PDGF-BB下调血管平滑肌标志物SM22α表达的机制

血管平滑肌细胞（vascular smooth muscle cell,VSMC）表型转化是血管重塑性疾病的细胞病理学基础,血小板源性生长因子（platelet-derived growth factor,PDGF）-BB抑制平滑肌分化标志基因表达、加速其降解,是VSMC表型转化的关键。该研究用PDGF-BB刺激VSMC诱导细胞发生表型转化,利用Western blot和免疫共沉淀等技术,检测PDGF-BB对早期分化相关基因平滑肌22 alpha（smooth muscle 22 alpha,SM22α）磷酸化与泛素化的影响。实验结果显示,PDGF-BB促进VSMC增殖;上调增殖相关蛋白PCNA的表达,下调分化相关蛋白SM22α与SMα-actin的表达;诱导SM22α发生磷酸化和泛素化,而且,该过程与SM22α水平下调具有时相相关性;抑制剂阻断分析证实,ERK和PKC参与介导了PDGF-BB诱导的SM22α磷酸化。以上结果提示,在VSMCs表型转化中,PDGF-BB可能是通过激活ERK-PKC信号通路,促进SM22α的磷酸化和泛素依赖的蛋白质降解。     

血管外膜成纤维细胞诱导BMSCs向平滑肌细胞定向分化

观察大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)与血管外膜成纤维细胞(ad-ventitial fibroblasts,AF)直接接触培养后,BMSCs向血管成分细胞分化的情况.将BMSCs(DAPI标记)与外膜成纤维细胞按一定的比例混合培养7 d,BMSCs单独培养作对照,显微镜下观察细胞形态变化;用免疫荧光染色检测BMSCs的血管平滑肌细胞表型标志物肌动蛋白(SMα-actin)表达的情况;RT-PCR检测SMα-actin mRNA表达的情况.BMSCs与血管外膜成纤维细胞共培养后可见细胞核蓝染的BMSCs与SMα-actin表达阳性(红色)的双标细胞出现,且随培养时间的延长,BMSCs的SMα-actin表达阳性率增高.结果可见:与血管外膜成纤维细胞直接接触有诱导BMSCs向血管平滑肌细胞分化的趋势.     

人妊娠子宫平滑肌细胞的原代培养Transgelin蛋白的检测

为了建立最佳的人妊娠子宫平滑肌细胞的原代培养方法和初步检测子宫平滑肌细胞中Transgelin蛋白的表达,采用组织块贴壁法和酶消化法进行人妊娠子宫平滑肌原代培养.发现组织贴块培养的细胞呈典型的梭状肌细胞样生长,经过传代纯化,通过免疫细胞化学方法检测平滑肌肌动蛋白(smooth muscle acting,SMA)进行细胞鉴定及检测Trangsgelin(smooth muscle 22 alpha,SM22-α)蛋白,得到SMA、snd2-α荧光免疫细胞化学染色为阳性.结果表明组织贴块法对妊娠子宫平滑肌细胞损伤小,可获得状态良好的纯净的子宫平滑肌细胞,子宫平滑肌细胞中大量表达sm2-α,为进一步研究sm22-α在子宫平滑肌细胞中作用打下基础.     

SM22α对血管平滑肌细胞骨架及收缩功能的影响

SM22α(smooth muscle 22 alpha,SM22α)是血管平滑肌细胞(vascular smooth muscle cells,VSMC)的标志蛋白,为了探讨该蛋白与VSMC表型和功能的关系,利用血清饥饿法诱导VSMC由合成型向收缩型转变,用RT—PCR对不同表型VSMC的SM22α表达活性进行检测,并通过转染反义SM22α表达载体,观察SM22α表达对VSMC细胞骨架和收缩功能的影响。结果显示,在VSMC由合成型逆转为收缩型的过程中,SM22α和平滑肌α-肌动蛋白(smooth muscle α—actin,SMα—actin)的表达分别被显诱导和轻度上调,与此同时,细胞骨架由稀疏的网格状变成均匀、致密的束状,VSMC重新获得收缩功能。用反义SM22α抑制该基因表达后,血清饥饿诱导的VSMC细胞骨架重构受阻,乙酰胆碱刺激引发的细胞收缩消失。结果提示,SM22α参与VSMC细胞骨架的构成及调节细胞的收缩功能,对维持VSMC处于收缩表型具有重要作用。     

血管损伤的新靶点：平滑肌22 alpha

血管平滑肌细胞(vascular smooth muscle cell,VSMC)表型转化是血管损伤性疾病动脉粥样硬化、高血压和血管成形术后再狭窄等的共同病理生理过程.平滑肌22 alpha (smooth muscle 22 alpha, SM22α) 是一种VSMC分化标志物,其表达具有平滑肌组织特异性和细胞表型特异性. 该蛋白不仅作为一种肌动蛋白细胞骨架相关蛋白参与VSMC骨架组构和收缩调节,它还参与VSMC的增殖、炎症和氧化应激等进程. 本文就SM22α 的结构特征及其在VSMC血管损伤中的作用机制进行综述.     

平滑肌22α蛋白在血管稳态和血管重构中的作用

有关血管稳态和重构的分子机制一直是近年来的研究热点,也被视为治疗血管损伤性疾病的突破点。大量研究证实,血管损伤修复及病理性重构过程与血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的表型转化、异常增殖与迁移、细胞衰老关系密切。平滑肌22α(smooth muscle 22α,SM22α)蛋白是一种在收缩型VSMCs中大量表达的细胞骨架相关蛋白,可通过与F-actin相互作用促进应力纤维形成,维持VSMCs收缩性,还可作为信号调节分子参与血管稳态和重构。本文综述了近年SM22α在血管稳态和血管重构中作用的研究进展。     

胚胎干细胞来源的拟胚体分化早期血管平滑肌标志物的表达时相

目的:研究胚胎血管发育早期SMα-actin、SM22α、myocardin、平滑肌肌球蛋白重链(SMMHC)的表达规律,并初步探讨在此阶段血小板源性生长因子-BB(PDGF-BB)对血管平滑肌细胞(VSMCs)分化的影响。方法:采用转染平滑肌特异性蛋白SM22α启动子控制下表达增强型绿色荧光蛋白(GFP)报告基因载体的胚胎干细胞制备拟胚体(EBs),用免疫荧光染色、RT-PCR、Western blot分析SMα-actin、SM22α、myocardin、SMMHC的表达时相;然后分别用0μmol/L(对照组)、10μmol/L、50μmol/L AG1296(血小板源性生长因子受体抑制剂)处理EBs,观察三组SMα-actin、SM22α、myocardin、SMMHC在基因及蛋白水平上的表达变化。结果:胚胎血管发育早期SMα-actin、myocardin、SM22α、SMMHC分别在EBs第0(胚胎干细胞)、8、11、13d开始有表达。AG1296三种浓度处理后SMα-actin、myocardin、SM22α、SMMHC蛋白表达及myocardin、SM22α和SMMHC mRNA表达均无明显差异。结论:EBs发育过程中存在着自发的VSMCs分化,SMα-actin表达最早,依次为myocardin、SM22α、SMMHC;PDGF-BB对EBs分化早期VSMCs标志物表达的调控可能不是必要的。     

胚胎干细胞SM22α-EGFP表达克隆的建立及平滑肌细胞体外发育的动态观察

为探讨血管发育早期血管平滑肌细胞(VSMCs)募集和增殖特点,构建了含有SM22α启动子序列和增强型绿色荧光蛋白(EGFP)编码序列的质粒,建立了平滑肌特异性蛋白SM22α启动子控制下稳定表达EGFP的胚胎干细胞株(ESCs),以研究VSMCs的发育特点.实验发现,起源于SM22α-EGFPESCs形成的胚胎小体(EBs)在第11天开启SM22α启动子并表达EGFP.此后EGFP阳性细胞持续增加,在第30天达到高峰.VSMCs多起源于EBs中细胞密集处,应用免疫荧光染色及RT-PCR观察到EGFP阳性细胞表达多种平滑肌特异性标志物.在贴壁培养的胚胎小体中VSMCs形态可分为纺锤形及上皮样的多角形,慢速视频显微摄像测得纺锤形细胞迁移速度较上皮形细胞快.以上结果表明,SM22α-EGFPESCs分化形成的EBs可以模拟体内早期胚胎血管形成过程,从形态学上获得VSMCs募集分化的证据.     

细胞骨架相关蛋白SM22α与肿瘤

平滑肌22α(smooth muscle 22 alpha,SM22α)蛋白是一种细胞骨架相关蛋白,其在种属间的高度同源性和进化上的高度保守性提示了其重要的生物学意义.最新研究发现,SM22α在多种肿瘤组织中表达异常,该蛋白除可通过与肌动蛋白相互作用参与细胞骨架重构外,还可作为信号分子参与细胞生长,细胞外基质降解和血管生成.其作为一种新型抑癌基因,在肿瘤发生,发展中的作用日益成为人们关注的焦点.本文就SM22α的结构特征、表达特点及其与肿瘤的关系进行综述.     

细胞骨架相关蛋白SM22α与肿瘤

平滑肌22α（smooth muscle 22 alpha,SM22α）蛋白是一种细胞骨架相关蛋白,其在种属间的高度同源性和进化上的高度保守性提示了其重要的生物学意义。最新研究发现,SM22α在多种肿瘤组织中表达异常,该蛋白除可通过与肌动蛋白相互作用参与细胞骨架重构外,还可作为信号分子参与细胞生长,细胞外基质降解和血管生成。其作为一种新型抑癌基因,在肿瘤发生、发展中的作用日益成为人们关注的焦点。本文就SM22α的结构特征、表达特点及其与肿瘤的关系进行综述。     

Production of hematopoietic cells from ES cells

Murine embryonic stem (ES) cells are cell lines established from blastocyst which can contribute to all adult tissues, including the germ-cell lineage, after reincorporation into the normal embryo. ES cell pluripotentiality is preserved in culture in the presence of LIF. LIF withdrawal induces ES cell differentiation to nervous, myocardial, endothelial and hematopoietic tissues. The model of murine ES cell hematopoietic differentiation is of major interest because ES cells are non transformed cell lines and the consequences of genomic manipulations of these cells are directly measurable on a hierarchy of synchronized in vitro ES cell-derived hematopoietic cell populations. These include the putative hemangioblast (which represents the emergence of both hematopoietic and endothelial tissues during development), myeloid progenitors and mature stages of myeloid lineages. Human ES cell lines have been recently derived from human blastocyst in the USA. Their manipulation in vitro should be authorized in France in a near future with the possibility of developing a model of human hematopoietic differentiation. This allows to envisage in the future the use of ES cells as a source of human hematopoietic cells.     

胚胎干细胞向造血细胞分化研究

胚胎干（embryonic stem,ES）细胞是来源于囊胚的内细胞团（inner cell mass,ICM）,具有发育的全能性或多能性,能嵌合到早期胚胎,在体内可以参与各种组织发育甚至包括生殖细胞;在体外分化培养条件下,可以顺序分化出各种组织细胞,与体内完整胚胎发育过程相符合,而且可以通过调节ES细胞某些基因的表达而调节其分化。因此,ES细胞是研究哺乳动物早期胚胎发育、细胞分化及其关键基因鉴定的理想模型。另外,胚胎生殖脊（embryonic germ,EG）细胞系也具有同样的生物学特性,它是由早期胚胎的原始生殖脊（primordial germ,PG）细胞建株而来。最近研究显示：ES细胞在体外不但可以分化为所有造血细胞系,而且还可以分化为具有长期增殖能力的造血干细胞。作者就胚胎干细胞向造血细胞和造血干细胞分化及其诱导因子和调控基因的表达作一综述。     

Homing of annexin-labeled stem cells to apoptotic cells

Ischemic diseases are characterized by the presence of pro-apoptotic stimuli, which initiate a cascade of processes that lead to cell injury and death. Several molecules and events represent detectable indicators of the different stages of apoptosis. Among these indicators is phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane, which can be detected by annexinV (ANXA5) conjugation. This is a widely used in vivo and in vitro assay marking the early stages of apoptosis. We report here on an original method that employs PS-ANXA5 conjugation to target stem cells to apoptotic cells. Mesenchymal stem cells (MSCs) from GFP-positive transgenic rats were biotinylated on membrane surfaces with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biot) and then bound to avidin. The avidin-biotinylated MSCs were labeled with biotin conjugated ANXA5. Bovine aortic endothelial cells (BAE-1 cells) were exposed to UVC to induce caspasedependent apoptosis. Finally, we tested the ability of ANXA5-labeled MSCs to bind BAE-1 apoptotic cells: suspended ANXA5-labeled MSCs were seeded for 1 hour on a monolayer of UV-treated or control BAE-1 cells. After washing, the number of MSCs bound to BAE-1 cells was evaluated by confocal microscopy. Statistical analysis demonstrated a significant increase in the number of MSCs tagged to apoptotic BAE-1 cells. Therefore, stem cell ANXA5 tagging via biotin-avidin bridges could be a straightforward method of improving homing to apoptotic tissues. A. Gerasimou, R. Ramella and A. Brero contributed equally to this paper.     

Progenitor cells of the testosterone-producing Leydig cells revealed

The cells responsible for production of the male sex hormone testosterone, the Leydig cells of the testis, are post-mitotic cells with neuroendocrine characteristics. Their origin during ontogeny and regeneration processes is still a matter of debate. Here, we show that cells of testicular blood vessels, namely vascular smooth muscle cells and pericytes, are the progenitors of Leydig cells. Resembling stem cells of the nervous system, the Leydig cell progenitors are characterized by the expression of nestin. Using an in vivo model to induce and monitor the synchronized generation of a completely new Leydig cell population in adult rats, we demonstrate specific proliferation of vascular progenitors and their subsequent transdifferentiation into steroidogenic Leydig cells which, in addition, rapidly acquire neuronal and glial properties. These findings, shown to be representative also for ontogenetic Leydig cell formation and for the human testis, provide further evidence that cellular components of blood vessels can act as progenitor cells for organogenesis and repair.     

Phosphatidylserine-dependent adhesion of T cells to endothelial cells

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.     

Transdifferentiation of mouse BM cells into hepatocyte-like cells

BACKGROUND: During the past few years multiple studies have revealed that adult stem cells, including BM origin stem cells, can be transdifferentiated into various cell types, including hepatocyte-like cells, under proper treatments or in a suitable microenvironment. However, little is known about the mechanism of the transdifferentiation, and the treatments employed seem to be very complicated and require simplification. It is important to determine the suitable conditions in which BM cells would be efficiently differentiated into hepatocytes. METHODS: Mouse BM cells were isolated from femurs and tibias and cultured in IMDM supplemented with 10% FBS. Hepatic differentiation was induced in a differentiation medium containing 20 ng/mL HGF, 10 ng/mL FGF-4, 10 ng/mL Oncostatin M (OSM) and different concentrations of liver-injured mouse sera. The differentiated hepatic cells were characterized by the expression of liver-associated mRNA and proteins and morphologic and functional features. RESULTS: BM cell-derived polygonal cell colonies appeared after several days of culture, and these hepatocyte-like cells expressed AFP, HNF-3beta, CK19, CK18, ALB, TAT and G-6-Pase at mRNA and protein levels, and the cells also had some hepatic cellular functions, such as glycogen storage and urea production. Interestingly, suitable concentrations of sera from liver-injured mice added to this system showed strong stimulation on the in vitro transdifferentiation of mouse BM cells into hepatocytes. DISCUSSION: In the present study we have established an effective hepatic differentiation system by a combination of HGF, FGF-4, OSM and liver-injured mouse sera in vitro. Accordingly, it will be a useful resource not only for understanding the mechanisms of transdifferentiation but also for efficient amplification of hepatocyte progenitor cells of BM origin.     

Stabilization of apoptotic cells: generation of zombie cells

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn²⁺ (a caspase inhibitor) and coenzyme Q₁₀ (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.Apoptosis, also known as programmed cell death, is central to homoeostasis and normal development and physiology in multicellular organisms, including humans.¹ The dysregulation of apoptosis can lead to the destruction of normal tissues in a variety of disorders, including autoimmune and neurodegenerative diseases (increased apoptosis) or cancer (reduced apoptosis). In addition, effective therapy of tumors requires the iatrogenic induction of apoptosis by radiation, chemotherapy or both. In particular, many antineoplasic drugs such as campothecin, a topoisomerase I inhibitor, kill tumor cells by inducing apoptosis.Apoptosis is thought to be physiologically advantageous because apoptotic cells are removed by phagocytosis before they lose their permeability barrier, thus preventing induction of an inflammatory response to the dying cells and potential harmful secondary effects. However, when massive cell death overwhelms macrophage clearance, as for example in early postchemotherapy or viral infection,² apoptotic cells may progress to secondary necrosis characterized by cell membrane degradation with spillage of intracellular contents to the extracellular milieu.³ Similarly, cells undergoing apoptosis in vitro cannot usually be cleared by phagocytes and undergo a late process of secondary necrosis.⁴In the execution phase of apoptosis, effector caspases cleave vital cellular proteins, leading to the morphological changes that characterize apoptosis. These changes include destruction of the nucleus and other organelles, DNA fragmentation, chromatin condensation, cell shrinkage, cell detachment and membrane blebbing.⁵ In apoptosis, all the degradative processes are isolated from the extracellular space by the plasma membrane that remains impermeable. However, the mechanisms involved in plasma membrane and associated protein protection from the action of caspases are not completely understood. In contrast, necrosis is accompanied by disruption of plasma membrane integrity with the subsequent release of all intracellular compounds to the intercellular space, thus inducing inflammation and more toxic effects to adjacent cells.^{6, 7}To allow the dramatic morphological changes that accompany the execution phase, an apoptotic cell undergoes a series of profound cytoskeletal breakdowns/rearrangements. Previous evidence suggests that the actomyosin cytoskeleton plays an essential role in apoptotic cell remodeling during the early events of the execution phase, whereas all other cytoskeleton elements (microtubules and intermediate filaments) are dismantled.⁸ However, during the course of the execution phase and after actininomyosin ring contraction, the actomyosin filaments are also depolymerized by a caspase-dependent mechanism. In this situation, the apoptotic cell forms a network of apoptotic microtubules that becomes the main cytoskeleton element of the apoptotic cell. The presence of microtubules in apoptotic cells has previously been reported.^{9, 10} Moreover, more recent results indicate that microtubules during apoptosis assist in the dispersal of nuclear and cellular fragments,^{11, 12} and may help to preserve the integrity of plasma membrane of the dying cell.¹³Reactive oxygen species (ROS) are also important mediators of apoptosis. ROS have been shown to play a major role in apoptosis signaling.^{14, 15, 16} Electron leak in the presence of oxygen during the process of oxidative phosphorylation make mitochondria the major endogenous source of ROS in the cell. Although mitochondria have been identified as a key player, the mechanism connecting ROS and apoptosis remains unclear.¹⁷ It has been debated whether increased ROS during apoptosis is a cause or a consequence of impaired mitochondrial function, and whether ROS are a death signal to the mitochondria or are produced as effector molecules by the mitochondria in response to apoptosis signal.^{18, 19} Hyperproduction of ROS in execution stages of apoptosis is thought to be caused by the disruption of the mitochondrial respiratory chain after release of cytochrome c into the cytosol.²⁰The main objective of this work was to develop a method for the stabilization of apoptotic cells for proper apoptosis detection or safer potential therapeutic applications. Our results show that apoptotic cells can be stabilized by a cocktail of a microtubule stabilizer (taxol), a caspase inhibitor such (Zn²⁺) and an antioxidant (coenzyme Q₁₀ (CoQ)).     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.