利用单细胞RT-PCR体外克隆个体化乙型肝炎表面抗体重链

目的建立一种利用单细胞RT-PCR技术克隆乙型肝炎表面抗体重链的方法,为人源化、个体化的乙肝治疗性抗体研制奠定基础。方法利用单细胞分选技术从乙肝疫苗接种志愿者外周血中分选得到单个抗体分泌细胞（antibody-secreting cells,ASC）,用单细胞RT-PCR克隆其IgG重链全长到pEGFP-N1载体,转染COS-7细胞,上清液用乙肝表面抗原（hepatitis B surface antigen,HBsAg）通过Western blot进行结合力验证。结果克隆得到1 500 bp左右的重链全长,重链表达产物经验证能与乙肝表面抗原特异结合。结论克隆得到表达产物能与HBsAg特异结合的重链基因,为我们后期制备抗体轻链、连接轻重链和抗体亲和力鉴定奠定基础。     

Cell lineage analysis of cerebellar Purkinje cells in mouse chimeras

Murine chimeras provide an experimental system in which cell lineage analysis of the mammalian central nervous system (CNS) can be accomplished. Utilizing a cell marker system that permits the identification of cells of each genotype in various cell populations present in histologic sections of the CNS at different developmental periods, fate maps of the mammalian CNS can be constructed. Thus, the presence or persistence of clones of cells can be readily visualized in simply organized CNS regions, like the cerebellar cortex. The electrophoretic variants of the glycolytic enzyme, glucosephosphate isomerase (GPI, E.C. 5.3.1.9; GPI-1A, GPI-1B), are the genotype-specific cell markers most commonly used by experimental mammalian embryologists in studies of cell lineage utilizing mammalian chimeras. We have adapted this cell marker system to permit the visualization and unequivocal identification of cells containing the GPI-1B variant throughout the CNS of adult $\text{BALB}\text{cBy}{}^{\mathrm{J}}\text{a3}\text{C57BL}\text{6J}$ chimeric mice. Utilizing allozyme-specific anti-GPI-1B antisera in immunocytochemical (PAP) staining techniques, we can score small as well as large cell populations, neurons as well as glia. We have reconstructed and statistically analyzed the location and distribution of chimerism present in the Purkinje cell population of four of these chimeric mice. We found the Purkinje cells in each of these animals existed as small (3–8) cell patches of like genotype that were not randomly arranged. This suggests that clones of cells may persist as contiguous groups of cells throughout mammalian cerebellar development.     

Kinetic analysis of single sodium channels from canine cardiac Purkinje cells

Single sodium channel events were recorded from cell-attached patches on single canine cardiac Purkinje cells at 10-13 degrees C. Data from four patches containing two to four channels and one patch with one channel were selected for quantitative analysis. The channels showed prominent reopening behavior at voltages near threshold, and the number of reopenings declined steeply with depolarization. Mean channel open time was a biphasic function of voltage with the maximum value (1-1.5 ms) occurring between -50 and -40 mV and lower values at more and at less hyperpolarized levels. Inactivation without opening was also prominent near threshold, and this occurrence also declined with depolarization. The waiting time distributions and the probability of being open showed voltage and time dependence as expected from whole-cell current studies. The results were analyzed in terms of a five-state Markovian kinetic model using both histogram analysis and a maximum likelihood method to estimate kinetic parameters. The kinetic parameters of the model fits were similar to those of GH3 pituitary cells (Horn, R., and C. A. Vandenberg. 1984. Journal of General Physiology. 84:505-534) and N1E115 neuroblastoma cells (Aldrich, R. W., and C. F. Stevens. Journal of Neuroscience. 7:418-431). Both histogram and maximum likelihood analysis implied that much of the voltage dependence of cardiac Na current is in its activation behavior, with inactivation showing modest voltage dependence.     

Aging of cerebellar Purkinje cells

Cerebellar Purkinje cells (PCs), the sole output neurons in the cerebellar cortex, play an important role in the cerebellar circuit. PCs appear to be rather sensitive to aging, exhibiting significant changes in both morphology and function during senescence. This article reviews such changes during the normal aging process, including a decrease in the quantity of cells, atrophy in the soma, retraction in the dendritic arborizations, degeneration in the subcellular organelles, a decline in synapse density, disorder in the neurotransmitter system, and alterations in electrophysiological properties. Although these deteriorative changes occur during aging, compensatory mechanisms exist to counteract the impairments in the aging PCs. The possible neural mechanisms underlying these changes and potential preventive treatments are discussed.     

Integrated analysis of multimodal single-cell data

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A single-cell view of spermatogonial stem cells

Spermatogonial stem cells (SSCs) are essential for long-term spermatogenesis and are the subject of considerable clinical interest, as ‘SSC therapy’ has the potential to cure some forms of male infertility. Recently, we have learned more about SSCs and spermatogenesis in general from a plethora of studies that performed single-cell RNA sequencing (scRNAseq) analysis on dissociated cells from human, macaque, and/or mice testes. Here, we discuss what scRNAseq analysis has revealed about SSC precursor cells, the initial generation of SSCs during perinatal development, and their heterogeneity once established. scRNAseq studies have also uncovered unexpected heterogeneity of the larger class of cells that includes SSCs — undifferentiated spermatogonia. This raises the controversial possibility that multiple SSC subsets exist, which has implications for mechanisms underlying spermatogenesis and future SSC therapeutic approaches.     

Variable stainability of Purkinje cells]

The author refers about different staining of the Purkinje-cells with luxol-fast-blue, gallocyanin, thionin and toluidin blue, chrom-alum-hematoxylin-phloxin, impregnation according to Palmgren, lithium and iron-hematoxylin, combination of the staining with phloxin and the Palmgren-impregnation and about the different activity on the acid phosphatase. The phenomenon that in the same histological specimen the positive (dark, chromophile) and negative (light, chromophobe) cells are situated beside, is true for normal animals too, but the number of the dark Purkinje cells is conspicuous higher after stress situations (96-h. immobilisation, intermittent hypoxia). This finding interprets the author by the occurence of phospholipids by binding on the granulated endoplasmatic reticulum, but also as a property of the neuroplasm. The author emphasizes that the staining dualism "light -- dark" of the ganglion cells does not refer only to the ganglion cells of the spinal ganglions (et on some epithelial cells), but also on the Purkinje cells.     

Different stainability of Purkinje cells.

Regarding the different stainability when using the Luxol fast blue methods, two kinds of Purkinje cells of the rat are described: Luxol-positive and Luxol-negative cells. Since, by this method, phospholipids are demonstrated, the author suggests the prospective varying functional conditions of these cells. Different tinction of Purkinje cells has been confirmed also by other methods (gallocyanin-chromalaun, thionine, toluidine blue, lithium-haematoxylin, chromalaun-haematoxylin-phloxine and acid phosphatase) in both animal and human material. After 96 h of immobilization the different stainability of Purkinje cells becomes more marked, which penomenon can be as well explained with regard to the functional point of view. Similar differences, though less marked, were found also in neurosecretory cells of the nucelus supra-opticus of the rat and in the nuclear region of the ganglion semilunare Gasseri cells in man. Finally, the author refers to the relations between the Luxol blue staining method and Baker's method employing acid haematoxylin for demonstration of phospholipids in certain kinds of nervous system cells, taking into consideration Kroon's findings.     

Differential appearance of dynamin in constitutive and regulated exo-endocytosis: a single-cell multiplex RT-PCR study

Neurons in the central nervous system establish, via their axons and dendrites, an extended network that allows synaptic transmission. During developmental maturation and process outgrowth, membrane turnover is necessary for the enlargement and subsequent growth of axons and dendrites from the perikarya to the target cell (constitutive exocytosis/endocytosis). After targeting and synapse formation, small synaptic vesicles are needed for the quantal release of neurotransmitters from the presynaptic terminal with subsequent recycling by regulated exocytosis/endocytosis. An investigation of the onset of the appearance of mRNA and protein in dissociated cultures of neurons from mouse hippocampus or from chick retina has shown an early abundance of proteins involved in exocytosis, such as syntaxin 1, SNAP-25, and synaptotagmin 1, whereas dynamin 1, a protein necessary for clathrin-mediated endocytosis, can be detected only after neurons have established contacts with neighboring cells. The results reveal that constitutive membrane incorporation and regulated synaptic transmitter release is mediated by the same neuronal proteins. Moreover, the data exclude that dynamin 1 takes part in constitutive recycling before synapse formation, but dynamin 2 is present at this stage. Thus, dynamin 2 may be the constitutive counterpart of dynamin 1 in growing neurons. Synapse establishment is linked to an upregulation of dynamin 1 and thereby represents the beginning of the regulated recycling of membranes back into the presynaptic terminal.     

Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis

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单细胞转录组高通量测序分析新进展

细胞异质性是生物组织的普遍特征。常规转录组测序(RNA-Seq)技术需要上万个细胞,所测结果实际上是一群细胞基因表达的平均值,所以难以鉴别细胞之间基因表达的异质性。单细胞RNA-Seq技术的分辨率精确至单个细胞,为辨别异质性群体中各种细胞类型的转录组特征提供了有力的工具。近年来单细胞RNA-Seq技术发展迅速,在方法学上包括cDNA扩增方法的多样化、对灵敏度和技术噪声的定量分析、浅覆盖高通量单细胞RNA-Seq方法和原位RNA-Seq技术等;在技术应用方面应用范围从早期胚胎发育扩大到组织器官发育、免疫和肿瘤等多个领域。文章对单细胞RNA-Seq在方法学和技术应用两方面的研究进展进行了详细阐述。     

Microtechnologies and nanotechnologies for single-cell analysis

Many efforts are currently underway to try and mimic the properties of single cells with the aim of designing chips that are as efficient as cells. However, cells are nature's nanotechnology engineering at the scale of atoms and molecules, and it might be better to envision a microchip that utilizes a single cell as an experimentation platform. A novel, so-called laboratory-in-a-cell concept has been described, where advantage is taken of micro- and nanotechnological tools to enable precise control of the biochemical cellular environment; these tools also offer the possibility to analyse the composition of single cells. Methods for single-cell handling and analysis are being developed and will be required for this concept to progress further.     

单细胞测序分析人类胚胎干细胞神经分化的分子机制

目的 探讨人类胚胎干细胞(ESCs)分化为神经细胞的关键性靶基因及分子机制,为临床靶向治疗神经康复患者提供分子理论依据.方法 基于GEO数据平台芯片,采用单细胞测序方法(scRNA-seq),利用R语言从多分子维度(单细胞差异基因、蛋白互作网络和基因通路等)分析人类ESCs分化过程中的关键Marker基因并利用质控和数...