内源性电场及其生物学意义

早在18世纪,人们就在肌肉损伤处检测到电流的存在,但直到近些年这种内源性电场的生物学意义才得到阐释.研究表明,内源性电场广泛存在于体内各种组织,并在系统发育、组织再生等生物学过程中有重要的生物学意义.本文综述了内源性电场产生的机理及其在伤口愈合、组织再生、胚胎发育以及肿瘤生成过程中的作用,并对与内源性电场紧密相关的组织水平的离子流动进行综述,以期为内源性电场的研究提供参考.     

视网膜感光细胞对仿生电场刺激的非矢量应答

正常状态下眼组织的细胞暴露于生物体内生电场中,故电场可以调控眼组织细胞一系列的生物学性能.细胞对电场信号的应答可分为两个方面,细胞对电场矢量的应答(迁移、定向生长等)和细胞对电场刺激的非矢量应答(增殖、凋亡等),而细胞对电场矢量的应答是最常见的应答现象.同时,细胞外的电场也能引起细胞内的信号转导,进而调控细胞的行为,但是对细胞内的应答机制目前知之甚少,所以对细胞非矢量性应答的机制也了解甚少.而感光细胞对电场刺激非矢量性应答的分子机制目前研究处于空白阶段,故本研究着重于探究感光细胞对于电场刺激的非矢量性应答的机制.为了探究电场刺激对感光细胞的影响,本课题组自主研发了一款电场刺激仪,研究发现,60和90 m V/mm强度的电场刺激能提高细胞活性,促进细胞增殖.检测基因表达谱,利用通路分析发现,Ca~(2+)离子依赖的ERK通路在细胞对电场刺激的应答中扮演重要角色.实验结果进一步证明,电场刺激促进Ca~(2+)离子内流进而改变了感光细胞的生长平衡,可以预见电场影响感光细胞的离子转运在感光细胞对电场刺激的应答过程中具有重要的意义.总而言之,本课题组发现,电场影响细胞内外的Ca~(2+)离子流,引发细胞内一系列的级连反应,使细胞的生长状态发生了改变.     

MiRNA 与皮肤伤口愈合

MiRNA是真核生物体内约由22个核苷酸组成的内源性非编码单链RNA,可调节基因转录。它通过其5’非翻译区(UTR)与目标mRNA的3’端非翻译区相结合,从而抑制后者的转录后翻译和降解,进而调节一系列生物学过程,包括生物体生长、发育和疾病等。研究表明,miRNA在干细胞分化、肿瘤形成、血管发生、内耳形成等过程中均发挥重要作用,已成为调节生物学过程的核心因子。伤口愈合是一个与多种类型细胞、细胞因子及细胞外基质相关的过程,它受机体多种因素紧密调控。伤口愈合过程一般被分为三个阶段:炎症反应期,肉芽生长期和组织重建期。已有大量证据证实miRNA在皮肤创伤愈合过程中发挥重要作用,并且miRNA在不同的愈合阶段发挥不同的作用。本文就miRNA在皮肤形态、胎儿无痕愈合及成人伤口愈合各环节中的作用做一综述。     

活性氧在创伤愈合中作用的研究

：创伤愈合是一个复杂的生物学过程,包括出血与凝血、炎症渗出、血管和肉芽组织的形成、再上皮化、纤维化和瘢痕改建等,在这一系列的生物学活动过程中都需要能量支持;高等动物使用氧气作为终端氧化剂,通过对碳水化合物的氧化作用为愈合过程中的各种生命活动提供能量,但该过程却可以产生大量的活性氧,这些活性氧在创伤愈合的过程中扮演着重要的角色,在低浓度情况下可以促进伤口的愈合,而在高浓度时会抑制伤口愈合,而活性量浓度的过高过低都会影响创口的正常愈合过程。     

Hippo信号通路生物学作用研究进展

Hippo信号通路是20世纪末在黑腹果蝇中进行基因筛选时发现的,该通路受各种生化、物理和结构信号的影响,调控细胞生长、分化,组织和器官发育以及内环境稳态等基本生物学过程。研究表明Hippo信号通路失调会引起一系列疾病的发生。本文阐述了目前Hippo信号通路在胚胎发育、器官和组织稳态调节、肿瘤的发生发展和细胞自噬等一系列生物学过程以及靶向治疗中的研究进展,其中Hippo信号通路通过细胞自噬来维持机体细胞内环境稳态成为新的研究热点。对该通路的功能和调控机制的深入研究也为组织器官修复再生医学及癌症治疗提供参考。     

人表皮生长因子的研究进展

人表皮生长因子是激活表皮生长因子受体的生长因子家族的典型成员,由人体的多个组织器官合成与分泌,通过结合受体激活一系列信号途径,调控细胞的增殖、分化和迁移等。近年来,有关人表皮生长因子的研究已扩展到其在人类生理和病理作用的领域,尤其在组织再生和伤口愈合方面成为研究热点。文中综述了人表皮生长因子的研究进展,简要描述了其基因和蛋白的结构与特点、作用机制与生物学效应,重点介绍该生长因子在胃肠溃疡愈合、皮肤伤口修复和肿瘤病理过程中的作用与影响,从而为相关研究提供辅助信息。     

原核基因组的进化方向和癌／正常细胞的熵产生

本文评述了理论生物学的两个基础实验。一是用离子束辐照加速大肠杆菌进化的方法研究原核基因组的进化方向,证明进化中的缺失偏好性和编码信息量扩增律不矛盾。二是提出用导热法测量细胞的熵产生,比较研究了癌细胞和正常细胞的熵产生和外加电场的关系,证明在一定强度的电场作用下正常细胞的熵产生可以明显超过癌细胞,从而实现改变两类细胞间熵流方向的目的。     

PIEZO1影响HaCaT细胞的趋电性迁移

目的 伤口中心产生的内源性电场是指导细胞定向迁移促进伤口愈合的重要因子。PIEZO1是机械门控阳离子通道家族成员之一,它参与细胞迁移,影响细胞的趋化迁移,并且受到电压的调节,在伤口愈合过程中发挥重要作用。但PIEZO1是否参与影响电场指导的细胞定向迁移过程尚不清晰,本文以HaCaT细胞为模型探讨PIEZO1及其下游相关蛋白质对细胞趋电性迁移的影响。方法 应用活细胞工作站追踪HaCaT细胞在微直流电场中的迁移,使用抑制剂及RNAi技术调控PIEZO1功能和表达,研究PIEZO1对于细胞趋电性迁移的影响;用蛋白质印迹（Western blot）检验细胞的整合素（integrin） β1与FAK磷酸化水平在电场作用下的变化情况,探讨PIEZO1与integrin β1及FAK等对电场信号的响应及细胞趋电性迁移的影响。结果 PIEZO1广谱抑制剂钌红、GsMTx4和RNAi处理显著抑制了HaCaT细胞向正极趋电性迁移的能力;电场和GsMTx4单独作用升高FAK磷酸化和integrin β1表达,GsMTx4阻止电场进一步升高FAK的磷酸化水平和integrin β1表达;siRNA干扰PIEZ...     

不同强度高压电场对A549肺癌细胞肿瘤生物学特性的影响

目的：探讨不同强度高压电场对A549肺癌细胞肿瘤转移生物学特性的影响。方法：选择处于生长周期的A549细胞,共分为7个组进行研究,其中A-F组为实验组,G组为不施加电场的空白对照组,A施加500V/cm强度高压电场,F组施加1750V/cm的高压电场,电压场强间隔为250V/cm。采取粘附实验、侵袭及转移实验,检验A549细胞在不同强度高压电场中,其肿瘤生物学转移特性的改变。结果：①各实验组与对照组、各实验组之间的细胞粘附能力,均存在显著性差异（P〈0.05）;②电场强度≥750V/cm时,各实验组之间、及其与对照组之间的细胞迁移能力,存在显著性差异（P〈0.05）;③电场强度≥1000V/cm时,各组与对照组间的细胞侵袭能力,存在显著性差异（P〈0.05）;④电场强度为1000-1250V/cm的各组与1500-1750V/cm各组间的细胞侵袭能力,存在显著性差异,有统计学意义（P〈0.05）。结论：不同强度的电场抑制A549肺癌细胞的程度不同,随着强度的增加,A549细胞粘附、迁移和侵袭能力的抑制现象依次出现,并随着电场强度的增加其抑制程度也持续增加。     

FGF8在脊椎动物早期胚胎发育中的调控作用

成纤维细胞生长因子8 (fibroblast growth factor 8,FGF8)是成纤维细胞生长因子家族的成员之一,是一种组织发育过程中的重要分泌性调控信号分子,参与脊椎动物的多种组织器官的发生与发育.早期胚胎细胞通过表达FGF8在组织和器官发育、血管发生、血细胞生成、附肢发生和伤口愈合等方面发挥着重要作用.FGF8不但可以在细胞外通过胞内信号通路,而且也可以进入细胞内部发挥生物学功能.本文就FGF8在脊椎动物神经系统、内脏器官、肢体发育及不对称发育等组织、器官发育中的调控作用予以阐述.     

Airway epithelial wounds in rhesus monkey generate ionic currents that guide cell migration to promote healing

Damage to the respiratory epithelium is one of the most critical steps to many life-threatening diseases, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. The mechanisms underlying repair of the damaged epithelium have not yet been fully elucidated. Here we provide experimental evidence suggesting a novel mechanism for wound repair: endogenous electric currents. It is known that the airway epithelium maintains a voltage difference referred to as the transepithelial potential. Using a noninvasive vibrating probe, we demonstrate that wounds in the epithelium of trachea from rhesus monkeys generate significant outward electric currents. A small slit wound produced an outward current (1.59 μA/cm(2)), which could be enhanced (nearly doubled) by the ion transport stimulator aminophylline. In addition, inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) with CFTR(Inh)-172 significantly reduced wound currents (0.17 μA/cm(2)), implicating an important role of ion transporters in wound induced electric potentials. Time-lapse video microscopy showed that applied electric fields (EFs) induced robust directional migration of primary tracheobronchial epithelial cells from rhesus monkeys, towards the cathode, with a threshold of <23 mV/mm. Reversal of the field polarity induced cell migration towards the new cathode. We further demonstrate that application of an EF promoted wound healing in a monolayer wound healing assay. Our results suggest that endogenous electric currents at sites of tracheal epithelial injury may direct cell migration, which could benefit restitution of damaged airway mucosa. Manipulation of ion transport may lead to novel therapeutic approaches to repair damaged respiratory epithelium.     

Electrical fields in wound healing—An overriding signal that directs cell migration

Injury that disrupts an epithelial layer instantaneously generates endogenous electric fields (EFs), which were detected at human skin wounds over 150 years ago. Recent researches combining molecular, genetic and imaging techniques have provided significant insights into cellular and molecular responses to this “unconventional” signal. One unexpected finding is that the EFs play an overriding guidance role in directing cell migration in epithelial wound healing. In experimental models where other directional cues (e.g., contact inhibition release, population pressure etc.) are present, electric fields of physiological strength override them and direct cell migration. The electrotaxis or galvanotaxis is mediated by polarized activation of multiple signaling pathways that include PI3 kinases/Pten, membrane growth factor receptors and integrins. Genetic manipulation of PI3 kinase/Pten (Phosphoinositide 3-kinases/phosphatase and tensin homolog) and integrin β4 demonstrated the importance of those molecules. The electric fields are therefore a fundamental signal that directs cell migration in wound healing. One of the most challenging question is: How do cells sense the very weak electric signals? Clinically, it is highly desirable to develop practical and reliable technologies for wound healing management exploiting the electric signaling.     

Beta-adrenergic receptor agonists delay while antagonists accelerate epithelial wound healing: evidence of an endogenous adrenergic network within the corneal epithelium

Wound healing is a complex and well-orchestrated biological process. Corneal epithelial cells (CECs) must respond quickly to trauma to rapidly restore barrier function and protect the eye from noxious agents. They express a high level of beta2-adrenergic receptors but their function is unknown. Here, we report the novel finding that they form part of a regulatory network in the corneal epithelium, capable of modulating corneal epithelial wound repair. Beta-adrenergic receptor agonists delay CEC migration via a protein phosphatase 2A-mediated mechanism and decrease both electric field-directed migration and corneal wound healing. Conversely, beta-adrenergic receptor antagonists accelerate CEC migration, enhance electric field-mediated directional migration, and promote corneal wound repair. We demonstrate that CECs express key enzymes required for epinephrine (beta-adrenergic receptor agonist) synthesis in the cytoplasm and can detect epinephrine in cell extracts. We propose that the mechanism for the pro-motogenic effect of the beta-adrenergic antagonist is blockade of the beta2-adrenergic receptor preventing autocrine catecholamine binding. Further investigation of this network will improve our understanding of one of the most frequently prescribed class of drugs.     

The role of electrical signals in murine corneal wound re-epithelialization

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.     

小鼠皮肤黑色素瘤细胞在三种不同基质上的趋电性比较

微小直流电场具有指导细胞进行定向迁移的作用。各种细胞外基质的物理、化学性质会影响细胞的迁移。该研究以小鼠皮肤黑色素瘤细胞（B16-F10）为模型,比较微直流电场（250mV／mm）指导下细胞在平滑基底与两种不同市售基质Matrigel及FNC上的趋电性。结果显示,黑色素瘤细胞在三种基底上均有明显的向电场阴极迁移的趋电运动,但在不同基质上细胞趋电的方向性无显著差异,但细胞迁移速度及在细胞沿电场进行定向迁移的持续性有显著差异。     

Cyclic AMP-dependent protein kinase A plays a role in the directed migration of human keratinocytes in a DC electric field.

Skin wound healing requires epithelial cell migration for re-epithelialization, wound closure, and re-establishment of normal function. We believe that one of the earliest signals to initiate wound healing is the lateral electric field generated by the wound current. Normal human epidermal keratinocytes migrate towards the negative pole, representing the center of the wound, in direct currents of a physiological strength, 100 mV/mm. Virtually nothing is known about the signal transduction mechanisms used by these cells to sense the endogenous electric field. To elucidate possible protein kinase (PK) involvement in the process, PK inhibitors were utilized. Two important findings have been described. Firstly, addition of 50 nM KT5720, an inhibitor of PKA, resulted in a 53% percent reduction in the directional response of keratinocytes in the electric field, while not significantly affecting general cell motility. The reduction was dose-dependent, there was a gradual decrease in the directional response from 5 to 50 nM. Secondly, addition of 1 microM ML-7, a myosin light chain kinase inhibitor, resulted in an approximate 31% decrease in the distance the cells migrated without affecting directional migration. The PKC inhibitors GF109203X at 4 microM and H-7 at 20 microM and W-7, a CaM kinase inhibitor, did not significantly alter either directed migration or cell migration, although they all resulted in a slight reduction in directional migration. D-erythro-sphingosine at 15 microM, a PKC inhibitor, had virtually no effect on either migration distance or directed migration. These findings demonstrate that divergent kinase signaling pathways regulate general cell motility and sustained directional migration and highlight the complexity of the signal transduction mechanisms involved. The inhibitor studies described in this paper implicate a role for PKA in the regulation of the directional migratory response to applied electric fields, galvanotaxis.     

Microfluidic devices for studying chemotaxis and electrotaxis

Directed cell migration plays important roles in physiological processes such as host defense, wound healing, cancer metastasis and embryogenesis. Many organisms are capable of directional migration, which can be guided by diverse cellular factors including chemical and electrical cues. Recently, microfluidic devices that consist of small channels with micrometer dimensions are being developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus can be used to study the complex guiding mechanisms for cell migration. In this paper we highlight recent applications of microfluidic devices for cell migration research, with a focus on electric field-directed cell migration, to provide important and timely updates of this rapidly developing research field.     

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations¹. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF^2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab''s techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.     

Extracellular electrical fields direct wound healing and regeneration

Endogenous DC electric fields (EFs) are important, fundamental components of development, regeneration, and wound healing. The fields are the result of polarized ion transport and current flow through electrically conductive pathways. Nullification of endogenous EFs with pharmacological agents or applied EFs of opposite polarity disturbs the aforementioned processes, while enhancement increases the rate of wound closure and the extent of regeneration. EFs are applied to humans in the clinic, to provide an overwhelming signal for the enhancement of healing of chronic wounds. Although clinical trials, spanning a course of decades, have shown that applied EFs enhance healing of chronic wounds, the mechanisms by which cells sense and respond to these weak cues remains unknown. EFs are thought to influence many different processes in vivo. However, under more rigorously controlled conditions in vitro, applied EFs induce cellular polarity and direct migration and outgrowth. Here we review the generation of endogenous EFs, the results of their alteration, and the mechanisms by which cells may sense these weak fields. Understanding the mechanisms by which native and applied EFs direct development and repair will enable current and future therapeutic applications to be optimized.