微流控芯片技术在细胞生物学研究中的应用进展

20世纪90年代以来,微流控芯片技术得到了快速发展。由于具有小型化、集成化、高通量、低消耗、分析快速等特点,微流控芯片作为一种新型的生物学研究平台,能够提供传统方法不具备的精细和可控制的细胞研究条件,在细胞生物学研究领域中得到了广泛关注。该文主要介绍其在细胞培养、分选、裂解、计数、凋亡检测、迁移、单细胞捕获、细胞间作用等方面的研究进展。     

Efficient and Innocuous Live‐Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry

The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry. Herein, the author reviews pioneering examples of such new cell‐based assays, provides evidence that challenges the paradigm that cell penetration is a necessarily damaging and stressful event for cells, and highlights some of the challenges that should be addressed to fully unlock the potential of this nascent field.     

Ultrastructural evidence for two-cell and three-cell neural pathways in the tentacle epidermis of the sea anemone Aiptasia pallida.

Sensory and ganglion cells in the tentacle epidermis of the sea anemone Aiptasia pallida were traced in serial transmission electron micrographs to their synaptic contacts on other cells. Sensory cell synapses were found on spirocytes, muscle cells, and ganglion cells. Ganglion cells, in turn, synapsed on sensory cells, spirocytes, muscle cells, and other neurons and formed en passant axo-axonal synapses. Axonal synapses on nematocytes and gland cells were not traced to their cells of origin, i.e., identified sensory or ganglion cells. Direct synaptic contacts of sensory cells with spirocytes and sensory cells with muscle cells suggest a local two-cell pathway for spirocyst discharge and muscle cell contraction, whereas interjection of a ganglion cell between the sensory and effector cells creates a local three-cell pathway. The network of ganglion cells and their processes allows for a through-conduction system that is interconnected by chemical synapses. Although the sea anemone nervous system is more complex than that of Hydra, it has similar two-cell and three-cell effector pathways that may function in local responses to tentacle contact with food.     

The role of glutamine, serum and energy factors in growth of enterocyte-like cell lines

Background: Glutamine is routinely added to most cell cultures. Glutamine has been found to be the preferential nutrient to the rapidly replicating intestinal mucosa, but whether this is a metabolic effect or due to other properties of this amino acid is not determined. To study the importance of glutamine on the growth of two enterocyte-like cell lines, the effects of depriving the media or supplementing it with glutamine were assessed in media with different serum and energy supplements. Methods: CaCo-2 and HT-29 cells were grown in serum-free medium, with fetal bovine or synthetic serum, and with or without glucose or galactose. The glutamine content was varied between 0 and 4 mM. All growth assays were performed in triplicate by counting in a hemocytometer. Results: Both cell lines were dependent of serum factors for growth, but displayed distinct requirements on glutamine supplementation. Glutamine was an obligate supplement with dose-dependent correlation to growth (r=0.87, p<0.01) for CaCo-2 cells cultured in synthetic, but not in fetal bovine serum. In HT-29 cells, the correlation between glutamine and growth was significant (r=0,68, p<0,05) only in fetal bovine serum in the absence of galactose. Conclusion: This study shows that glutamine has different growth stimulating effects on two enterocyte-like cell lines studied. This could reflect different modes of action of glutamine on proliferation and differentiation in an enterocyte cell population.     

Of mice, frogs and flies: generation of membrane asymmetries in early development

Embryonic development begins with cleavage of the fertilized egg. Cleavage comprises two major processes: cytokinesis and formation of a polarized epithelial cell layer. The focus of this review is comparison of the generation of membrane polarity during embryonic cleavage in three different developmental model systems. In mammalian embryos, as exemplified by analysis of the mouse, generation of distinct membrane domains is uncoupled from cleavage divisions and is initiated in a specific developmental phase, called compaction. In Xenopus laevis embryos, generation of polarized blastomeres occurs simultaneously with cytokinesis. The origin of specific membrane domains of X. laevis polar blastomeres, however, can be traced back to oogenesis. Finally, in Drosophila melanogaster, generation of polarized cells occurs at cellularization. The relevance of cell adhesion, cell junctions and cytocortical scaffolds will be discussed for each of the model systems. Despite enormous morphologic differences, the three models share many common features; in particular, many important molecular interactions are conserved.     

哺乳动物体细胞核移植中供体细胞的研究进展

在哺乳动物体细胞核移植中,供体细胞是影响其效率的主要因素之一。供体细胞的类型、细胞周期、细胞的培养代数、冷藏与冷冻处理,以及供体动物的性别、年龄等都可能影响核移植胚胎的发育。根据现有资料,简要综述了在哺乳动物体细胞核移植中有关供体细胞的研究进展。     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

Engineering Cardiac Cell Junctions In Vitro to Study the Intercalated Disc

Abstract

This review article discusses a recent work using engineered cardiac cells to study the function of the intercalated disc putting emphasis on mechanical and electrical coupling.     

Mammalian testis: a target of in vivo electroporation

Mammalian spermatogenesis consists of three biologically significant processes: stem cell self-renewal and differentiation, meiosis, and haploid cell morphogenesis. Understanding the molecular mechanisms behind these processes might provide clues to the puzzle of species preservation and evolution, and to treatments for male infertility. However, few useful in vitro systems exist to investigate these processes at present. To elucidate these mechanisms, in vivo electroporation of the testis might be a convenient option. Since DNA solution can be injected into the seminiferous tubule via the rete testis, similar to germ cell transplantation, it is easy to transfect expression vectors into various differentiated germ cells and supporting Sertoli cells with adequate electric shock. Unfortunately, it is difficult to create transgenic animals using this method because of its low efficiency. However, gain- and loss-of-function assays, promoter assays, and tagged-protein behavior assays can be conducted with this technique, as in in vitro culture systems.     

Besides Purkinje cells and granule neurons: an appraisal of the cell biology of the interneurons of the cerebellar cortex

Ever since the groundbreaking work of Ramon y Cajal, the cerebellar cortex has been recognized as one of the most regularly structured and wired parts of the brain formed by a rather limited set of distinct cells. Its rather protracted course of development, which persists well into postnatal life, the availability of multiple natural mutants, and, more recently, the availability of distinct molecular genetic tools to identify and manipulate discrete cell types have suggested the cerebellar cortex as an excellent model to understand the formation and working of the central nervous system. However, the formulation of a unifying model of cerebellar function has so far proven to be a most cantankerous problem, not least because our understanding of the internal cerebellar cortical circuitry is clearly spotty. Recent research has highlighted the fact that cerebellar cortical interneurons are a quite more diverse and heterogeneous class of cells than generally appreciated, and have provided novel insights into the mechanisms that underpin the development and histogenetic integration of these cells. Here, we provide a short overview of cerebellar cortical interneuron diversity, and we summarize some recent results that are hoped to provide a primer on current understanding of cerebellar biology.     

Perivascular cells for regenerative medicine

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.     

Uniform straw-like cell architecture for three-dimensional cell–cell communication assay

ABSTRACT

We fabricated uniform straw-like cell architecture with central lumen using a suture thread within 1 h. The architecture consisting of cancer cells and mature adipocyte was used for cell–cell communication assay, although mature adipocyte could not form spontaneous multi-cellular spheroids. Using the system, it is possible to investigate three-dimensional cell–cell communication as an alternative to animal experiments.     

牛脂肪间充质干细胞的分离、培养与鉴定

为了给组织工程提供种子细胞,对牛间充质干细胞(Adipose-derived stem cells,ADSCs)进行体外分离培养。首先应用胶原酶消化法分离牛ADSCs,进行体外培养、连续传代,并观察细胞的形态变化,通过细胞计数绘制生长曲线,细胞压片进行染色体分析,采用细胞免疫荧光化学方法检测细胞表面标记,利用成骨分化和成脂分化检测其分化能力。结果显示牛ADSCs体外培养时细胞形态呈成纤维细胞样,增殖稳定;Vimentin、CD49d、CD13表达呈阳性,CD34表达呈阴性;成骨诱导条件下的细胞碱性磷酸酶活性高,茜素红染色呈阳性;成脂诱导条件下细胞周围脂滴明显,油红-O染色呈阳性。结果证明牛ADSCs体外生长稳定、增殖速度快、定向分化能力强,简易的体外分离培养及诱导方法为其在组织工程中的应用奠定了基础。     

Primordial germ cell-somatic cell partnership: a balancing cell signaling act

Recent studies demonstrate that the normal progression of the germ cell lineage during gonadogenesis involves a delicate balance of primordial germ cell survival and death factors generated by surrounding somatic cells. This balance operates in a different fashion in females and males. The fine tuning primordial germ cell specification in the wall of the yolk sac, migration through the hindgut and dorsal mesentery, and colonization in the urogenital ridges involves the temporal and spatial activation of the following signaling pathways: Primordial germ cell specification involves bone morphogenetic proteins 2, 4 and 8b, and their migration is facilitated by the c-kit receptor-ligand duet. When colonization occurs: (1) neuregulin-beta ligand is expressed and binds to an ErbB2-ErbB3 receptor tyrosine kinase heterodimer on primordial germ cells; (2) Vasa, an ortholog of the Drosophila gene vasa, member of an ATP-dependent RNA helicase of the DEAD (Asp-Glu-Ala-Asp)-box family protein is also expressed by primordial germ cells; (3) Bcl-x (cell survival factor) and Bax (cell death factor) join forces to modulate the first burst of primordial germ cell apoptosis; (4) Cadherins, integrins, and disintegrins bring together primordial germ cells and somatic cells to organize testis and ovary. Information on other inducers of primordial cell survival, such as TER (teratoma) factor, is beginning to emerge.     

干细胞理论及研究进展

干细胞是目前细胞工程研究最活跃的领域,通过对各种干细胞的界定,胚胎干细胞和成体干细胞的比较研究、以及干细胞的技术应用,揭示出干细胞尤其是胚胎干细胞在医学以及整个生命科学中的巨大潜势,乃至于引发医学领域的重大变革。     

以T细胞受体为基础的免疫疗法研究进展

T细胞是机体抗肿瘤免疫的核心,以T细胞功能调控为基础的免疫检查点疗法已经在多种肿瘤的临床治疗中取得了重大突破,以基因工程化T细胞为基础的过继性免疫细胞疗法在血液瘤治疗中取得了重要进展,免疫治疗已经对肿瘤的临床治疗产生了深刻变革,成为肿瘤临床治疗策略的重要组成部分。T细胞受体（T cell receptor,TCR）赋予了T细胞识别肿瘤抗原的特异性,能够识别由主要组织相容性复合体（major histocompatibility complex,MHC）呈递的包括胞内抗原在内的广泛肿瘤抗原,具有高度的抗原敏感性,因而具有广泛的抗肿瘤应用前景。2022年第一款TCR药物的上市开启了TCR药物开发的新纪元,多项TCR药物临床研究表现出潜在的肿瘤治疗价值。本文综述了以TCR为基础的免疫治疗策略研究进展,包括T细胞受体工程化T细胞（T cell receptor-engineered T cell,TCR-T）和TCR蛋白药物,以及基于TCR信号的其他免疫细胞疗法,以期为以TCR为基础的免疫治疗策略开发提供参考。     

BMP signaling and stem cell regulation

Stem cells play an essential role in cellular specialization and pattern formation during embryogenesis and in tissue regeneration in adults. This is mainly due to a stem cell's ability to replenish itself (self-renewal) and, at the same time, produce differentiated progeny. Realization of these special stem cell features has changed the prospective of the field. However, regulation of stem cell self-renewal and maintenance of its potentiality require a complicated regulatory network of both extracellular cues and intrinsic programs. Understanding how signaling regulates stem cell behavior will shed light on the molecular mechanisms underlying stem cell self-renewal. In this review, we focus on comparing the progress of recent research regarding the roles of the BMP signaling pathway in different stem cell systems, including embryonic stem cells, germline stem cells, hematopoietic stem cells, and intestinal stem cells. We hope this comparison, together with a brief look at other signaling pathways, will bring a more balanced view of BMP signaling in regulation of stem cell properties, and further point to a general principle that self-renewal of stem cells may require a combination of maintenance of proliferation potential, inhibition of apoptosis, and blocking of differentiation.     

Phenotypic Modulation of Hamster Acinar Cells by Culture in Collagen Matrix

The aim of this study was to assess the effect of different culture conditions on the survival and morphological phenotype of cultured acinar cells. Acinar fragments isolated from hamster pancreas were embedded in rat-tail collagen. Four groups were established: Medium 1—5% NuSerum + basic medium (basic MEDIUM = DMEM/F12 supplemented with dexamethasone, 3-isobutyl-2-methylxanthine, and antibiotics); Medium 2—10% NuSerum + basic medium. Medium 3—Medium 2 supplemented with epidermal growth factor and cholera toxin; and Medium 4:—Medium 3 supplemented with soybean trypsin inhibitor. Freshly isolated acinar cells were retrieved morphologically intact. In Medium 1, more than 80% of cells retained a normal histological appearance at 34 days in culture. Immunostaining for amylase was observed at the apical pole of the cells. The remaining cells showed variable degrees of degeneration. In Medium 2, approximately 50% of acinar cells appeared normal at 34 days in culture, while the remainder were severely degenerated. A few cystic structures were also observed. Positive immunostaining for amylase was limited to the cells with a normal histological appearance. The cells grown in Media 3 and 4 had similar courses of morphological changes. After 8 days in culture, most acinar fragments disappeared and were replaced by cystic structures, lined by a single layer of cuboidal cells. Some amylase-positive immunoreactive cells were integral components of the cystic wall. Cellular amylase activity was a function of the different culture media, a more rapid decrease in amylase activity being observed in Media 3 and 4. Uptake of [³H]thymidine did not show any significant differences between the media. It was also found that the ductlike cells cultured in Medium 4 had a limited capacity to redifferentiate into acinar cells. This study shows that the acinar cell phenotype can be maintainedin vitrofor more than 1 month. This study also suggests that ductal-like epithelial structures arise from transformation of acinar cells.     

Trout sertoli and leydig cells: Isolation,separation, and culture

Trout testes at various stages of maturation were dissociated by perfusion at 12°C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin. This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes). Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml). Sertoli and Leydig cells were prepared by a two-step separation method: (1) the testis cell suspension was separated by sedimentation at unit gravity into “isolated cell” and “cell cluster” populations; (2) these populations were fractionated by isopyknic centrifugation in Percoll gradients. In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes). These various cell populations were cultured in modified Leibovitz L15 medium for 10–15 days. When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin. Leydig cells remained 3β-HSD positive and produced progesterone and 17α, 20β-OH progesterone for at least 11 days. This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.