上皮间质转化与干细胞自我更新和分化

上皮间质转化(epithelial-mesenchymal transition,EMT)是指上皮细胞失去连接和极性转变为间质细胞的过程,这一现象普遍存在于胚胎发育、创伤愈合、器官纤维化以及肿瘤转移.在胚胎早期发育和晚期发育过程,例如着床、原肠运动、心血管发育等事件中有EMT和间质上皮转化(mesenchymal-ep...     

Planar Cell Polarity Signaling in Collective Cell Movements During Morphogenesis and Disease

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders.     

微小RNA-30e调控EMT对胃癌侵袭和迁移影响的研究

目的:探讨微小RNA-30e(miR-30e)对胃癌细胞迁移和侵袭能力的影响及可能的作用机制。方法:利用Transwell实验和细胞划痕实验检测胃癌细胞系BGC823侵袭和迁移的能力;以脂质体包裹合成miR-30e转染至BGC823细胞,并设空白载体作为对照组;Real-time PCR分别检测实验组和对照组细胞中miR-30e的表达。RT-PCR检测过表达miR-30e后对上皮细胞间充质转化(EMT)相关标记分子Snail、Vimentin、N-cadherin和E-cadherin表达的影响。结果:miR-30e转染至胃癌细胞后,抑制EMT通路主要因子Snail,Vimentin和N-cadherin m RNA和蛋白质表达,而增加E-cadherin的mRNA和蛋白质表达;miR-30e通过TGF-β对BGC823细胞的侵袭和迁移能力有明显的抑制作用。结论:miR-30e可能是肿瘤细胞EMT过程的关键靶标靶点,阻断EMT过程,可以抑制胃癌细胞的侵袭和迁移能力。     

Fibroblast Growth Factor (FGF) Signaling during Gastrulation Negatively Modulates the Abundance of MicroRNAs That Regulate Proteins Required for Cell Migration and Embryo Patterning

FGF signaling plays a pivotal role in regulating cell movements and lineage induction during gastrulation. Here we identify 44 microRNAs that are expressed in the primitive streak region of gastrula stage chicken embryos. We show that the primary effect of FGF signaling on microRNA abundance is to negatively regulate the levels of miR-let-7b, -9, -19b, -107, -130b, and -218. LIN28B inhibits microRNA processing and is positively regulated by FGF signaling. Gain- and loss-of-function experiments show that LIN28B negatively regulates the expression of miR-19b, -130b, and let-7b, whereas negative modulation of miR-9, -107, and -218 appears to be independent of LIN28B function. Predicted mRNA targets of the FGF-regulated microRNAs are over-represented in serine/threonine and tyrosine kinase receptors, including ACVR1, ACVR2B, PDGFRA, TGFBR1, and TGFBR3. Luciferase assays show that these and other candidates are targeted by FGF-regulated microRNAs. PDGFRA, a receptor whose activity is required for cell migration through the primitive streak, is a target of miR-130b and -218 in vivo. These results identify a novel mechanism by which FGF signaling regulates gene expression by negatively modulating microRNA abundance through both LIN28B-dependent and LIN28B-independent pathways.     

miR-448通过抑制上皮间质转化抑制肺癌细胞增殖和运动能力

探讨mi R-448对肺癌细胞增殖和运动的影响及其分子机制。采用实时荧光定量PCR(polymerase chain reaction)检测原发肺癌组织和癌旁正常组织mi R-448表达水平。转染mi R-448 mimic和inhibitor至肺癌A549细胞系,通过MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide)、平板克隆形成和Transwell实验观察mi R-448表达对A549增殖和运动能力的影响;利用Western blot检测EMT(epithelial-mesenchymal transition)标志物蛋白表达水平,通过实时荧光定量PCR检测EMT相关转录因子m RNA表达水平。实时荧光定量PCR显示较癌旁正常组织相比,mi R-448在原发肺癌组织中表达降低。MTT和平板克隆形成实验显示,过表达mi R-448抑制A549细胞增殖和运动能力;降表达mi R-448增强A549细胞增殖和运动能力。Western blot显示降表达mi R-448能下调上皮标志物E-cadherin,上调间质标志物Vimentin表达水平。实时荧光定量PCR显示降表达mi R-448能上调EMT相关转录因子Twist1和ZEB1 m RNA表达水平。mi R-448可通过抑制EMT抑制肺癌进展。     

热疗对SW1990 胰腺癌细胞上皮间质转化（EMT）的影响及其作用机制

目的：探讨热疗对化疗诱导的人胰腺癌细胞株SW1990 细胞上皮间质转化（EMT）的影响及其可能的作用机制。方法：分别 用不同浓度吉西他滨(0, 5, 10, 20,30 滋M)作用于SW1990 细胞不同时间(24, 48, 72 小时)及30 滋M 吉西他滨作用24h 联合热疗 43℃ 1h,观察细胞的形态学变化,通过四甲基偶氮唑蓝(MTT) 法检测细胞的增殖情况。采用Western blot 方法检测细胞内 E-cadherin 蛋白和剪切的Notch1 蛋白的表达。结果：①吉西他滨在一定浓度范围内可明显抑制SW1990细胞的增殖（P<0.05）,并 呈浓度和时间依赖性。吉西他滨作用前24 h 给予43℃ 1h热疗预处理可显著增强吉西他滨对SW1990细胞的抑制作用（P<0.05） ②吉西他滨作用SW1990 细胞24 小时后,细胞数目减少,细胞形态变大,细胞呈梭形,且细胞间连接减少;而热疗预处理的联合 能够逆转此种形态学变化。③吉西他滨作用24 小时后,细胞内E-cadherin蛋白的表达下调、Cleaved Notch1 的蛋白表达上调,热 疗预处理可明显上调吉西他滨诱导的E-cadherin 蛋白表达下调、并下调Cleaved Notch1 蛋白表达的上调。结论：热疗预处理显著 逆转吉西他滨所诱导的人胰腺癌细胞株SW1990 细胞的EMT 现象,其机制可能与Notch 信号通路有关。     

On the Mechanical Interplay Between Intra- and Inter-Synchronization During Collective Cell Migration: A Numerical Investigation

Collective cell migration is a fundamental process that takes place during several biological phenomena such as embryogenesis, immunity response, and tumorogenesis, but the mechanisms that regulate it are still unclear. Similarly to collective animal behavior, cells receive feedbacks in space and time, which control the direction of the migration and the synergy between the cells of the population, respectively. While in single cell migration intra-synchronization (i.e. the synchronization between the protrusion-contraction movement of the cell and the adhesion forces exerted by the cell to move forward) is a sufficient condition for an efficient migration, in collective cell migration the cells must communicate and coordinate their movement between each other in order to be as efficient as possible (i.e. inter-synchronization). Here, we propose a 2D mechanical model of a cell population, which is described as a continuum with embedded discrete cells with or without motility phenotype. The decomposition of the deformation gradient is employed to reproduce the cyclic active strains of each single cell (i.e. protrusion and contraction). We explore different modes of collective migration to investigate the mechanical interplay between intra- and inter-synchronization. The main objective of the paper is to evaluate the efficiency of the cell population in terms of covered distance and how the stress distribution inside the cohort and the single cells may in turn provide insights regarding such efficiency.     

Activities of Enzymes Involved in the Metabolism of Platelet-Activating Factor in Neural Cell Cultures During Proliferation and Differentiation

Platelet-Activating Factor (PAF) is a potent lipid mediator involved in physiological and pathological events in the nervous tissue where it can be synthesized by two distinct pathways. The last reaction of the de novo pathway utilizes CDPcholine and alkylacetylglycerol and is catalyzed by a specific phosphocholinetransferase (PAF-PCT) whereas the remodelling pathway ends with the reaction catalyzed by lyso-PAF acetyltransferase (lyso-PAF AcT) utilizing lyso-PAF, a product of phospholipase A₂ activity, and acetyl-CoA. The levels of PAF in the nervous tissue are also regulated by PAF acetylhydrolase that inactivates this mediator. We have studied the activities of these enzymes during cell proliferation and differentiation in two experimental models: 1) neuronal and glial primary cell cultures from chick embryo and 2) LA-N-1 neuroblastoma cells induced to differentiate by retinoic acid (RA). In undifferentiated neuronal cells from 8-days chick embryos the activity of PAF-PCT was much higher than that of lyso-PAF AcT but it decreased during the period of cellular proliferation up to the arrest of mitosis (day 1–3). During this period no significant changes of lyso-PAF AcT activity was observed. Both enzyme activities increased during the period of neuronal maturation and the formation of cellular contacts and synaptic-like junctions. The activity of PAF acetylhydrolase was unchanged during the development of the neuronal cultures. PAF-PCT activity did not change during the development of chick embryo glial cultures but lyso-PAF AcT activity increased up to the 12th day. RA treatment of LA-N-1 cell culture in proliferation decreased PAF-PCT activity and had no significant effect on lyso-PAF AcT and PAF acetylhydrolase indicating that the synthesis of PAF by the enzyme catalyzing the last step of the de novo pathway is inhibited when the LA-N-1 cells are induced to differentiate. These data suggest that: 1) in chick embryo primary cultures, both pathways are potentially able to contribute to PAF synthesis during development of neuronal cells particularly when they form synaptic-like junctions whereas, during development of glial cells, only the remodelling pathway might be particularly active on synthesizing PAF; 2) in LA-N-1 neuroblastoma cells PAF-synthesizing enzymes coexist and, when cells start to differentiate the contribution of the de novo pathway to PAF biosynthesis might be reduced.     

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.     

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.     

壳聚糖薄膜成球培养对脐带间充质干细胞迁徙趋化的影响

基于细胞实验研究壳聚糖（chitosan,CS）薄膜成球培养技术对间充质干细胞(mesenchymal stem cells, MSCs)迁徙趋化特性的影响。从脐带组织中分离原代MSCs采取CS成球法培养,以常规贴壁培养MSCs作为对照,72 h后收集两组细胞分别进行划痕实验、Tranthwell迁徙实验观察并拍照记录,RT-PCR方法检测两种培养方式中MSCs迁徙相关基因表达水平的差异。研究结果显示,相较常规贴壁培养方式,CS培养组MSCs体外迁徙趋化能力增强,差异具有显著统计学意义（P<0.01）;CS成球培养组MSCs 中CXCR4、CXCR7、MCP-1、MMP-1、MMP-2、MMP-9、TIMP-2等迁徙相关基因表达均明显上调（P<0.01）。实验表明CS成球培养可显著促进MSCs的迁移趋化特性。     

Cell Migration and Cell-Cell Interaction in the Presence of Mechano-Chemo-Thermotaxis

Although there are several computational models that explain the trajectory that cells take during migration, till now little attention has been paid to the integration of the cell migration in a multi-signaling system. With that aim, a generalized model of cell migration and cell-cell interaction under multisignal environments is presented herein. In this work we investigate the spatio-temporal cell-cell interaction problem induced by mechano-chemo-thermotactic cues. It is assumed that formation of a new focal adhesion generates traction forces proportional to the stresses transmitted by the cell to the extracellular matrix. The cell velocity and polarization direction are calculated based on the equilibrium of the effective forces associated to cell motility. It is also assumed that, in addition to mechanotaxis signals, chemotactic and thermotactic cues control the direction of the resultant traction force. This model enables predicting the trajectory of migrating cells as well as the spatial and temporal distributions of the net traction force and cell velocity. Results indicate that the tendency of the cells is firstly to reach each other and then migrate towards an imaginary equilibrium plane located near the source of the signal. The position of this plane is sensitive to the gradient slope and the corresponding efficient factors. The cells come into contact and separate several times during migration. Adding other cues to the substrate (such as chemotaxis and/or thermotaxis) delays that primary contact. Moreover, in all states, the average local velocity and the net traction force of the cells decrease while the cells approach the cues source. Our findings are qualitatively consistent with experimental observations reported in the related literature.     

miR-181a通过调控上皮间质转化过程影响卵巢癌细胞的迁移和侵袭

目的:探讨mi R-181a在卵巢癌细胞中的表达及对卵巢癌细胞的迁移和侵袭的影响和可能机制。方法:采用细胞免疫荧光检测卵巢癌细胞中抗波形蛋白和E-钙粘蛋白的表达,Western blotting检测mi R-181a对抗波形蛋白和E-钙粘蛋白的表达情况的调控;划痕愈合实验检测mi R-181a对卵巢癌细胞迁移能力的影响;Transwell侵袭实验检测mi R-181a对卵巢癌细胞侵袭能力的影响。结果:增加mi R-181a的表达后,EMT过程中的相关蛋白激活水平下调,mi R-181a一定程度上可以抑制卵巢癌细胞的EMT过程;过表达mi R-181a后可以明显影响Vimentin[D1(4.58±0.85)vs(0.29±0.02),P0.05;D5(4.16±0.79)vs(0.29±0.02),P0.05]和E-Cadherin[D1(4.75±0.41) vs.(4.56±0.38),P0.05;D5(2.19±0.18) vs.(4.56±0.38),P0.05]蛋白的表达;COC1细胞的迁移能力随mi R-181a的表达的增高而降低[(19.24±4.31)%vs.(25.95±6.02)%,P0.05;(51.25±8.75)%vs.(73.49±12.54)%,P0.05];过表达mi R-181a后可以一定程度上减弱卵巢癌COC1细胞的侵袭能力[(74.64±8.21)vs(231.98±21.72),P0.05]。结论:mi R-181a通过调控上皮间质转化过程影响卵巢癌细胞的迁移和侵袭行为。     

Distribution of Keratan Sulphate and Chondroitin Sulphate in Wild Type and White Mutant Axolotl Embryos During Neural Crest Cell Migration

In embryos of the white mutant axolotl, prospective pigment cells are unable to migrate from the neural crest (NC) due to a deficiency in the subepidermal extracellular matrix (ECM). This raises the question of the molecular nature of this functional defect. Some PGs can inhibit cell migration on ECM molecules in vitro, and an excess of this class of molecules in the migratory pathways of neural crest cells might cause the restricted migration of prospective pigment cells seen in the white mutant embryo. In the present study, we use several monoclonal antibodies against epitopes on keratan sulphate (KS) and chondroitin sulphate (CS) and LM immunofluorescence to examine the distribution of these glycosaminoglycans at initial (stage 30) and advanced (stage 35) stages of neural crest cell migration. Most KS epitopes are more widely distributed in the white mutant than in the wild type embryo, whereas CS epitopes show very similar distributions in mutant and wild type embryos. This is confirmed quantitatively by immunoblotting: certain KS epitopes are more abundant in the white mutant. TEM immunogold staining reveals that KS as well as CS are present both in the basal lamina and in the interstitial ECM in both types of embryos. It remains to be investigated whether the abundance of certain KS epitopes in the white mutant embryo might contribute to the deficiency in supporting pigment cell migration shown by its ECM.     

HGF/SF Induces Mesothelial Cell Migration and Proliferation by Autocrine and Paracrine Pathways

Mesothelial repair differs from that of other epithelial-like surfaces as healing does not occur solely by centripetal in-growth of cells as a sheet from the wound margins. Mesothelial cells lose their cell-cell junctions, divide, and adopt a fibroblast-like morphology while scattering across and covering the wound surface. These features are consistent with a cellular response to hepatocyte growth factor/scatter factor (HGF/SF). In this study, we examined the ability of mesothelial cells to secrete HGF/SF and investigated its possible role as an autocrine regulator of mesothelial cell motility and proliferation. We found that human primary mesothelial cells expressed HGF/SF mRNA and secreted active HGF/SF into conditioned medium as determined by ELISA and in a scattering bioassay. These cells also expressed the HGF/SF receptor, Met, as shown by RT-PCR and by Western blot analysis and immunofluorescence. Incubation of mesothelial cells with neutralizing antibodies to HGF/SF decreased cell migration to 25% of controls, whereas addition of HGF/SF disrupted cell-cell junctions and induced scattering and enhanced mesothelial cell migration. Furthermore, HGF/SF showed a small but significant mitogenic effect on all mesothelial cell lines examined. In conclusion, HGF/SF is produced by mesothelial cells and induces both motility and proliferation of these cells. These data are consistent with HGF/SF playing an autocrine role in mesothelial healing.