斑马鱼心脏再生的研究

人类心脏损伤后不可再生,使得心脏疾病成为威胁人类生命的主要疾病之一。斑马鱼等其他非哺乳动物类脊椎动物的心脏在受损伤后可以再生,再加上其遗传操作已十分成熟,使其成为研究脊椎动物心脏再生的一个极好模型。本文总结了国内外关于斑马鱼心脏再生的最新进展、并分析了心脏再生特异的分子机制以及涉及再生现象的保守机制,进一步提出了这些研究对于人类心脏疾病的治疗的可借鉴之处。     

Differential effects of genotoxic stress on both concurrent body growth and gradual senescence in the adult zebrafish

Among vertebrates, fish and mammals show intriguing differences in their growth control properties with age. The potential for unlimited or indeterminate growth in a variety of fish species has prompted many questions regarding the senescent phenomena that appear during the aging process in these animals. Using zebrafish as our model system, we have attempted in our current study to examine the growth phenomena in fish in relation to the onset of senescence-associated symptoms, and to evaluate the effects of genotoxic stress on these processes. We observed in the course of these analyses that the zebrafish undergoes continuous growth, irrespective of age, past the point of sexual maturation with gradually decreasing growth rates at later stages. Animal population density, current body size and chronological age also play predominant roles in regulating zebrafish growth and all inversely influence the growth rate. Interestingly, the induction of genotoxic stress by exposure to ionizing radiation (IR) did not adversely affect this body growth ability in zebrafish. However, IR was found to chronically debilitate the regeneration of amputated caudal fins and thereby induce high levels of abnormal fin regeneration in the adult zebrafish. In addition, by resembling and mimicking the natural course of aging, IR treatments likewise enhanced several other symptoms of senescence, such as a decline in reproductive abilities, increased senescence-associated beta-galactosidase activity and a reduction in melatonin secretion. Our current data thus suggest that during the lifespan of zebrafish, the onset of senescence-associated symptoms occurs in parallel with continuous growth throughout mid-adulthood. Moreover, our present findings indicate that genotoxic DNA damage may play a role as a rate-limiting factor during the induction of senescence, but not in the inhibition of continuous, density-dependent growth in adult zebrafish.     

Fat tissue,aging, and cellular senescence

Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age‐related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro‐inflammatory, tissue‐remodeling, senescent‐like state. Other mesenchymal progenitors also can acquire a pro‐inflammatory, adipocyte‐like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro‐inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress‐responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent‐like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro‐inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role.     

构建斑马鱼心脏损伤-再生模型的手术方法

斑马鱼心脏再生是近年来心血管再生医学研究的新热点之一, 也是以斑马鱼为模式进行脊椎动物遗传发育研究的一个新的重要方向。通过了解斑马鱼成体心脏再生的过程和研究其分子和细胞机制有可能为诱导哺乳动物成体心脏再生、治疗心肌梗塞等人类心脏疾病提供理论依据。文章主要介绍通过简单的手术切除成体斑马鱼约20%心室造成成体心脏损伤、诱导心脏再生的操作方法与经验。其基本流程主要包括麻醉成鱼、在体视镜下用尖镊撕开斑马鱼心脏腹面的皮肤和心包膜以暴露心脏、用剪刀切除心尖区域的部分心室。这种方法的手术成功率可达90%以上, 操作简便且重复性好, 是目前研究斑马鱼成体心脏损伤-再生的最常用的方法。     

成年斑马鱼脊髓损伤修复中脑gdnf和nos基因的表达

成年斑马鱼(Danio rerio)具有很强的脊髓损伤后自主修复的能力, 但目前其机制不明。为了研究斑马鱼中脑组织对脊髓再生的影响, 文章应用成年斑马鱼脊髓损伤模型, 采用实时定量PCR方法和原位杂交技术, 检测了斑马鱼脑中胶质细胞源性神经营养因子(gdnf)和一氧化氮合酶(nos)基因在脊髓损伤后4 h、12 h、6 d、11 d的表达情况, 展示了这两种基因在斑马鱼脑内不同核团的动态表达变化。结果显示, 成年斑马鱼脊髓损伤后, 神经营养因子gdnf基因在损伤急性期(4 h、12 h)和神经修复期(6 d、11 d)于斑马鱼脑内呈现显著性升高(P<0.05),而一氧化氮合酶基因nos的表达于损伤急性期显著性升高 (P<0.05), 随后下降, 并在修复期 (11 d)显著降低(P<0.05)。这表明, 脊髓损伤后, 高表达gdnf基因同时低表达nos基因的脑环境给脊髓损伤提供了良好的神经再生微环境, 从而可能促进轴突的再生长及运动能力的恢复。     

The tumor suppressor ING1 contributes to epigenetic control of cellular senescence

Cellular senescence is an effective tumor-suppressive mechanism that causes a stable proliferative arrest in cells with potentially oncogenic alterations. Here, we have investigated the role of the p33ING1 tumor suppressor in the regulation of cellular senescence in human primary fibroblasts. We show that p33ING1 triggers a senescent phenotype in a p53-dependent fashion. Also, endogenous p33ING1 protein accumulates in chromatin in oncogene-senescent fibroblasts and its silencing by RNA interference impairs senescence triggered by oncogenes. Notably, the ability to induce senescence is lost in a mutant version of p33ING1 present in human tumors. Using specific point mutants, we further show that recognition of the chromatin mark H3K4me3 is essential for induction of senescence by p33ING1. Finally, we demonstrate that ING1-induced senescence is associated to a specific genetic signature with a strong representation of chemokine and cytokine signaling factors, which significantly overlaps with that of oncogene-induced senescence. In summary, our results identify ING1 as a critical epigenetic regulator of cellular senescence in human fibroblasts and highlight its role in control of gene expression in the context of this tumor-protective response.     

Autophagy is required for zebrafish caudal fin regeneration

Regeneration is the ability of multicellular organisms to replace damaged tissues and regrow lost body parts. This process relies on cell fate transformation that involves changes in gene expression as well as in the composition of the cytoplasmic compartment, and exhibits a characteristic age-related decline. Here, we present evidence that genetic and pharmacological inhibition of autophagy – a lysosome-mediated self-degradation process of eukaryotic cells, which has been implicated in extensive cellular remodelling and aging – impairs the regeneration of amputated caudal fins in the zebrafish (Danio rerio). Thus, autophagy is required for injury-induced tissue renewal. We further show that upregulation of autophagy in the regeneration zone occurs downstream of mitogen-activated protein kinase/extracellular signal-regulated kinase signalling to protect cells from undergoing apoptosis and enable cytosolic restructuring underlying terminal cell fate determination. This novel cellular function of the autophagic process in regeneration implies that the role of cellular self-digestion in differentiation and tissue patterning is more fundamental than previously thought.     

Extranuclear DNA accumulates in aged cells and contributes to senescence and inflammation

Systemic inflammation is central to aging‐related conditions. However, the intrinsic factors that induce inflammation are not well understood. We previously identified a cell‐autonomous pathway through which damaged nuclear DNA is trafficked to the cytosol where it activates innate cytosolic DNA sensors that trigger inflammation. These results led us to hypothesize that DNA released after cumulative damage contributes to persistent inflammation in aging cells through a similar mechanism. Consistent with this notion, we found that older cells harbored higher levels of extranuclear DNA compared to younger cells. Extranuclear DNA was exported by a leptomycin B‐sensitive process, degraded through the autophagosome–lysosomal pathway and triggered innate immune responses through the DNA‐sensing cGAS‐STING pathway. Patient cells from the aging diseases ataxia and progeria also displayed extranuclear DNA accumulation, increased pIRF3 and pTBK1, and STING‐dependent p16 expression. Removing extranuclear DNA in old cells using DNASE2A reduced innate immune responses and senescence‐associated (SA) β‐gal enzyme activity. Cells and tissues of Dnase2a^?/? mice with defective DNA degradation exhibited slower growth, higher activity of β‐gal, or increased expression of HP‐1β and p16 proteins, while Dnase2a^?/?;Sting^?/? cells and tissues were rescued from these phenotypes, supporting a role for extranuclear DNA in senescence. We hypothesize a direct role for excess DNA in aging‐related inflammation and in replicative senescence, and propose DNA degradation as a therapeutic approach to remove intrinsic DNA and revert inflammation associated with aging.     

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16^Ink4a or p21^Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.     

p38α MAPK regulates myocardial regeneration in zebrafish

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.     

Using zebrafish to study podocyte genesis during kidney development and regeneration

During development, vertebrates form a progression of up to three different kidneys that are comprised of functional units termed nephrons. Nephron composition is highly conserved across species, and an increasing appreciation of the similarities between zebrafish and mammalian nephron cell types has positioned the zebrafish as a relevant genetic system for nephrogenesis studies. A key component of the nephron blood filter is a specialized epithelial cell known as the podocyte. Podocyte research is of the utmost importance as a vast majority of renal diseases initiate with the dysfunction or loss of podocytes, resulting in a condition known as proteinuria that causes nephron degeneration and eventually leads to kidney failure. Understanding how podocytes develop during organogenesis may elucidate new ways to promote nephron health by stimulating podocyte replacement in kidney disease patients. In this review, we discuss how the zebrafish model can be used to study kidney development, and how zebrafish research has provided new insights into podocyte lineage specification and differentiation. Further, we discuss the recent discovery of podocyte regeneration in adult zebrafish, and explore how continued basic research using zebrafish can provide important knowledge about podocyte genesis in embryonic and adult environments. genesis 52:771–792, 2014. © 2014 Wiley Periodicals, Inc.     

p38α MAPK regulates myocardial regeneration in zebrafish

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.     

干细胞参与组织再生的一种机制:细胞融合

干细胞的研究开创了生命科学革命性的进程,人们将通过这一研究找到攻克诸如心血管疾病、神经退行性疾病、癌症、糖尿病等顽症的方法,因而这一工程又被称之为治疗人类疾病的“希望工程”。然而,人们对干细胞在组织修复和再生过程中的作用机制还知之甚少,目前认为干细胞横向分化能力及干细胞的融合是其主要的,也是最有争议的两种可能的机制。细胞融合在发育过程中是不可或缺的,如果人体内不能进行正常的细胞融合,将产生因精卵不融合造成的不孕、骨骼石化症、肌营养不良等疾病,而将干细胞融合应用于临床治疗的研究也日渐兴起。本文对干细胞参与组织再生和修复过程中与细胞融合相关的研究进展进行了综述,概述了干细胞融合的机制,分析了体内融合产生的原因及条件,以期为今后这方面的研究提供理论基础。     

Carbofuran accelerates the cellular senescence and declines the life span of spns1 mutant zebrafish

Carbofuran is a carbamate pesticide, widely used in agricultural practices to increase crop productivity. In mammals, carbofuran is known to cause several untoward effects, such as apoptosis in the hippocampal neuron, oxidative stress, loss of memory and chromosomal anomalies. Most of these effects are implicated with cellular senescence. Therefore, the present study aimed to determine the effect of carbofuran on cellular senescence and biological ageing. Spinster homolog 1 (Spns1) is a transmembrane transporter, regulates autolysosomal biogenesis and plays a role in cellular senescence and survival. Using senescence-associated β-galactosidase staining, we found that carbofuran accelerates the cellular senescence in spns1 mutant zebrafish. The yolk opaqueness, a premature ageing phenotype in zebrafish embryos, was accelerated by carbofuran treatment. In the survival study, carbofuran shortened the life span of spns1 mutant zebrafish. Autophagy is the cellular lysosomal degradation, usually up-regulated in the senescent cells. To know the impact of carbofuran exposure on autophagy progress, we established a double-transgenic zebrafish line, harbouring EGFP-tagged LC3-II and mCherry-tagged Lamp1 on spns1 mutant background, whereas we found, carbofuran exposure synergistically accelerates autolysosome formation with insufficient lysosome-mediated degradation. Our data collectively suggest that carbofuran exposure synergistically accelerates the cellular senescence and affects biological ageing in spns1 defective animals.     

生长激素促进斑马鱼尾鳍的再生

目的建立生长激素过表达的转基因斑马鱼,研究生长激素在斑马鱼尾鳍再生过程中的作用。方法利用Gateway技术构建表达质粒"pDestTol2CG2; ubi:GH-polyA",在一细胞期显微注射表达质粒和转座酶mRNA后,通过荧光显微镜和qPCR技术筛选鉴定GH过表达的转基因斑马鱼。将斑马鱼分为对照组(野生型)和生长激素过表达组,尾鳍横切后,记录分析斑马鱼尾鳍再生过程。结果转基因斑马鱼中心脏被绿色荧光蛋白标记。荧光定量PCR检测结果显示GH表达水平显著高于对照组(P<0.05)。斑马鱼尾鳍横断后,生长激素过表达组的再生速度显著提高(P<0.05)。结论建立稳定生长激素过表达的转基因斑马鱼品系,过表达生长激素能够提高斑马鱼尾鳍再生速度。     

Repair and regeneration: opportunities for carcinogenesis from tissue stem cells

This review will discuss the mechanisms of repair and regeneration in various tissue types and how dysregulation of these mechanisms may lead to cancer. Normal tissue homeostasis involves a careful balance between cell loss and cell renewal. Stem and progenitor cells perform these biologic processes as the functional units of regeneration during both tissue homeostasis and repair. The concept of tissue stem cells capable of giving rise to all differentiated cells within a given tissue led to the concept of a cellular hierarchy in tissues and in tumors. Thus, only a few cells may be necessary and sufficient for tissue repair or tumor regeneration. This is known as the hierarchical model of tumorigenesis. This report will compare this model with the stochastic model of tumorigenesis. Under normal circumstances, the processes of tissue regeneration or homeostasis are tightly regulated by several morphogen pathways to prevent excessive or inappropriate cell growth. This review presents the recent evidence that dysregulation of these processes may provide opportunities for carcinogenesis for the long-lived, highly proliferative tissue stem cell population. New findings of cancer initiating tissue stem cells identified in several solid and circulating cancers including breast, brain and hematopoietic tumors will also be reviewed. Finally, this report reviews the cellular biology of cancer and its relevance to the development of more effective cancer treatment protocols.     

Cellular senescence in osteoarthritis pathology

Cellular senescence is a state of stable proliferation arrest of cells. The senescence pathway has many beneficial effects and is seen to be activated in damaged/stressed cells, as well as during embryonic development and wound healing. However, the persistence and accumulation of senescent cells in various tissues can also impair function and have been implicated in the pathogenesis of many age‐related diseases. Osteoarthritis (OA), a severely debilitating chronic condition characterized by progressive tissue remodeling and loss of joint function, is the most prevalent disease of the synovial joints, and increasing age is the primary OA risk factor. The profile of inflammatory and catabolic mediators present during the pathogenesis of OA is strikingly similar to the secretory profile observed in ‘classical’ senescent cells. During OA, chondrocytes (the sole cell type present within articular cartilage) exhibit increased levels of various senescence markers, such as senescence‐associated beta‐galactosidase (SAβGal) activity, telomere attrition, and accumulation of p16ink4a. This suggests the hypothesis that senescence of cells within joint tissues may play a pathological role in the causation of OA. In this review, we discuss the mechanisms by which senescent cells may predispose synovial joints to the development and/or progression of OA, as well as touching upon various epigenetic alterations associated with both OA and senescence.