昆明小鼠胚胎成纤维细胞分离培养及应用

目的探讨昆明小鼠胚胎成纤维细胞培养及应用。方法取昆明小鼠胚胎分离成纤维细胞,用丝裂霉素C处理成纤维细胞为胚胎干细胞制备饲养层,消化贴壁成纤维细胞为核移植提供供体细胞。结果13.5d、14.5d、15.5d孕鼠均可分离出成纤维细胞,14.5d孕鼠制作的成纤维细胞形态规则,3-5代成纤维细胞经处理可作饲养层,可作核移植供体细胞。结论昆明小鼠可分离成纤维细胞,用于饲养层制作及核移植供体细胞。     

The growth of cochlear fibers and the formation of their synaptic endings in the avian inner ear: a study with the electron microscope

The developmental sequence of nerve-epithelial cell contacts, leading up to the formation of the mature receptoneuronal synapse, has been studied in the basilar papilla of chick embryos with electron microscopy. The receptor epithelium before innervation, on embryonic days 3-4, consists of a homogeneous population of primitive cells; hair cells and supporting cells cannot be distinguished. During innervation of the epithelium (embryonic days 5-7), the invading peripheral fibers of cochlear ganglion cells penetrate the basal lamina and form nerve-epithelial attachments with the epithelial cell bases. Once within the epithelium some fibers turn and spread in the transverse dimension across the basilar papilla through channels formed between the basal epithelial processes. Subsequently, nerve-epithelial attachments are observed more superficially within the epithelium. Hair cells and supporting cells differentiate during early synaptogenesis (embryonic days 8-9). Receptoneural synapses, possibly derived from the nerve-epithelial attachments formed during the innervation stage, are first seen during this period. They are characterized by symmetrical or asymmetrical membrane densities, separated by a cleft containing a dense material. At many of these junctions synaptic bodies, as well as dense-cored and coated vesicles, gather in the hair cells. During mid-synaptogenesis (embryonic days 11-13) the hair cells proliferate synaptic bodies, many of which are not located at receptoneural junctions. The preterminal portions of the sensory endings form large swellings, containing flocculent material, endoplasmic reticulum and vesicles. Late in synaptogenesis (embryonic days 15-17) the swellings disappear, while synaptic endings are transformed to foot-shaped terminals. In the hair cells, synaptic bodies not associated with junctions disappear. Efferent synapses are first seen during this period. This sequence of ultrastructural changes, which the developing sensory nerve endings and their target cells undergo in parallel, can be correlated with observations of Golgi preparations from a companion study. These correlations suggest that the innervation of the cochlea involves the following developmental processes. Initially the peripheral fibers of the ganglion cells grow directly toward the otocyst in fascicles. Having reached the base of the primitive receptor epithelium, the axonal endings, including some with growth cones, encounter a barrier in the basal lamina. When they enter some of the fibers attach to the basal end-feet of the primitive epithelial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Growth of syngeneic transplantable tumors in Syrian hamsters immunized with embryonic hamster tissue

The effect of immunization with embryonic tissue on growth of two syngeneic transplantable tumors was investigated in Syrian hamsters. Immunization of inbred hamsters with embryonic hamster tissue followed by transplantation of continuous lines of syngeneic tumors was shown neither to inhibit nor to stimulate growth of the tumors. It is suggested that embryonic antigens do not participate in the mechanism of transplantation antitumor immunity.Laboratory of Immunology of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 7, pp. 57–58, July, 1978.     

Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain

Macrophages and microglia in the developing and adult mouse brain have been identified by immunohistochemical localization of the macrophage-specific antigen F4/80 and monoclonal antibodies to the FcIgG1/2b (2.4G2) and type-three complement (Mac-1) receptors. In the adult mouse there are two classes of F4/80-positive cells; those associated with the choroid plexus, ventricles and leptomeninges and the microglia. The cells bearing Fc and complement receptors are indistinguishable, by their morphology and distribution, from those revealed by F4/80. During development macrophages invade the brain and can be followed through a series of transitional forms as they differentiate to become microglia. Macrophage invasion occurs when naturally dying cells are observed in large numbers and this is consistent with the idea that dying neurons and axons provide a stimulus for macrophage infiltration. Our results provide strong support for the hypothesis that the microglia are derived from monocytes and show that microglia possess receptors which would allow them to play a part in the immune defence of the nervous system.     

The development of innervation patterns in the avian cochlea

The sequence of developmental events leading to the innervation of the cochlea and the differentiation of its receptor cells has been studied in chick embryos with Golgi methods. We describe the morphogenesis of cochlear ganglion cell peripheral processes from their appearance in early embryos to the formation of their mature endings on hair cells in the basilar papilla (organ of Corti) of prehatching chicks. In the stage of peripheral fiber outgrowth, embryonic days 3-5, the fibers emerge from the ganglion cell bodies and grow, in a uniform fashion, toward the undifferentiated receptor epithelium of the otocyst. In the stage of the invasion of the otocyst by the peripheral fibers, embryonic days 6-7, some fibers enter the epithelium directly after reaching it, others enter after traveling some distance longitudinally beneath its basal lamina. The invading fibers appear to encounter resistance at the basal lamina, but, once within the epithelium, at embryonic days 8-9, they form a surfeit of branches in columnar zones oriented radially toward the surface. In early synaptogenesis (embryonic days 8-9) hair cells first become apparent. They differentiate from primitive epithelial cells. These cells withdraw their basal processes, which appear to accompany the growing fibers into the superficial epithelium. At embryonic days 11-13, the stage of mid-synaptogenesis, the fibers develop large, bulbous, preterminal and terminal swellings, which are located below the bases of the hair cells; the surplus branches atrophy or withdraw. Efferent axons are first seen in the epithelium at this time. In late synaptogenesis (embryonic days 14-17), the preterminal swellings disappear and the endings transform into mature foot-shapes at the bases of the hair cells. These morphological changes during the development of the peripheral endings are comparable to those of cochlear axons in nucleus magnocellularis (cochlear nucleus). During mid-synaptogenesis, when the ganglion cells develop swellings in the periphery, their central axons ramify extensively. Late in synaptogenesis, while the peripheral swellings disappear, there is a corresponding condensation of the central terminals to form the end-bulbs of Held. Thus, specific connections of the cochlear ganglion cells and their target cells in the ear and brain may result from two sequential developmental phases: (1) loosely organized and overabundant initial growth of branches from the fibers entering their target tissue; (2) reorganization of these fibers with the disappearance or resorption of the surplus branches during the transformation of their endings into mature synaptic arrangements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Trophic interactions between the cochleovestibular ganglion of the chick embryo and its synaptic targets in culture

The effect of the availability of synaptic targets on neuronal survival was tested by explanting the cochleovestibular ganglion from embryonic day 3-1/2 chick embryos and maintaining it in the presence or absence of appropriate synaptic target tissues for 14 days in culture. The targets were the inner ear, peripherally, and the myelencephalon, centrally. Light and electron microscopic observations showed that the ganglion cells in the explants with targets present had generally achieved a degree of differentiation comparable to that of their counterparts in embryonic day 14 embryos. The variety of cell types seen in the normal embryonic day 14 ganglia was also evident in vitro. In ganglia explanted without peripheral or central targets, few neurons survived. Ganglia explanted with either peripheral or central target intact showed considerably better survival than those explanted without any target. Ganglia explanted with only the peripheral target (the inner ear) survived equally as well as those with both central and peripheral targets. Ganglia cultured with the central target (myelencephalon) did not survive as well as those with peripheral targets. The effect of the peripheral target on the ganglion was less clear-cut when ganglia were first dissected from their targets and then recombined in culture. However, the results of such experiments in which nerve fascicles were traced in serial sections from ganglia to target areas, suggest that the actual innervation of target cells, as well as proximity of ganglia to target tissues, could influence neuronal survival. Establishment of innervation appeared to be selective, in that the closest available target area was not always the one contacted by the ganglionic fibers. The present findings are consistent with a role of neuron-target cell interactions in supporting neuronal survival in the cochleovestibular ganglion of the chick embryo. Both the central and the peripheral targets are implicated in trophic interactions with the sensory neurons.     

Binocular interaction in the fetal cat regulates the size of the ganglion cell population

During fetal development of the cat's visual system there is a marked overproliferation of optic nerve axons. In utero binocular interaction contributes to the severity of fiber loss since removal of an eye during gestation attenuates axon loss in the remaining optic nerve. The purpose of the present study was to determine whether this reduced loss of optic nerve fibers is due to a failure of retraction by supernumerary axon branches or to a reduction in ganglion cell death. To resolve this issue, we compared the number of ganglion cells and optic nerve fibers in adult cats which had one eye removed at known gestational ages. Retinal ganglion cells were backfilled with horseradish peroxidase and counts were made from retinal wholemounts. The axon complement was assessed with an electron microscopic assay. In the retinas of a normal cat we estimated 151,000 and 152,000 ganglion cells. The optic nerves of two other normal cats contained approximately 158,000 and 159,000 axons. In comparison, an animal enucleated on embryonic day 42 had 180,000 ganglion cells and 178,000 optic nerve fibers, while in an animal enucleated on embryonic day 51 the corresponding estimates were 182,000 and 190,000. The close agreement between cell and fiber counts indicates that axonal bifurcation does not contribute appreciably to the axon surplus in the optic nerve of prenatally enucleated cats. These results demonstrate that prenatal binocular interaction regulates the size of the mature retinal ganglion cell population.     

Efferents of frontal or occipital cortex grafted into adult rat''s motor cortex

Phaseolus vulgaris leucoagglutinin (PHA-L) was used to examine the efferent connectivity of embryonic (E16) frontal (homotopic) or occipital (heterotopic) neocortical transplants placed into — or in the vicinity of — lesion cavities made in the frontal cortex of adult recipients. Homotopic transplants projected towards the host sensorimotor cortex and, in most cases, into the lateral caudate-putamen (CPu). Heterotopic transplants projected into the anterior cingulate cortex and, in most cases, distributed terminals into the medial CPu. It is suggested that embryonic neocortical tissue placed into a damaged cortical site of an adult recipient develops a pattern of efferents corresponding to its cortical origin.     

上颌窦的胚胎发育

对46例(92侧)12～40周胎儿标本的上颌窦,行冠状切,HE染色,光镜观察:(1)原始上颌窦在胚胎第20～22周由原始筛漏斗底部扩展而成,上颌窦位于钩突和中鼻道外侧壁的外侧,窦腔与眼眶之间的骨板最薄,且骨化较晚.(2)窦口位于上颌窦的顶部(壁),随胎龄增长,”钩突角”变小.向外下的筛漏斗渐近水平位.(3)出生时,上颌窦大小约3.0×5.0×6.5mm.窦内粘膜上皮细胞呈立方形或矮柱状,其纤毛稀疏,,固有膜增厚、疏松,内含极少量血管和腺体.结果表明:出生时,上颌窦虽已出现,但粘膜厚,窦腔小,且与眼眶的毗邻关系密切,上颌窦粘膜的组织结构与鼻腔粘膜的结构有所不同.     

小鼠胚胎发育过程中SCE值的观察分析

提要:本文运用液体石蜡-IUdR、UV-Giemsa术,对小鼠的早、中、晚期胚胎细胞和母鼠骨髓细胞的SCE值进行了测定,其数值分别为2.96±1.53、3.21±1.48、3.05±1.31和3.29±1.71,P>0.05。表明小鼠胚胎细胞的SCE值是比较恒定的,不受胚胎发育的影响,同时与母鼠间也无显著差异。     

人胚胎干细胞体外培养并分化为心肌细胞

目的 :体外培养并鉴定人胚胎干细胞 (EG细胞 ) ,在不添加细胞因子的条件下 ,观察细胞集落分化情况。方法 :取 4～ 6周人胚胎生殖嵴、肠背系膜和中肾嵴 ,进行组织块培养 ,利用组织化学和免疫组织化学技术对培养的细胞进行鉴定。结果 :培养 7～ 8小时 ,在组织块周围出现成纤维细胞 ;5～ 7天后 ,在成纤维细胞上出现EG细胞集落 ,集落内细胞表达阶段特异性胚胎表面抗原SSEA 1和SSEA 3,并表达碱性磷酸酶 (ALP)活性 ;培养 1 8天后 ,有6个集落分化为有节律跳动的心肌细胞 ,这些细胞集落在继续培养 1 5天后 ,仍能有节律收缩。此时集落内细胞ALP表达呈阴性 ,但仍有少量细胞表达SSEA 1和SSEA 3。结论 :体外培养人胚生殖嵴、肠背系膜和中肾嵴组织块 ,以内源性的成纤维细胞作为饲养层 ,可以分离得到EG细胞集落 ,一些EG细胞集落可以自发地分化成心肌细胞。     

Ascidian embryonic development: An emerging model system for the study of cell fate specification in chordates

The ascidian tadpole larva represents the basic body plan of all chordates in a relatively small number of cells and tissue types. Although it had been considered that ascidians develop largely in a determinative way, whereas vertebrates develop in an inductive way, recent studies at the molecular and cellular levels have uncovered several similarities in the way developmental fates are specified. In this review, we describe ascidian embryogenesis and its cell lineages, introduce several characteristics of ascidian embryos, describe recent advances in understanding of the mechanisms of cell fate specification, and discuss them in the context of what is known in vertebrates and other organisms. Developmental Dynamics 236:1748–1757, 2007. © 2007 Wiley‐Liss, Inc.     

Cultures of hESM human embryonic stem cells: Chromosomal aberrations and karyotype stability

Cytogenetic analysis of karyotypes of hESM01-hESM04 human embryonic stem cells and substrains derived from these strains showed that all these strains retained normal karyotype during long-term culturing. Two substrains of embryonic stem cells with chromosome aberrations indicating clonal origin of these strains were detected. The potentialities of using analysis of chromosome variability of embryonic stem cells for evaluation of predisposition of the corresponding genotypes to the formation of chromosome abnormalities are discussed. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 3, pp. 143–146, March, 2007     

Ultrastructure of eggshell and embryological development of Salvator merianae (Squamata: Teiidae)

The objective of this study was to characterize the external morphology of Salvator meriane embryos in different stages of embryonic development and establish a relationship with the ultrastructure of the shell from oviductal transit to hatching. A total of 120 embryos were analyzed to describe their external morphology, and 78 eggs were used for the analysis of the shell. For embryonic development, the series was established according to the total length of the body. We established 40 embryonic stages from the primitive streak. In the early stages, the external morphological features are the C-shaped body, the maxillary, and mandibular fusion processes with the frontal process and the fusion of the forelimb with the digital plate. In the middle stages, the eyelid appears, and there are claws on the toes, cornification of fingers, and the onset of pigmentation. The last stage of embryonic development is characterized by the beginning of the formation of the scales, appear the toenails, and finalize the entire pigmentation. Regarding the relationship that exists with the ultrastructure of the egg during development, it was possible to observe a marked change in the composition of the shell and well-marked compaction during embryonic development, which may be related to the transport of calcium during embryonic ossification. Our results allowed us to show the complete sequence of embryonic development, determining the laying stage for this species. It was possible to establish a relationship with the ultrastructure of the eggshell from the oviductal transit to the moment of hatching.     

菘蓝对小鼠胚胎发育的保护作用

目的 :研究菘蓝叶氯仿提取物对环磷酰胺 (CP)引起的胚胎发育障碍的保护作用。方法 :采用小鼠胚胎致畸试验 ,观察了受孕母鼠的体重变化 ,活胎率 ,死胎率 ,吸收胎率 ,以及胎鼠的身长、尾长、体重等指标。并用骨骼染色法观察胎鼠有无骨骼畸形。结果 :菘蓝叶氯仿提取物本身对胚胎发育无毒性 ,而对CP引起的胚胎发育障碍有一定的保护性抑制作用。CP +菘蓝叶提取物 1 0 0mg·kg- 1·w- 1剂量组与阳性对照组相比可显著增加胎鼠的活胎率 (增加 5 9.60 % )、降低吸收胎率 (降低 86.91 % )、及胎鼠畸形率的发生 ,也可明显增加胎鼠的身长、尾长和体重等指标。结论 :中药菘蓝具有保护小鼠胚胎发育的作用。     

不同载体对癌胚抗原DNA疫苗诱导小鼠产生抗-CEA水平的影响

目的　将癌胚抗原（ＣＥＡ） 基因克隆入载体ｐｃＤＮＡ３ 及ＶＲ１０１２ 中,比较两套重组质粒在体内表达并诱导小鼠产生抗ＣＥＡ 的差别。方法　通过基因工程技术构建两套ＣＥＡ 真核表达载体ｐｃＤＮＡ３ＣＥＡ 及ＶＲ１０１２ＣＥＡ,将重组质粒肌注ＢＡＬＢ／ｃ 小鼠,运用ＥＬＩＳＡ 法检测不同时间肌肉组织表达ＣＥＡ 水平及血清中抗ＣＥＡ 的产生。结果　ｐｃＤＮＡ３ＣＥＡ 在肌肉内呈低表达ＣＥＡ,最高时为０－１８ｎｇ／ ｍｇ 肌肉（ 胫前肌） ,而ＶＲ１０１２ＣＥＡ 表达量为０－９３ｎｇ／ ｍｇ 肌肉,但两重组质粒诱导小鼠产生的抗ＣＥＡ 的水平相差不大,均大于１ ２００ 。结论　ＤＮＡ 疫苗诱导的免疫反应不仅取决于其在体内的表达抗原量,质粒结构中的免疫激活序列（ＩＳＳ） 对免疫反应也起重要的调节作用。     

Theoretical mechanisms for synthesis of carcinogen-induced embryonic proteins: IV. The viruses.

Speculations are developed for a mechanism by which oncogenic viruses can induce alterations in cells allowing them to express embryonic genes. It is suggested that if viral deoxyribonucleic acid, directly or via ribonucleic acid directed deoxyribonucleic acid polymerase activity becomes inserted at particular euchromatin - heterochromatin junctions of quasidifferentiated stem-like cells, then deheterochromatization may result, causing in turn derepression of genes for acidic protein phosphokinases. This sets into motion a series of events including altered acid protein repressors of embryonic genes which are repressed by uniquely weak type repressors. This explains how viruses can act as specific embryonic gene-inducing agents similar to chemical inducing agents such as the hepatocarcinogen ethinine.