阪崎杆菌侵袭人脑微血管内皮细胞对细胞骨架的影响

目的观察阪崎杆菌侵袭人脑血管内皮细胞后,细胞骨架中微丝和微管的变化。方法用荧光染色技术观察阪崎杆菌侵袭人脑微血管内皮细胞后,细胞骨架中的微丝和微管的变化,观察微丝和微管的抑制剂对阪崎杆菌侵袭人脑微血管内皮细胞的影响。结果阪崎杆菌侵袭人脑微血管内皮细胞后,细胞骨架结构发生明显的改变,微丝和微管部分断裂,模糊,散在分布,微丝抑制剂(细胞松弛素D)和微管抑制剂(秋水仙素)可以抑制阪崎杆菌侵袭人脑微血管内皮细胞。结论阪崎杆菌侵袭人脑血管内皮细胞破坏细胞骨架结构,微丝和微管抑制剂抑制阪崎杆菌侵袭。     

小鼠皮肤角质细胞在电场中的移动及其机理的研究

研究小鼠皮肤角质细胞在电场中的移动以及微管、微丝和钙离子通道在细胞移动中的作用.制作直流电场干预细胞装置,以不同强度的电流作用于小鼠皮肤角质细胞,并在培养基中加入L-型钙离子通道阻断剂硝苯地平、微管抑制剂秋水仙碱和微丝抑制剂细胞松弛素B观察小鼠皮肤角质细胞运动的变化.结果表明:小鼠皮肤角质细胞在1.6 mA电流刺激下向阴极方向移动,移动速率为29.966 μm/h.加入硝苯地平,对小鼠皮肤角质细胞移动的抑制作用不明显.而秋水仙碱和细胞松弛素B对小鼠皮肤角质细胞移动有明显抑制作用.小鼠皮肤角质细胞在电场中可以向阴极定向移动,微管和微丝参与调解这一运动,而L-型钙离子通道与此运动无关.     

用考马斯蓝R250(Coomassie Blue R250)显示人成纤维细胞内微丝的观察

用考马斯蓝显示人包皮成纤维细胞的微丝。使用微丝的特异性抑制剂细胞松弛素 B,对微丝进行抑制处理及恢复实验,进一步肯定了考马斯蓝染色效果的可靠性。     

微丝对豚鼠胃窦平滑肌细胞钾电流的影响

目的　观察微丝对豚鼠胃窦环行平滑肌细胞钾电流的影响 ,并且探讨肌动蛋白微丝是否参与低渗牵张增强豚鼠胃窦环行平滑肌细胞钾电流的过程。方法　本实验用Ⅱ型胶原酶急性分离单个豚鼠胃窦部环行平滑肌细胞 ,采用传统全细胞模式的膜片钳技术 ,以微丝的解聚剂细胞松弛素B(Cytochala sin -B)和微丝的稳定剂鬼笔环肽 (Phalloidin)作为工具药分别研究了在等渗和低渗条件下 ,微丝与钾电流之间的关系。统计学处理采用同体对照t检验和异体对照t检验 ,所有统计结果皆以均数±标准误来表示。结果　细胞松弛素B明显增强钙激活钾电流(IK(Ca) )和延迟…     

自噬对烧伤创面愈合的影响

细胞自噬是细胞在应激状态下应用溶酶体对自身损伤细胞器等物质进行分解,将产生的大分子物质予以回收利用,从而保留细胞活性的降解过程。自噬在细胞的发育和分化进程中起着至关重要的作用。烧伤创面经历炎症期、细胞增殖期和组织重塑期3个时期完成组织愈合。细胞自噬在烧伤创面愈合过程中,具有促进病原体清除,参与新生血管增殖、肉芽组织形成,改善上皮的角质化以及瘢痕重塑的作用。调控自噬,可影响烧伤创面愈合。本文综述细胞自噬在烧伤创面愈合中的研究进展。     

微丝抑制剂细胞松弛素D对ECa 109食管癌细胞的作用

经0.2～0.5微克/毫升细胞松弛素D处理后,ECa 109细胞以暗、亮细胞为主。在间期细胞中,出现了一种小细胞。其细胞间隙变大,微绒毛减少,出现了胞浆突。0.5～1微克/毫升细胞松弛素D处理后细胞表面出现芽胞,其中可含有内质网、高尔基复合器、线粒体等细胞器。在胞质中,可见到靶形线粒体、重膜夹片内质网与环形内质网;高尔基复合器分散。还出现一种多核巨细胞,一个细胞中可见2～6个核,核大小不一,形态各异,每个核的核膜变化很大,有些没有完整核膜。有丝分裂细胞仍可见到。根据细胞松弛素D所致ECa 109细胞超微结构的改变,提出了微丝在支持细胞结构与维持功能中的作用。并讨论了微丝与微管在维持细胞结构与功能的细胞骨架系统中作用的异同。     

内皮细胞微丝结构与血管内皮完整性关系的探讨

为证实内皮细胞微丝与血管内皮完整性之间的直接关系，用Ｒｈｏｄａｍｉｎｅ－ｐｈａｌｌｏｉｄｉｎ显示了体外培养的牛主动脉内皮细胞形成一微密单层细胞后的微丝形态及用细胞毒素Ｂ孵育单层内皮细胞后的细胞形态及微丝的变化。结果表明：内皮细胞内的微丝结构ＤＰＢ是维持内皮细胞之间连接的重要结构。文内还对内皮细胞微丝结构与动脉粥样硬化早期病变的关系进行了讨论。     

比较细胞松弛素A与B在胞质分裂阻断法微核试验中的应用

目的比较细胞松弛素A(Cyto A)及细胞松弛素B(Cyto B)对微核试验常用细胞系——中国仓鼠肺成纤维细胞(CHL)毒性及对体外微核试验结果的影响。方法在荧光显微镜下观察由微丝组成的应力纤维结构,并使用CHL开展胞质分裂阻断法微核试验,比较两者对多核细胞率及含微核双核细胞率的影响。结果不同浓度的Cyto A和Cyto B与细胞作用24 h后,与对照组(26%±7%)比较均可诱导多核细胞形成率的显著升高(Cyto A及Cyto B 3.0μg/ml浓度组分别为58%±12%和72%±9%,P 0.001),且Cyto A在3.0μg/ml浓度条件下诱导的平均多核细胞率与1.5μg/ml浓度组比较有所降低。Cyto A处理组在MMC浓度分别为0、0.1和0.3μg/ml时的微核率分别为0.25%±0.16%、 1.98%±0.59%和3.80%±0.90%;而Cyto B处理组在相同MMC浓度时的微核率分别为0.32%±0.16%、2.50%±0.61%和5.05%±1.09%。结论虽然Cyto B的多核细胞造模效果略优于Cyto A,两者均可有效用于胞质分裂阻断法微核研究。研究结果将为相关领域研究者提供可借鉴的实际经验。     

松弛素保护心肌微血管内皮细胞缺氧复氧损伤的机制

背景：研究报道松弛素可显著改善病理因素导致的心肾功能障碍,抑制心肌肥厚、抗纤维化以及改善缺血再灌注损伤等,但其对内皮细胞缺氧复氧损伤的保护机制尚不明确。目的：探讨松弛素保护心肌微血管内皮细胞缺氧复氧损伤的机制。方法：取生长状态良好的小鼠心肌微血管内皮细胞系(H5V),分3组处理：对照组常规培养33 h,模型组常规培养24 h后给予6 h缺氧+3 h复氧模拟心肌缺氧再灌注损伤,松弛素组常规培养(加180 ng/mL松弛素)24 h后给予6 h缺氧+3 h复氧(加180 ng/mL松弛素)模拟心肌缺氧再灌注损伤。检测细胞通透性与Caspase-3的表达,细胞上清中肿瘤坏死因子α、白细胞介素1β和白细胞介素6水平,RT-PCR检测细胞中钙黏蛋白、Akt1、GSK-3β mRNA表达;Western blot检测细胞中钙黏蛋白、Akt、GSK-3β蛋白表达。结果与结论：(1)与对照组比较,模型组细胞通透性增强(P <0.05),细胞内Caspase-3表达升高(P <0.05);与模型组比较,松弛素组细胞通透性降低(P <0.05),细胞内Caspase-3表达降低(P &l...     

与平滑肌细胞联合培养的内皮细胞的形态学和生长增殖

目的　探讨平滑肌细胞对内皮细胞形态和增殖的影响 ,为内皮细胞种植的组织工程学提供理论基础。　方法　模拟血管壁的内层结构及内皮细胞平滑肌细胞间的相互影响途径 ,建立内皮细胞与平滑肌细胞联合培养的模型 ,应用形态学、细胞计数对内皮细胞的结构和增殖进行研究。　结果　联合培养的新内皮细胞呈伸长性生长 ,沿细胞长轴排列的微丝增多 ,增殖受抑制。　结论　内皮细胞和平滑肌细胞联合培养的新模型是目前研究这两种细胞间相互影响的最佳方式。联合培养的内皮细胞的形态结构更接近于在体生理状态 ,这一变化可能更有利于其抗应力功能的调节     

Centrosomes, microtubules, and microfilaments in the reendothelialization and remodeling of double-sided in vitro wounds.

The maintenance of endothelial monolayer integrity is an important function of aortic endothelial cells. Our study was designed to test two hypotheses related to the repair of the wounded endothelial monolayer. First, that the reappearance of the highly ordered cobblestone monolayer after wound closure is associated with specific sequential changes in the cytoskeletal system. Second, that there are different patterns of reendothelialization depending on whether microfilaments or microtubules are disrupted. One and a half millimeter wide wounds were created down the middle of confluent endothelial monolayers so that there were two wound edges facing each other. During the initiation of repair, the centrosomes of the cells on both sides of the wound reorientated to the front of the cell. The dense peripheral band of actin microfilaments disappeared, the cells elongated and migrated as a uniform sheet with wound closure occurring within 60 hours. The rate of closure remained constant until the migrating fronts met. The cytoskeletal changes observed and the rate of closure were similar to those we reported in a single edge wound. At closure, however, there was a transient piling up of cells which disappeared after 24 to 36 hours. Within 36 to 48 hours after closure, the centrosomes became randomly distributed around the nucleus. By 40 to 48 hours, the dense peripheral band started to reappear and the cells returned to a cobblestone appearance 72 to 96 hours after closure. Thus, the remodeling of the confluent monolayer after wound closure occurs in association with a specific series of cytoskeletal changes. When the microfilaments were disrupted with cytochalasin B, cell migration still occurred but it took up to four times longer for closure. These cells were initially flatter than normal and contained only a few microfilament fibers; however, the microtubule system was intact and centrosome reorientation occurred, but at a slower rate. However, when the microtubules were disrupted with colchicine, either at the onset of wounding or during repair, neither centrosome reorientation nor cell migration occurred. Thus wound closure of small-sized wounds require the presence of intact microtubules, whereas the additional presence of microfilaments results in a more rapid and efficient system of reendothelialzation.     

Fibroblast growth factor 2 enhances early stages of in vitro endothelial repair by microfilament bundle reorganization and cell elongation.

As endothelial cells convert from quiescent to migrating cells over 8 h along a wound edge, actin microfilaments undergo well-defined sequential changes characterized by an initial random distribution followed by a parallel and then a perpendicular orientation of microfilaments with respect to the wound edge. The latter is associated with subsequent cell migration. We tested the hypothesis that fibroblast growth factor 2 (FGF-2) can enhance the very early stages of wound repair even prior to migration and that FGF-2 enhancement of wound repair is associated with changes in the endothelial actin cytoskeleton. Using an in vitro two-sided wound model, the addition of FGF-2 at the time of wounding enhanced the extent of wound closure over 8 h. Treatment with FGF-2 was associated with significantly longer cells along the wound edge at 4 and 8 h after wounding. When treated with increasing concentrations of neutralizing FGF-2 antibody, the extent of wound closure decreased over 8 h and was associated with a decrease in cell length along the wound edge. Actin microfilaments were localized using rhodamine phalloidin and viewed using laser confocal microscopy. At 4 h after wounding, FGF-2 treatment was associated with significantly more cells along the wound expressing perpendicular microfilaments compared to untreated cells, which suggested a more rapid transition of parallel to perpendicular microfilament distribution. Thus, FGF-2 affects the very early stages of wound repair prior to migration by enhancing wound closure due to the early appearance of perpendicular microfilaments and lengthening of cells along the wound edge.     

Acceleration of cutaneous wound healing by transient p53 inhibition

The increase of cell proliferation during early wound healing is thought to be regulated by a decrease of apoptosis. In contrast, the reduction of cellularity during final wound maturation may be controlled by an increase of apoptotic cell death. Herein we studied whether p53 is involved in wound healing-associated apoptosis and whether transient inhibition of p53 is effective to improve the early healing process of cutaneous wounds. Using intravital microscopic and immunohistochemical techniques in hairless mice, we demonstrated that in vivo inhibition of p53 by pifithrin-alpha (PFT-alpha; 2.2 mg/kg ip) accelerates early epithelialization and neovascularization of cutaneous wounds by (i) promoting leukocyte recruitment, (ii) increasing cell proliferation, and (iii) reducing apoptotic cell death. We further show that final wound closure with down-regulation of cell proliferation is not inhibited by PFT-alpha treatment, indicating that transient blockade of p53 function does not affect the process of wound maturation. Western blot analysis revealed that PFT-alpha lowered nuclear but not cytoplasmic p53, implying that cytoplasmic retention of p53 mediates the antiapoptotic effects of PFT-alpha. Furthermore, PFT-alpha significantly increased expression of proliferating cell nuclear antigen protein in whole extracts of cutaneous tissue and caused a rise in proliferation of wild-type, but not mutant, p53-expressing keratinocytes. From our study we conclude that transient inhibition of p53 supports the early cell proliferation required for rapid tissue repair and that this may represent an attractive approach in the treatment of delayed wound healing.     

A homeopathic remedy from arnica, marigold, St. John¿s wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts

ABSTRACT: BACKGROUND: Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan(R) Arnica plus Spray on wound closure in a controlled, blind trial in vitro. METHODS: We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2), its succussed hydroalcoholic solvent (0712-1) and unsuccussed solvent (0712-3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect TM Wound Healing Assay Kit which generated a defined "wound field". All assays were performed in three independent controlled experiments. RESULTS: None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712-2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712-1) at 1:100 dilutions (p<0.001). Unsuccussed solvent (0712-3) had no influence on cell migration (6.3%; p>0.05). Preparation (0712-2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p<0.001) from succussed solvent (0712-1), which caused 22.1% wound closure. CONCLUSION: Results of this study showed that the low potency homeopathic remedy (0712-2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis.     

Spreading, proliferation, and differentiation of the epidermis after wounding a cichlid fish, Hemichromis bimaculatus

A large superficial wound has been experimentally provoked in the cichlid fish Hemichromis bimaculatus to study the interactions between the epidermal cells and the substrate on which they spread, on the one hand, and the restoration of the subepidermal tissues and the epithelial-mesenchymal interactions preceding scale regeneration, on the other hand. The re-epithelialization process, e.g., migration, spreading, differentiation, and proliferation of the epidermal cells, has been followed step by step, using light, scanning and transmission electron microscopy, and tritiated thymidine incorporation, until complete reorganization of the healing epidermis. Wound healing is fast (500 microm/hr) and proceeds centripetally from the wound margins. The epidermal cells spread on a wound surface which is composed of two different matrices: the remains of basement membrane materials covering the scale-pockets, and collagen fibrils of cut dermal strips. Even though both matrices favour cell spreading and attachment, migrating cells show a different behaviour. The re-epithelialization of the wound follows an orderly sequence similar to amphibian and mammalian wound healing, i.e., a "leap frog" mechanism of cell locomotion involving three epidermal layers. The basal layer cells, which spread on the substrate, and the superficial layer cells which protect the epidermis, differentiate first. Whatever the type of substrate over which the epithelium spreads (basement membrane material or collagen fibrils), the epidermal basal layer cells differentiate as soon as they become attached. The incorporation of tritiated thymidine has revealed that there is no proliferation in the healing epidermis until after complete closure of the wound, but that the rapid re-epithelialization of the large surface requires the recruitment of epidermal cells at the wound margins. The present study offers new data on the dynamics of re-epithelialisation and on the resistance of cichlid skin to such wounds. It is also clearly shown that the epidermal basal layer cells differentiate rapidly, a step which is interpreted as the first stage of epithelial-mesenchymal interactions that will lead to scale regeneration.     

Characterization of avascular corneal wound healing fibroblasts. New insights into the myofibroblast.

The characteristics and derivation of corneal wound healing fibroblasts (myofibroblasts) were evaluated by studying the temporal changes in the cellular actin distribution of corneal fibrocytes following full thickness 3-mm diameter central corneal wounds in the rabbit. Under certain conditions these wounds heal without neovascularization, allowing for the detailed analysis of invading fibroblasts with minimal contamination by other cell types. The authors employed transmission electron microscopy to localize microfilaments, fluorescent microscopy using NBD-phallacidin, a mushroom toxin which binds specifically to f-actin and oligomeres of g-actin, to localize actin filaments, and isoelectric focusing gels to characterize actin isotypes. During the early stages of wound healing (1-7 days) there is a gradual change in the corneal fibrocytes adjacent to the wound margin characterized by the development of extensive rough endoplasmic reticulum, microtubules, a prominent Golgi apparatus, and a cortical microfilament network. This is in contrast to the normal fibrocyte, which, for the most part, lacks these structures. The development of microfilaments correlated with increased NBD-phallacidin fluorescence of fibrocytes adjacent to the wound as compared with fibrocytes farther removed from the site of injury. Fibroblasts appearing within the wound from 7 days to 2 months after injury had ultrastructural characteristics similar to those of myofibroblasts, including parallel arrays of microfilaments, stress fibers and cell-cell, cell-matrix attachments. Furthermore, these cells stained intensely with NBD-phallacidin, supporting the ultrastructural findings. At 1 month after injury, cells contained within the wound possessed predominantly nonmuscle isoactins (gamma) as seen by silver staining of isoelectric focusing gels, but little or no (smooth muscle) isoactins could be detected. Moreover, no significant differences could be detected between electrophoretic profiles obtained from wounded versus normal corneas. These morphologic and biochemical data suggest that the corneal fibrocyte may develop into a fibroblastlike cell similar to the myofibroblast, and is characterized by a marked increase in filamentous actin.     

Specific interactions between human fibroblasts and particular chondroitin sulfate molecules for wound healing

The chondroitin sulfates (CSs) constitute an important group of biomacromolecules in the extracellular matrix. However, limited information is available about their specific biological functions. This study aimed to define the interactions between cells and various types of CS. The effects of CSs on cellular activities and the cell cycle were evaluated using cell culture, RNA interference, real-time polymerase chain reaction, flow cytometry, wound healing and contraction models. The results showed that C-6-S promoted both cell proliferation and adhesion, while C-4-S promoted proliferation but inhibited adhesion. Moreover, knockdown of chondroitin inhibited cell proliferation and migration, as well as arresting cells in the G₂/M phase. Also, both C-4-S and C-6-S promoted wound closure in a two-dimensional wound model, whereas only C-6-S inhibited wound contraction in a three-dimensional wound model. This study illustrates that the interaction between cells and different CSs are specific and sulfate-group-dependent. These findings provide useful information for better applications of CSs for wound healing.     

The mevalonate pathway during acute tubular injury: selected determinants and consequences

Examination of thermally induced serosal lesions in mice displayed collections of inflammatory cells, predominantly macrophages, on and surrounding the wound within 48 hours of injury. Furthermore, by 2 days a large number of uninjured mesothelial cells adjacent to the wound were synthesizing DNA. From these findings, it was hypothesized that macrophages play a major role in serosal repair by stimulating mesothelial cell proliferation. Again, using a murine model of mesothelial regeneration, depletion of circulating monocytes significantly delayed serosal healing whereas addition of peritoneal exudate cells to the wound site 36 hours before injury increased the healing rate. In vivo assessment of mesothelial cell proliferation using tritiated thymidine incorporation and autoradiography demonstrated that peritoneal exudate cells stimulated mesothelial cell proliferation (12.44 +/- 1.63% labeling index, compared with controls in which medium only was used 4.48 +/- 0.71%). The mesothelial proliferation was predominantly because of macrophage-secreted products with molecular weights of 36 to 53 kd or 67 to 100 kd. These data support the hypothesis that macrophages play an important role in serosal healing by stimulating mesothelial cell proliferation.     

毛发生长周期对小鼠皮肤创伤愈合的影响

目的　探讨处于不同毛发生长周期的C57BL/6小鼠皮肤创伤愈合速度。 方法　制备小鼠皮肤创伤模型,计算术后0、3、7 d创面愈合率,愈合率=（原始创面面积－未愈合的创面面积）/原始创面面积×100%,比较毛发静止期（Hair telogen stages）小鼠和毛发生长期（Hair anagen stages）小鼠伤口愈合速度。采用HE染色比较伤口愈合的组织形态结构差异,利用BrdU检测伤口周围细胞增殖。 结果　毛发生长期的小鼠皮肤伤口愈合率显著高于毛发静止期的小鼠伤口愈合率。HE染色显示毛发生长期小鼠伤口周围表皮细胞层较多,且表皮细胞向伤口迁移增强;BrdU检测显示毛发生长期小鼠皮肤伤口周围表皮BrdU+ 细胞数多于毛发静止期小鼠。 结论　毛发生长期的小鼠皮肤创伤愈合率高于毛发静止期小鼠,这一结果为进一步探讨毛囊在创伤愈合过程中的作用提供研究基础,也为选择皮肤创伤愈合动物模型提供指导。     

Effects of modified collagen matrices on human umbilical vein endothelial cells

The most commonly used biomaterials fail to ensure sufficient angiogenesis for fast in vivo incorporation. This results in central necrosis and consequent infection. One way of obtaining a high angiogenic response is the application of vascular endothelial growth factor (VEGF). To obtain a sustained release of these cytokines, heparin was incorporated into collagen matrices using 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinmide (NHS). The functionality of the heparinized collagen matrices was then enhanced by immobilization of VEGF via its heparin-binding domain. This procedure changed in vitro degradation behavior and water-binding capacity. Accelerated endothelial cell proliferation was also achieved. A range of different heparin and EDC/NHS concentrations in combination with VEGF induced variation in endothelial cell growth and tubulogenic formation. Polymerized collagen scaffolds presented biointeractive systems with integrated angiogenic activity. This may become a useful tool in the clinical therapy of disorders connected with wound healing.