有鳞目动物显微皮纹学

简要介绍了近年来国内外对有鳞目动物表皮扫描电镜研究的动态。现已描述了多样化的鳞片感觉器官;明确了有鳞目动物表皮具有复杂的组织学层次结构;其中最重要的是证明了表皮几丁质双层排列的事实以及得杂的蜕皮循环。研究还表明由于鳞片表皮结构特征在高级分类阶元中具有保守性,因而可以提供有用的系统演化信息。至少某些表皮特征,如微饰类型反映了系统联系而非生态适应。     

纳米二次离子质谱技术(NanoSIMS)在微生物生态学研究中的应用

超高分辨率显微镜成像技术与同位素示踪技术相结合的纳米二次离子质谱技术(NanoSIMS)具有较高的灵敏度和离子传输效率、极高的质量分辨率和空间分辨率(＜ 50 nm),代表着当今离子探针成像技术的最高水平.利用稳定性或者放射性同位素在原位或者微宇宙条件下示踪目标微生物,然后将样品进行固定、脱水、树脂包埋或者导电镀膜处理,制备成可供二次离子质谱分析的薄片,进一步通过NanoSIMS成像分析,不仅能够在单细胞水平上提供微生物的生理生态特征信息,而且能够准确识别复杂环境样品中的代谢活跃的微生物细胞及其系统分类信息,对于认识微生物介导的元素生物地球化学循环机制具有重要意义.介绍了纳米二次离子质谱技术的工作原理和技术路线,及其与同位素示踪技术、透射电子显微镜(TEM)、扫描电子显微镜(SEM)、荧光原位杂交技术(FISH)、催化报告沉积荧光原位杂交技术(CARD-FISH)、卤素原位杂交技术(Halogen In Situ Hybridization,HISH)等联合使用在微生物生态学研究方面的应用.     

中华绒螯蟹蜕壳过程中肌肉的组织学、超微结构及主要蛋白质含量的变化

为探讨中华绒螯蟹(Eriocheir sinensis)蜕壳前后肌肉组织的形态特征变化, 采用石蜡切片、电镜及生物化学方法, 研究了中华绒螯蟹蜕皮过程中步行足和腹部肌肉的组织学、超微结构及主要蛋白质含量的变化。结果显示: 相对于蜕皮间期, 步行足在蜕皮前后组织学形态特征无明显变化; 超微结构在蜕皮前无明显变化, 蜕皮后可见肌原纤维纵裂及肌小节横裂现象, 表明蜕皮后外骨骼硬化的过程伴随着肌肉的生长。相对于蜕皮间期, 腹部肌肉在蜕皮前后组织学特征变化明显: 蜕皮前肌束间隙增大, 蜕皮后肌束内肌纤维间隙增大。电子显微镜观察显示, 蜕皮前肌原纤维在内部降解, 出现空洞, 肌原纤维边缘降解, 导致肌原纤维间隙增大; 蜕皮后肌原纤维重新组装、重建, 恢复到间期正常形态。生物化学研究发现, 蜕皮前后步行足和腹部肌肉中肌原纤维蛋白和可溶性蛋白含量的变化同其结构特征的变化相一致。以上研究结果表明, 中华绒螯蟹肌肉组织的结构特征同蜕皮周期密切相关。     

植物电子显微镜的负染和投影技术

电子显微镜技术是目前生物学研究中重要的手段之一。运用电镜开展研究,不仅要有高分辨本领的仪器,而且要有精心制备的样品。由于生物样品的电子散射力很低,如不经过特殊处理,样品的反差极弱,观察精细结构几乎不可能。因此,样品制备的质量在利用电镜的研究中具有极其重要的地位。制备样品的技术包括超薄切片、负染、投影、复型等,本文着重介绍负染和投影技术。     

PCR扩增循环数对细菌群落多样性测序分析的影响

运用高通量测序技术分析复杂样品中微生物群落组成及变化趋势,已经成为目前微生物研究领域的热点之一。本研究以复杂土壤样品和应用范围较广的瘤胃食糜样品为对象,选取20、25和30三个扩增循环数分别对样品的16S r RNA基因的V3区进行扩增,然后进行文库构建和测序。最后通过数据分析比较不同的扩增循环数对细菌多样性测定结果的影响。结果表明,扩增循环数越多,捕获到的细菌数量和种类越多;但并非循环数越多,群落中的微生物组成比例最优。整体来看,当扩增循环数为25时,样品中物种的数量和组成是最优的。     

家蚕蜕皮液羧肽酶A的表征及免疫荧光定位分析

蜕皮是许多变态发育昆虫的一种重要生理现象,昆虫通过蜕皮液中的酶对新旧表皮进行分离。已有相关蛋白组学的研究证明,家蚕蜕皮液中具有一种含量丰富的羧肽酶A(Bombyx mori-carboxypeptidase A, Bm-CPA),目前对其作用功能尚不清楚。为了更好地了解Bm-CPA在家蚕蜕皮发育过程的作用,本研究通过生物信息学分析、实时荧光定量PCR、抗体制备、免疫荧光染色和毕赤酵母表达等方法对Bm-CPA进行了研究。结果显示,Bm-CPA具有保守的M14锌羧肽酶结构域和糖基化位点,并且受蜕皮激素(20-hydroxyecdysone, 20E)调控,在眠期和上簇期的表皮中大量表达;免疫荧光染色显示Bm-CPA在眠期的表皮中富集,Bm-CPA抑制剂会导致幼虫因无法蜕皮而死亡;通过毕赤酵母表达系统在体外成功获得大量的重组Bm-CPA蛋白。这些结果为深入了解家蚕蜕皮发育过程提供了一定的参考。     

绿安乐蜥微皮纹和皮肤感受器形态学与组织学研究

运用扫描电镜、组织切片、显微拍照等实验方法对绿安乐蜥Anolis carolinensis背部未蜕皮和蜕皮皮肤微皮纹及皮肤感受器形态、组织学进行了研究,发现未蜕皮和蜕皮皮肤的微皮纹和皮肤感受器特征、大小没有显著区别,因此蜕皮组织是研究这类结构的理想实验材料;鳞片表面的棘突与支柱构成了网状的三维结构,凹窝处有1～2个皮肤感受器;皮肤感受器真皮层内有真皮小体,绞合区域和皮肤感受器β-角质层和中层明显变薄。这些特点表明,绿安乐蜥背部表皮结构与栖息地及变色能力密切相关,在保护皮肤、实现自身伪装和调节体温等方面具有重要作用,研究结果有助于进一步了解其变色机理,能够为开发自主变色材料提供生物学依据,对于研究珍稀物种皮肤微观结构具有重要借鉴意义。     

甲壳动物蜕皮抑制激素调控机制的研究进展

甲壳动物的蜕皮过程主要是由Y器(Y-organ)分泌的蜕皮类固醇激素与X器-窦腺复合体(X-organ-sinus gland,XO-SG)分泌的蜕皮抑制激素MIH相互拮抗而进行调控的。而MIH调控机制较为复杂,且存在争议。本文就MIH调控机制的研究进展,包括研究方法,以及目前调控机制中争议最大的3个问题:MIH受体、cAMP与cGMP功能以及Ca2+功能作一综述。     

灭幼脲引起两种幼虫表皮组织病变的显微观察

本文研究了灭幼脲引起黄粉(虫甲)(Tenebrio mclitor)和粘虫(Mythimna separata)幼虫的中毒征象和组织学病变.低剂量能引起幼虫蜕皮障碍,但看不到明显的组织学病变.高剂量处理,不仅引起了严重的中毒征象,而且伴有明显的组织学病变:内表皮生长停滞,真皮细胞排列异常,在内表皮和真皮细胞之间出现附加层和球状颗粒.对这些现象进行了较细致的讨论.     

非损伤微测技术在细胞生物学研究中的应用系列讲座(一)——技术简介

非损伤微测技术是一种选择性微电极技术,可以不损伤样品而获得进出样品的离子和分子信息,具有非损伤性,长时间、多电极、多角度测量等优势.本文介绍非损伤微测技术原理和技术特点及其在细胞生物学不同领域中的应用.     

Scanning electron microscopy of changes in epidermal structure occurring during the shedding cycle in squamate reptiles

Previous studies of squamate epidermal structure have focused on either histology and ultrastructure or oberhautchen surface texture as revealed by scanning electron microscopy (SEM). Using SEM data drawn from a variety of lizard taxa (primarily iguanids, but also agamids, chamaeleonids, and scincids), as well as amphisbaenians and colubrid snakes, we relate the surfaces encountered in gross dissection of squamate skin to histologically identifiable layers, and characterize their surface structure. Only the oberhautchen bears the repeating pattern of ornamentation noted by previous authors. Because the clear layer is a perfect template of the oberhautchen surface, it is the only layer with which the oberhautchen might be confused. However, the clear layer can be identified by its tendency to curl and crack during preparation. All other surfaces encountered were relatively featureless, except for impressions left by dermal “papillae” associated with mechanoreceptors. Using a method for examining preserved specimens to determine the stage in the shedding cycle, we assess two sources of variation in epidermal surface structure: stage in the shedding cycle and wear. Examination of immature renewal-phase epidermis suggests that the oberhautchen does not mature synchronously across a single scale or across body regions. Comparing inner- and outer-generation oberhautchen in sheddingphase epidermis, we conclude that changes in surface appearance caused by natural wear fall into two categories: discrete scratches and accumulation of debris. We see no evidence of overall “buffing” on a microscopic level, though surface structure may be obscured by scratches and gouges. Many squamate taxa show a gradient from low relief surface structure on elevated regions such as keels to high relief patterns at scale edges. This gradient is not due to wear; its significance is unknown.     

Formation of adherens and communicating junctions coordinate the differentiation of the shedding‐layer and beta‐epidermal generation in regenerating lizard epidermis

In the lizard epidermis, the formation of a stratified alpha‐ and beta‐layer, separated by a shedding complex for molting, suggests that keratinocytes communicate in a coordinated manner after they leave the basal layers during the shedding cycle. I have therefore studied the localization of cell junctional proteins such as beta‐catenin and connexins 43 and 26 during scale regeneration in lizard using immunocytochemistry. Beta‐catenin is also detected in nuclei of basal cells destined to give rise to the Oberhäutchen and beta‐cells suggesting activation of the Wnt‐pathway during beta‐cell differentiation. The observations show that cells of the entire shedding layer (clear and Oberhäutchen) and beta‐layer are connected by beta‐catenin (adherens junctions) and connexins (communicating junctions) during their differentiation. This likely cell coupling determines the formation of a distinct shedding and beta‐layer within the regenerating epidermis. The observed pattern of cell junctional stratification suggests that after departing from the basal layer Oberhäutchen and beta‐cells form a continuous communicating compartment that coordinates the contemporaneous differentiation along the entire scale. While the beta‐layer matures the junctions are lost while other cell junctions are formed in the following mesos‐ and alpha‐cell layers. This process determines the formation of layers with different texture (harder or softer) and the precise localization of the shedding layer within lizard epidermis. J. Morphol. 275:693–702, 2014. © 2014 Wiley Periodicals, Inc.     

Enzymes in the epidermis of Natrix piscator during its sloughing cycle

Acid phosphatase, non-specific esterase, alkaline phosphatase, monoamine oxidase and true lipase activities, in the epidermis of Natrix piscator in different stages of the sloughing cycle, have been localized using various histochemical techniques.
Different layers in scale epidermis have staining properties similar to corresponding layers in hinge epidermis.
Acid phosphatase and non-specific esterase activity in cell layers undergoing keratinization, and the lacunar tissue undergoing disintegration are associated with hydrolytic and catabolic wasting processes involving cell death. The activity of these enzymes in the clear layer is associated with the breaking down of the cementing substance resulting in the separation of clear layer from underlying tissue and facilitating the shedding of old slough.
Alkaline phosphatase activity in the stratum germinativum and undifferentiated epidermal cells has been associated with cell proliferation and differentiation. The presence of alkaline phosphatase in the lacunar tissue and clear layer has been correlated with the synthesis of mucopolysaccharides in these layers.
Monoamine oxidase and true lipase activity could not be located in the epidermis at any stage of the sloughing cycle.     

Comparative immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) supports a new explanation for the cyclical process of keratinocyte differentiation in lizard epidermis

Lizard epidermis is made of beta‐ and alpha‐layers. Using Western blot tested antibodies, the ultrastructural immunolocalization of specific keratin‐associated beta‐proteins in the epidermis of different lizard species reveals that glycine‐rich beta‐proteins (HgG5) localize in the beta‐layer, while glycine–cysteine‐medium‐rich beta‐proteins (HgGC10) are present in oberhautchen and alpha‐layers. This suggests a new explanation for the formation of different epidermal layers during the shedding cycle in lepidosaurian epidermis instead of an alternance between beta‐keratins and alpha‐keratins. It is proposed that different sets of genes coding for specific beta‐proteins are activated in keratinocytes during the renewal phase of the shedding cycle. Initially, glycine–cysteine‐medium‐rich beta‐proteins with hydrophilic and elastic properties accumulate over alpha‐keratins in the oberhautchen but are replaced in the next cell layer with glycine‐rich hydrophobic beta‐proteins forming a resistant, stiff, and hydrophobic beta‐layer. The synthesis of glycine‐rich proteins terminates in mesos and alpha‐cells where these proteins are replaced with glycine–cysteine‐rich beta‐proteins. The pattern of beta‐protein deposition onto a scaffold of intermediate filament keratins is typical for keratin‐associated proteins and the association between alpha‐keratins and specific keratin‐associated beta‐proteins during the renewal phase of the shedding cycle gives rise to epidermal layers possessing different structural, mechanical, and texture properties.     

Differentiation of the epidermis of scutes in embryos and juveniles of the tortoise Testudo hermanni with emphasis on beta-keratinization

The sequence of differentiation of the epidermis of scutes during embryogenesis in the tortoise Testudo hermanni was studied using autoradiography, electron microscopy and immunocytochemistry. The study was mainly conducted on the epidermis of the carapace, plastron and nail. Epidermal differentiation resembles that described for other reptiles, and the embryonic epidermis is composed of numerous cell layers. In the early stages of differentiation of the carapacial ridge, cytoplasmic blebs of epidermal cells are in direct contact with the extracellular matrix and mesenchymal cells. The influence of the dermis on the formation of the beta‐layer is discussed. The dermis becomes rich in collagen bundles at later stages of development. The embryonic epidermis is formed by a flat periderm and four to six layers of subperidermal cells, storing 40–70‐nm‐thick coarse filaments that may represent interkeratin or matrix material. Beta‐keratin is associated with the coarse filaments, suggesting that the protein may be polymerized on their surface. The presence of beta‐keratin in embryonic epidermis suggests that this keratin might have been produced at the beginning of chelonian evolution. The embryonic epidermis of the scutes is lost around hatching and leaves underneath the definitive corneous beta‐layer. Beneath the embryonic epidermis, cells that accumulate typical large bundles of beta‐keratin appear at stage 23 and at hatching a compact beta‐layer is present. The differentiation of these cells shows the progressive replacement of alpha‐keratin bundles with bundles immunolabelled for beta‐keratin. The nucleus is degraded and electron‐dense nuclear material mixes with beta‐keratin. In general, changes in tortoise skin when approaching terrestrial life resemble those of other reptiles. Lepidosaurian reptiles form an embryonic shedding layer and crocodilians have a thin embryonic epidermis that is rapidly lost near hacthing. Chelonians have a thicker embryonic epidermis that accumulates beta‐keratin, a protein later used to make a thick corneous layer.     

Morphogenesis of the digital pad lamellae in the embryo of the lizard Anolis lineatopus

The present study in the embryo of the lizard Anolis lineatopus describes the modality of cell proliferation responsible for the morphogenesis of the digital pad lamellae and of the epidermal stratification. After tritiated thymidine and 5-bromodeoxy-uridine administration, autoradiographic and immunocytochemical methods have been used. The lamellae originate as long, slightly slanted, undulations of the epidermis of fingers and toes. At an early stage, the epidermis consists of an outer periderm and a basal layer. Cell hypertrophy, and the prevalent cell proliferation in the longer side of the undulation with respect to the shorter side, generate the surface of the outer lamella. Under the peridermis, a shedding complex, composed by clear and oberhautchen layers, is formed and later determines the first intraepidermal shed. The first subperidermal layer derived from the basal layer is a clear layer and the first shed epidermis in the embryo is represented by periderm and clear layer. The heavily granulated clear layer in Anolis lineatopus represents the first epidermal layer produced in the embryonic epidermis, and is connected with the process of shedding. The spinulae of the underlying oberhautchen in the outer scale surface become long setae which grow toward the upper clear layer. Under the shedding complex a β-layer is produced. Autoradiographical study shows that the radioactivity stays in the basal layer for about 4 days before cells move to upper layers. At 6–8 days post-injection labelled cells are visible in the differentiated clear, oberhautchen and β-layers. Under the β-layer differentiating mesos cells are visible before the embryo hatches.     

The shift from aquatic to terrestrial phenotype in Lissotriton italicus: larval and adult remodelling of the skin

Morphology and ultrastructure of the skin of Lissotriton italicus (previously named Triturus italicus) have been described in different phases of its biological cycle: larval stage, metamorphic stage and adult stage with emphasis on modifications occurring between aquatic and terrestrial adults. In the present study, light microscopy and both scanning and transmission electron microscopy were employed to analyze the histological and cytological remodelling that occurs in the skin of L. italicus during metamorphosis. The ultrastructure of the larval epidermis is arranged into three principal layers comprising an external layer of pavement cells, a basal layer and 1-3 intermediate layers consisting of Leydig cells along with accessory cells and mitochondria-rich cells. By the onset of metamorphosis, morphological changes of the skin include stratification and flattening of epidermal layers and disappearance of typical larval cells. In both aquatic and terrestrial adult phases the thin, cornified epidermis shows the same general arrangement as found in other vertebrates with an external stratum corneum and a variable number of intermediate cell layers. During the terrestrial adult phase, the skin is characterized by the presence of numerous tubercles; moreover, the lower epithelium is thicker than in the aquatic phase. Ultrastructural analysis revealed no substantial differences in the cellular composition of the skin between aquatic and terrestrial phases.     

A whole lamella perspective on the origin of the epidermal free margin of Anolis (Reptilia: Dactyloidae) toe pads

Anoline lamellae terminate in an epidermal free margin carrying the majority of its setae. How the free margin is extruded from the body of the scale is not well understood. Two hypotheses have been advanced to account for it, one advocating distal migration of the outer epidermal layers relative to the body of the lamella, and the other proposing regression of the dermal core. Available evidence provides partial support for both. We assembled a series of specimens of Anolis grahami representing all shedding cycle stages, and prepared histological sections of the toe pads to allow measurement of appropriate lamellar components through the shedding cycle. Through its proliferative phases the lamellae increase markedly in length, with the distance between the distal tip of the dermal core and that of the lamella accounting for most of this, indicating that epidermal extrusion is responsible for production of the new free margin. The dermal core showed no evidence of regression. Concomitant with epidermal extrusion, the lacunar cells on the inner lamellar face hypertrophy and keep pace with the increasing thickness of the outer lamellar face resulting from the lengthening of the replacement setae. The integrated changes observed are consistent with continuity of functioning and alignment of the exposed setal carpet of the outer epidermal generation while ensuring that the new setal carpet is fully aligned and functional immediately after shedding. At shedding the original proportions of the lamellae are restored. Development of the new free margin results from a combination of distal displacement of Oberhäutchen cells along with arrested maturation of the epidermis in this region. Changes in length of the lamellae during the proliferative stages may impact the overall size of the adhesive toe pad, which may have consequences for assessments of the relationship between whole animal clinging ability and adhesive pad area. J. Morphol. 278:360–368, 2017. © 2017 Wiley Periodicals, Inc.     

Histidine uptake in the epidermis of lizards and snakes in relation to the formation of the shedding complex

Mammalian epidermis utilizes histidine-rich proteins (filaggrins) to aggregate keratin filaments and form the stratum corneum. Little is known about the involvement of histidine-rich proteins during reptilian keratinization. The formation of the shedding complex in the epidermis of snakes and lizards, made of the clear and the oberhautchen layers, determines the cyclical epidermal sloughing. Differently from snakes, keratohyalin-like granules are present in the clear layer of lizards. The uptake of tritiated histidine into the epidermis of two lizards and one snake has been studied by autoradiography in sections at progressive post-injection periods. At 40 min and 1 hr post-injection keratohyalin-like granules were not or poorly labeled. At 3-22 hr post-injection most of the labeling was present over suprabasal cells destined to form the shedding complex, in keratohyalin-like granules of the clear layer, and in the forming a-layer but was low in the forming b-layer, and in superficial keratinized layers. The analysis of the shedding complex in the pad lamellae (a specialized scale used for climbing) of a gecko showed that the setae and the cytoplasm of clear cells among them are main sites of histidine uptake at 4 hr post-injection. In the snake most of the labeling at 4 hr post-injection was localized in the shedding complex along the boundary between the clear and oberhautchen layers. The present study suggests that, in the epidermis of lepidosaurian reptiles, the synthesis of a histidine-rich protein is involved in the formation of the shedding layer and, as in mammals, in a-keratinization.     

Cell adhesion and junctional proteins in the developing skin of snakes indicate they coordinate the differentiation of the epidermis

The development of scales and the sequence of epidermal layers during snake embryogenesis has been studied by immunofluorescence for the localization of cell adhesion, adherens, and communicating cell junctional proteins. At about 2nd/3rd of embryonic development in snakes the epidermis forms symmetric bumps at the beginning of scale formation, and they rapidly become asymmetric and elongate forming outer and inner surfaces of the very overlapped scales seen at hatching. The dermis separates a superficial loose from a deeper dense part; the latter is joined to segmental muscles and nerves, likely acting on scale orientation during snake movements. N-cam is present in the differentiating epidermis and mesenchyme of forming scales while L-cam is only/mainly detected in the periderm and epidermis. Mesenchymal N-cam is associated with the epidermis of the elongating dorsal scale surface and with the beta-differentiation that occurs in the overlapping outer surface of scales. Beta-catenin and Connexin-43 show a similar distribution, and they are mainly present in the periderm and differentiating suprabasal keratinocytes likely forming an intense connectivity during epidermal differentiation. Beta-catenin also shows nuclear localization in differentiating cells of the shedding and beta-layers at late stages of scale morphogenesis, before hatching. The study suggests that intensification of adhesion and gap junctions allows synchronization of the differentiation of suprabasal cells to produce the ordered sequence of epidermal layers of snake scales, starting from the shedding complex and the beta-layer.