值得进一步探究。     

水螅AChE和NPY类似物的定位

乙酰胆碱脂酶(AChE)组织化学定位方法常用于研究动物的神经系统,但迄今未见水螅AChE分布定位的相关报道。本文使用免疫细胞化学、组织化学和亚甲基蓝活染方法,探索AChE、NPY类似物在水螅上的连续分布状态,对阳性标本进行内胚层、外胚层人工分离手术,并与亚甲基蓝染色结果进行比较。本文首次成功地在水螅上定位出AChE,展示了AChE、NPY类似物在水螅上的分布特点。AChE定位在水螅体柱和触手基部外胚层的近外表,呈网状结构,正常水螅上没有发现明显的阳性神经细胞,外胚层表面受损部位内有阳性神经细胞,我们认为是一种临时的生理现象。成熟刺丝囊的刺针内有AChE 产物。NPY免疫组织化学反应(NPYIR)在水螅外胚层近外表处上均有分布,呈网状结构。垂唇和近基盘处NPYIR最强,NPYIR神经细胞仅存在于垂唇和近基盘处的外胚层基部。刺丝囊分化、发育初期NPYIR较强。随刺丝囊发育成熟而逐渐减弱。最后,进行了分析和比较,认为NPY、AChE来源于上皮细胞。水螅刺丝囊的分化和发育与NPY的作用密切相关;刺丝囊发射刺丝的机理与胆碱能的作用可能有联系     

水螅的无性生殖

淡水水螅的无性繁殖方式是出芽生殖。水螅胃区的皮肌细胞摄取的营养物质,经细胞间传递方式,转移到芽体的内、外胚层,为芽体的发育提供了能量。胃区的干细胞与增殖的细胞不断迁移到芽体,保障芽体发生时的细胞数。水螅的头部与基盘对芽体的发生,同时存在着激活与抑制2对位置信息素,共同控制并决定了芽体在体柱上的发生位置。芽体先发生垂唇与触手芽,其发生位置必需远离母体头部抑制素的作用。芽体发育后期,发生基盘时,必须远离母体基盘抑制素的作用。     

光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶活力的影响

单细胞真核绿藻在中国水螅(Hydra sinensis)内胚层皮肌细胞中共生是有较高科研价值的特殊生物学现象。水螅宿主细胞为共生藻提供CO2、氮源及矿物质,而共生藻通过光合作用可能为宿主提供碳水化合物等有机物营养,因此水螅与共生藻间代谢流是以共生藻光合作用为中心,但基于代谢流二者间的互作机制目前尚未阐明。水螅通过营养积累进行出芽生殖,从母体脱落的芽体数量间接反映水螅营养积累的相对量。而光暴露时长能影响共生绿藻光合作用,如果共生藻的确能向水螅细胞转移光合作用产物,那光暴露时长应该能间接影响水螅的营养积累、从而进一步影响水螅无性出芽生殖。为证实该假说,本研究应用种群累积培养法,观察了光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶(SOD和CAT)活力的影响。结果显示,光周期对中国水螅种群增长具有明显的影响。培养15 d后,所有实验组水螅的种群密度均为正增长,其中8L∶16D(在一个24h周期内光暴露8 h、黑暗16 h)实验组种群密度最大、而0L∶24D(持续黑暗)实验组种群密度最小。另外,随着光暴露时长的增加,中国水螅SOD及CAT活力整体均呈下降趋势。结果表明,从光周期对中国水螅无性出芽生殖及两种抗氧化酶活力的影响来看,中国水螅对光周期的生理学响应较为敏感,这个现象可能源于共生绿藻能通过向宿主细胞转移光合作用产物的方式为水螅提供营养补充。     

盐地碱蓬(Suaeda salsa)APX 基因的克隆及盐胁迫下的表达

从盐地碱蓬 (Suaedasalsa)中克隆了抗坏血酸过氧化物酶 (ascorbateperoxidase ,APX)的全长cDNA(SsAPX) ,基因注册号为AY0 34 893。SsAPX全长 1.1kb ,推导的氨基酸序列长为 2 5 0个氨基酸残基。BLAST同源性分析表明 ,该cDNA与已报告的菠菜(Spinaciaoleracea)细胞质抗坏血酸过氧化物酶基因同源性最高 ,在核苷酸水平上一致性为 87% ,在氨基酸水平上一致性为 89%。Southern杂交表明APX基因在盐地碱蓬基因组中只有 1个拷贝。盐 (NaCl 40 0mmol/L)处理不同时间后的Northern杂交分析表明盐地碱蓬中SsAPX基因在盐胁迫下表达量增加 ,而且在盐胁迫下抗坏血酸过氧化物酶的活性也显著地增加 ,说明该基因受盐诱导。推测抗坏血酸过氧化物酶可能在保护盐地碱蓬免受氧化损伤的过程中起到一定作用     

大乳头水螅增殖细胞核抗原基因的克隆及再生进程中的表达分析

研究以增殖细胞标记物-增殖细胞核抗原(Proliferating cell nuclear antigen, PCNA)蛋白为切入点, 采用RACE(Rapid amplification of cDNA ends)方法克隆了大乳头水螅(Hydra magnipapillata)PCNA基因的cDNA序列。该cDNA序列包含1240 bp, 其中837 bp为编码区, 编码的蛋白质预测分子量为30.93 kD。把PCNA基因ORF中的部分序列克隆到原核表达质粒pET-28b(+)中, 重组质粒转化E. coli BL21 (DE3)菌株, IPTG诱导后成功表达重组PCNA蛋白, 再用该重组蛋白免疫新西兰兔制备多克隆抗体用于PCNA蛋白的免疫印迹分析(Western blotting assay, WB)。采用实时定量PCR (quantitative Real-time PCR, qPCR)及WB方法检测水螅头部再生进程中其PCNA表达水平的动态变化, 结果表明在再生进程的中后期阶段水螅PCNA表达量呈现一定程度的上调。结果暗示水螅头部再生进程的中后期阶段其伤口及附近区域可能存在细胞分裂活动。     

中国绿水螅抗坏血酸过氧化物酶基因的克隆、抗体制备及表达分析

为探讨中国绿水螅(Hydra sinensis)抗坏血酸过氧化物酶(Ascorbate peroxidase, APX)基因的起源及功能, 研究采用RACE方法克隆了中国绿水螅APX基因的全长cDNA序列。该cDNA序列总长1357 bp, 包括5′非编码区107 bp, 3′非编码区146 bp及开放阅读框(Open reading frame, ORF) 1104 bp, 共编码367个氨基酸, 预测蛋白质分子量为40.79 kD。BLAST结果表明中国绿水螅APX蛋白同源序列绝大部分来自植物界; 通过最大似然法(Maximum-likelihood)和贝叶斯分析(Bayesian inference)进行的系统发生分析显示植物界及动物界物种的APX序列各自形成单系群。把APX基因ORF全长序列克隆到原核表达质粒pET-GST中, 重组质粒转化E. coli BL21 (DE3)菌株, IPTG诱导后成功表达重组融合蛋白GST-APX, 再使用纯化的重组蛋白免疫新西兰兔制备多克隆抗体用于APX蛋白的免疫印迹分析(Western blotting assay, WB)。在不同光照时长梯度(光强度2000 lx, 每天分别光照0、4h、8h、12h、16h、20h及24h)下培养中国绿水螅30d, 实时定量PCR (Quantitative real-time PCR, qPCR)及WB检测结果均表明光照时间较长时(每天光照12h以上)绿水螅APX表达呈现一定程度的上调。在长时间光辐射下水螅体内共生绿藻连续进行光合作用所累积的大量活性氧能够扩散到水螅细胞内, 此时水螅体内表达上调的APX可能参与清除其细胞内的活性氧。     

Expression of a novel receptor tyrosine kinase gene and a paired-like homeobox gene provides evidence of differences in patterning at the oral and aboral ends of hydra

Axial patterning of the aboral end of the hydra body column was examined using expression data from two genes. One, shin guard, is a novel receptor protein-tyrosine kinase gene expressed in the ectoderm of the peduncle, the end of the body column adjacent to the basal disk. The other gene, manacle, is a paired-like homeobox gene expressed in differentiating basal disk ectoderm. During regeneration of the aboral end, expression of manacle precedes that of shin guard. This result is consistent with a requirement for induction of peduncle tissue by basal disk tissue. Our data contrast with data on regeneration of the oral end. During oral end regeneration, markers for tissue of the tentacles, which lie below the extreme oral end (the hypostome), are detected first. Later, markers for the hypostome itself appear at the regenerating tip, with tentacle markers displaced to the region below. Additional evidence that tissue can form basal disk without passing through a stage as peduncle tissue comes from LiCl-induced formation of patches of ectopic basal disk tissue. While manacle is ectopically expressed during formation of basal disk patches, shin guard is not. The genes examined also provide new information on development of the aboral end in buds. Although adult hydra are radially symmetrical, expression of both genes in the bud's aboral end is initially asymmetrical, appearing first on the side of the bud closest to the parent's basal disk. The asymmetry can be explained by differences in positional information in the body column tissue that evaginates to form a bud. As predicted by this hypothesis, grafts reversing the orientation of evaginating body column tissue also reverse the orientation of asymmetrical gene expression.     

Epidermal growth factor promotes chick embryonic angiogenesis

The response of the early extraembryonic vasculature to epidermal growth factor (EGF) was studied. Millipore filter discs containing 10ng-1 microgram of EGF were placed onto the advancing edge of the area vasculosa of 3-day chick embryos, and the effect examined macroscopically and histologically 24 hours after disc application. The capillary density at the site of application increased significantly, and the effect was seen to be dose-dependent; a similar but more marked response was observed in the vessel cross-sectional area per unit length. This change in vascularity was accompanied by tortuous folding of the mesoderm and endoderm (which normally lie parallel to the ectoderm in a trilaminar arrangement) into the yolk substance: this may indicate precocious development of all three layers (ectoderm, mesoderm and ectoderm) of the membrane, and the proliferative effects of EGF may not be confined to the vascular endothelium.     

Epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases

As a member of the phylum Cnidaria, the body wall of hydra is organized as an epithelium bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM). Previous studies have established the general molecular structure of hydra ECM and indicate that it is organized as two subepithelial zones that contain basement membrane components such as laminin and a central fibrous zone that contains interstitial matrix components such as a unique type I fibrillar collagen. Because of its simple structure and high regenerative capacity, hydra has been used as a developmental model to study cell-ECM interaction during epithelial morphogenesis. The current study extends previous studies by focusing on the relationship of ECM biogenesis to epithelial morphogenesis in hydra, as monitored during head regeneration or after simple incision of the epithelium. Histological studies indicated that decapitation or incision of the body column resulted in an immediate retraction of the ECM at the wound site followed by a re-fusion of the bilayer within 1 hour. After changes in the morphology of epithelial cells at the regenerating pole, initiation of de novo biogenesis of an ECM began within hours while full reformation of the mature matrix required approximately 2 days. These processes were monitored using probes to three matrix or matrix-associated components: basement membrane-associated hydra laminin beta1 chain (HLM-beta1), interstitial matrix-associated hydra fibrillar collagen (Hcol-I) and hydra matrix metalloproteinase (HMMP). While upregulation of mRNA for both HLM-beta1 and Hcol-I occurred by 3 hours, expression of the former was restricted to the endoderm and expression of the latter was restricted to the ectoderm. Upregulation of HMMP mRNA was also associated with the endoderm and its expression paralleled that for HLM-beta1. As monitored by immunofluorescence, HLM-beta1 protein first appeared in each of the two subepithelial zones (basal lamina) at about 7 hours, while Hcol-I protein was first observed in the central fibrous zone (interstitial matrix) between 15 and 24 hours. The same temporal and spatial expression pattern for these matrix and matrix-associated components was observed during incision of the body column, thus indicating that these processes are a common feature of the epithelium in hydra. The correlation of loss of the ECM, cell shape changes and subsequent de novo biogenesis of matrix and matrix-associated components were all functionally coupled by antisense experiments in which translation of HLM-beta1 and HMMP was blocked and head regeneration was reversibly inhibited. In addition, inhibition of translation of HLM-beta1 caused an inhibition in the appearance of Hcol-I into the ECM, thus suggesting that binding of HLM-beta1 to the basal plasma membrane of ectodermal cells signaled the subsequent discharge of Hcol-I from this cell layer into the newly forming matrix. Given the early divergence of hydra, these studies point to the fundamental importance of cell-ECM interactions during epithelial morphogenesis.     

SELECTIVE INHIBITION OF REGENERATION IN HYDRA VULGARIS BY ACTINOMYCIN D

Twelve-hour continuous pretreatment of regenerating Hydra with 60 μg/ml actinomycin D inhibited the synthesis of RNA by 98%. In such Hydra, hypostome regeneration was found more affected than basal disc regeneration since a complete blockage of development of oral structures occurred. It is assumed that the hypostome regeneration requires new DNA-dependent RNA synthesis. Better differentiation of the basal disc is explained on the basis of a stable variety of messenger RNA (mRNA), which would become activated at the time of determination. The formation of mesoglea and the development of fibrous materials in the basal disc are attributed to new DNA-dependent RNA synthesis.     

Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.     

损伤视神经后斑马鱼视网膜神经节细胞数量和分布的变化

中枢神经系统的再生是神经科学领域的一个重要课题。鱼类和两栖类的视神经作为中枢神经系统的一部分,具有再生的能力。已知在损伤视神经后,对与视神经纤维直接相连的视网膜神经节细胞的形态结构,数量和分布等产生一系列的影响。视神经再生过程中细胞学研究在很大程度上依赖于示踪方法和其它技术的发展,结合光镜和电镜,它们仅对神经细胞末梢的精细结构和神经细胞间突触连接构筑等研究较准确详实,但对视网膜神经节细     

Hydra pattern is controlled by two distinct but interacting morphogen sets

Summary Temporal course of regeneration of the hypostome and basal disc along the body length of the hydra is studied both in the presence and absence of the other determined centre. The regeneration times vary nonlinearly with distance from the original position indicating that the underlying processes are of non-linear nature. The presence of hypostome influences the regeneration of basal disc in an inhibitory manner throughout the body length, whereas, basal disc influences the regeneration of hypostome only in the lower portion of the body in a positive manner. A scheme in terms of the activators and inhibitors specific to hypostome and basal disc, is given. The implication of these results is that the two inhibitors are functionally distinct.