机械牵张导致肺上皮细胞损伤的信号转导机制研究进展

临床上机械通气导致急性肺损伤(ALI)已成事实，但机械张力转化为生物学损伤(biotmuma)的机制尚不清楚。现就近年来机械张力对离体肺上皮细胞的结构形态和功能改变以及细胞内炎性因子产生的上游信号转导可能机制作一综述。     

肺保存过程中损伤机制的研究进展

肺保存(ｌｕｎｇｐｒｅｓｅｒｖａｔｉｏｎ)技术虽然有了很大的进步,但目前临床也仅能保存4～6ｈ,即缺血时间最长不得超过6ｈ。近年来,虽然,动物实验中肺保存可长达18～24ｈ,甚至更长,但是临床尚无此报道〔1,2〕。研究肺保存的一个重要目的是扩大供体库,使供肺的转送、手术的安排乃至手术操作不被肺保存的时间所限制。第二个重要意义是,肺保存的研究不仅仅着眼于术前的肺保存,而且影响到术中和术后的肺损伤程度及术后的并发症和预后〔3〕。因此,肺保存在肺移植术中有着越来越重要的地位,近年来在此方面的发展速度比较快。研究肺保存,首先面…     

机械牵张诱导肺上皮细胞转化——肺纤维增殖的新机制

背景 机械通气是纠正低氧血症的重要治疗手段之一,在急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)的支持措施中拥有无可替代的地位.但机械通气治疗在提供有效呼吸支持治疗的同时,还可能导致或进一步加重肺损伤甚至引起肺部纤维化.有研究报道,在ARDS致死原因中,难以控制的肺纤维化占40％ ～70％. 目的 就机械性牵拉致肺纤维化的发生机制的最新研究进行综述,以期人们深化对肺纤维化发生机制的认识. 内容 机械通气对肺组织产生的机械牵拉作用可诱导肺上皮细胞表型转化,这是形成肺纤维化的重要基础. 趋向 一系列刺激因素均可诱发肺纤维化,包括致敏源、化学毒素和机械牵拉等.而机械牵拉导致的肺纤维化已成为重症监护过程中的重要致死原因之一,这就需要我们的高度重视,在充分了解其发病机制的基础上,研究出有效的预防和干预措施.     

异丙酚对肺缺血再灌注损伤大鼠肺上皮细胞凋亡的影响

目的拟通过观察肺上皮细胞凋亡的变化,探讨异丙酚减轻大鼠肺缺血再灌注损伤的作用机制。方法雄性SD大鼠136只,随机分为3组:假手术组(S组,n=24)、缺血再灌注组(I/R组,n=56)、异丙酚组(P组,n=56)。I/R组、P组制备大鼠肺原位热缺血模型,缺血1h。P组于缺血前30min静脉输注异丙酚20mg·kg~(-1)·h~(-1)至再灌注4h。S组除不作缺血处理外,其余处理与I/R组相同。分别于再灌注0.5、1、2、4h随机处死大鼠(S组每个时点处死6只,I/R组、P组每个时点分别处死14只大鼠),每组每个时点的一半大鼠用于测定支气管肺泡灌洗液(BALF)中中性粒细胞百分比、蛋白浓度,另外一半大鼠用于测定肺组织丙二醛(MDA)含量、湿,干重比(W/D)及肺上皮细胞凋亡指数(TUNEL法),并在光镜下观察肺组织的病理学改变;I/R组、P组肺上皮细胞凋亡指数与W/D进行直线相关分析。结果与S组相比,I/R组和P组再灌注各时点BALF中中性粒细胞百分比、蛋白浓度及肺组织MDA含量、W/D、肺上皮细胞凋亡指数均增加(P<0.05或0.01);与I/R组比较,P组上述指标均降低(P<0.05或0.01),肺组织病理学损伤减轻;I/R组、P组肺上皮细胞凋亡指数与W/D之间的相关系数分别为0.784、0.830(P<0.01)。结论异丙酚抑制肺上皮细胞凋亡可能是减轻大鼠肺缺血再灌注损伤的机制之一。     

单肺通气时炎性反应机制及预防的研究进展

背景 开胸手术需要长时间维持单肺通气(one-lung ventilation,OLV),这一过程可激活炎性细胞并释放大量炎性因子,导致肺部炎症反应及并发症. 目的 着重探讨OLV所致炎性因子释放的机制,保护性通气策略,促炎症消退等相关进展.内容 OLV时,过度机械牵张刺激作用于肺细胞膜表面机械感受器,激活细胞内各种传导通路,导致各种致炎因子及炎症介质的上调表达.术中小潮气量、低气道压、低呼气末正压通气(end-expiratory positive pressure,PEEP)为策略的保护性通气模式的应用及吸入麻醉药、局部麻醉药、糖皮质激素等药物的使用可显著减少炎性因子的释放,减少胸科患者术后肺损伤的发病率.趋向 内源性炎症消退机制及基因和生物靶向治疗已成为近年来研究炎症的新的热点.     

肾小管上皮细胞损伤与草酸钙肾结石形成机制的研究进展

肾小管上皮细胞损伤被认为是草酸钙结石形成的重要原因,与细胞-晶体相互作用、细胞分子结构变化、细胞差异蛋白分子表达、晶体的粘附等过程密切相关.因此,上皮细胞损伤机制及结石微晶体粘附分子机制的深入研究将为草酸钙肾结石的防治提供理论基础和理论依据.     

周期性牵张对肺泡Ⅱ型上皮细胞IL-8表达的影响

目的研究周期性牵张肺泡Ⅱ型上皮细胞株A549细胞对炎性介质白细胞介素-8（IL8）产生和释放的影响。方法对融合的肺泡Ⅱ型上皮细胞株A549细胞施加周期性机械牵张应力。实验一：加载频率0．5Hz，加载时间4h，加载应力分别为5％、15％、30％；实验二：加载应力为15％，加载频率0．5Hz，加载时间分别为1h、2h、4h、8h；实验三：加载应力为15％，加载时间4h，加载频率分别为0．2Hz、0．5Hz、1．0Hz。每个实验均设立空白对照即不给予机械应力。用实时荧光定量PCR法测定IL-8 mRNA的表达，用ELISA法测定IL-8蛋白的浓度。结果随着加载应力的增加和加载时间的延长，IL-8蛋白浓度增加、IL-8 mRNA表达上调（P〈0．05）；施加不同加载频率后，IL-8蛋白浓度增加、IL-8 mRNA表达上调（P〈0．05）；不同加载频率间IL-8蛋白浓度和IL-8 mRNA表达比较差异无统计学意义（P〉0．05）。结论肺泡Ⅱ型上皮细胞IL-8的产生和释放与周期性的机械牵张应力呈强度和时间依赖性，而加载频率对其无影响。     

单肺通气时机械通气模式的研究进展

背景 单肺通气(one lung ventilation,OLV)实施过程中最常见的并发症是低氧血症,也是麻醉医师遇到的最严重的挑战. 目的 近来研究表明OLV本身能够引起低氧血症和急性肺损伤(acute lung injury,ALI).因此,如何实施OLV时机械通气模式,降低肺内分流率(pulmonary shunt fraction,Qs/Qt)、预防低氧血症一直是临床研究的热点. 内容 综述提高吸入氧分数(fraction of inspiration O2,FiO2)、控制通气模式、高频通气(high frequency ventilation,HFV)、潮气量(tidal volume,Vt)、反比通气、部分液体通气(partial liquid ventilation,PLV)、持续气道正压通气(continuous positive airway pressure,CPAP)、呼气末正压通气(positive end-expiratory pressure,PEEP)等通气模式,以及实施联合多种模式的保护性肺通气策略. 趋向 综合运用多种预防OLV期间低氧血症的通气模式取得良好的效果,但应针对患者和手术情况制定OLV时机械通气模式.     

Signal Transduction Pathways Involved in Brain Death-Induced Renal Injury

Kidneys derived from brain death organ donors show an inferior survival when compared to kidneys derived from living donors. Brain death is known to induce organ injury by evoking an inflammatory response in the donor. Neuronal injury triggers an inflammatory response in the brain, leading to endothelial dysfunction and the release of cytokines in the circulation. Serum levels of interleukin-6, -8, -10, and monocyte chemoattractant protein-1 (MCP-1) are increased after brain death. Binding with cytokine-receptors in kidneys stimulates activation of nuclear factor-kappa B (NF-κB), selectins, adhesion molecules and production of chemokines leading to cellular influx. Mitogen-activated protein kinases (MAP-kinases) mediate inflammatory responses and together with NF-κB they seem to play an important role in brain death induced renal injury. Altering the activation state of MAP-kinases could be a promising drug target for early intervention to reduce cerebral injury related donor kidney damage and improve outcome after transplantation.     

Electrotaxis of alveolar epithelial cells in direct-current electric fields

PurposeThis study aims to elucidate the electrotaxis response of alveolar epithelial cells (AECs) in direct-current electric fields (EFs), explore the impact of EFs on the cell fate of AECs, and lay the foundation for future exploitation of EFs for the treatment of acute lung injury.MethodsAECs were extracted from rat lung tissues using magnetic-activated cell sorting. To elucidate the electrotaxis responses of AECs, different voltages of EFs (0, 50, 100, and 200 mV/mm) were applied to two types of AECs, respectively. Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs. Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration. To further demonstrate the impact of EFs on the pulmonary tissue, the human bronchial epithelial cells transformed with Ad12-SV40 2B (BEAS-2B cells) were obtained and experimented under the same conditions as AECs. To determine the influence on cell fate, cells underwent electric stimulation were collected to perform Western blot analysis.ResultsThe successful separation and culturing of AECs were confirmed through immunofluorescence staining. Compared with the control, AECs in EFs demonstrated a significant directionality in a voltage-dependent way. In general, type Ⅰ alveolar epithelial cells migrated faster than type Ⅱ alveolar epithelial cells, and under EFs, these two types of cells exhibited different response threshold. For type Ⅱ alveolar epithelial cells, only EFs at 200 mV/mm resulted a significant difference to the velocity, whereas for, EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference. Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11.ConclusionEFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects, which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.     

Signal Transduction of Opioid-induced Cardioprotection in Ischemia-Reperfusion

Background: Morphine reduces myocardial ischemia-reperfusion injury in vivo and in vitro. The authors tried to determine the role of opioid [delta]1 receptors, oxygen radicals, and adenosine triphosphate-sensitive potassium (KATP) channels in mediating this effect.

Methods: Chick cardiomyocytes were studied in a flow-through chamber while pH, flow rate, oxygen, and carbon dioxide tension were controlled. Cell viability was quantified by nuclear stain propidium iodide, and oxygen radicals were quantified using molecular probe 2',7'-dichlorofluorescin diacetate.

Results: Morphine (1 [mu]m) or the selective [delta]-opioid receptor agonist BW373U86 (10 pm) given for 10 min before 1 h of ischemia and 3 h of reoxygenation reduced cell death (31 +/- 5%, n = 6, and 28 +/- 5%, n = 6 [P < 0.05], respectively, 53 +/- 6%, n = 6, in controls) and generated oxygen radicals before ischemia (724 +/- 53, n = 8, and 742 +/- 75, n = 8 [P < 0.05], respectively, vs. 384 +/- 42, n = 6, in controls, arbitrary units). The protection of morphine was abolished by naloxone, or the selective [delta]1-opioid receptor antagonist 7-benzylidenenaltrexone. Reduction in cell death and increase in oxygen radicals with BW373U86 were blocked by the selective mitochondrial KATP channel antagonist 5-hydroxydecanoate or diethyldithiocarbamic acid (1000 [mu]m), which inhibited conversion of O2- to H2O2. The increase in oxygen radicals was abolished by the mitochondrial electron transport inhibitor myxothiazol. Reduction in cell death was associated with attenuated oxidant stress at reperfusion.     

Reoxygenation results in cell death of human alveolar epithelial cells.

BACKGROUND: The functional response of isolated alveolar epithelial cells (AECs) to ischemia/reperfusion injury (I/R) is incompletely understood. Using a cell culture model, we investigated the tolerance of human type II alveolar cells (ATII) to hypoxia and subsequent reoxygenation. METHODS: Cell cultures of A549 cells (human lung adenocarcinoma) and primary ATII were incubated in 95% N(2)/5% CO(2) saturated medium at 37 degrees C for 48 hours or 72 hours. The hypoxic medium was subsequently exchanged to normoxic medium at 37 degrees C. Lactate dehydrogenase (LDH) release and mitochondrial viability, as assessed by WST-1 metabolism, were determined during both hypoxia and reoxygenation. A549 cells and ATII maintained under normoxic conditions served as controls. RESULTS: Before reoxygenation, after 48 or 72 hours of hypoxia, WST-1 metabolism in A549 cells was significantly reduced (p < 0.05), but LDH release remained low in both cell types. Reoxygenation after 48 h of hypoxia was associated with recovery of WST-1 metabolism and an only minimal increase in LDH release. Reoxygenation after 72 hours of hypoxia, in contrast, induced marked injury in both A549 cells and primary ATII as indicated by significantly reduced WST-1 metabolism and a dramatic increase of LDH release compared with normoxic controls (p < 0.05). CONCLUSIONS: Viability of alveolar cell lines and primary ATII is maintained during hypoxia for up to 72 hours. Reoxygenation after 72 hours of hypoxia results in rapid development of injury and cell death in both cell types.     

VEGF regulates the proliferation of acid-exposed alveolar lining epithelial cells

BACKGROUND: Acid induced pneumonitis resulting in acute respiratory distress syndrome (ARDS) is characterised by increased alveolar permeability and accumulation of neutrophils. It is hypothesised that vascular endothelial growth factor (VEGF) is involved in the development of lung oedema. Furthermore, lower levels of VEGF are detected in bronchoalveolar lavage fluid from patients with ARDS than from non-ARDS patients. We hypothesised that VEGF acts cytoprotectively and have investigated this possibility in vitro with A549 cells. METHODS: A549 cells were incubated in 24 well culture dishes 24 hours before exposure to acid, then incubated with serum free medium containing various concentrations of HCl for 30 minutes at 37 degrees C in 5% CO(2). The acidified medium was changed to normal complete medium; at specified incubation periods the supernatants were collected and the VEGF concentration measured and the number of adherent cells counted. RESULTS: Proliferation of A549 cells and VEGF production were suppressed for at least 48 hours in HCl at a concentration of 50 mM. Restoration of cellular proliferation occurred following exogenous administration of VEGF (concentration of 1-250 ng/ml) and was inhibited by co-incubation with neutralising anti-VEGF antibody, indicating an interaction between VEGF molecules and A549 cells. Control cells were not influenced by administration of exogenous VEGF or anti-VEGF antibody. Treatment with neutralising anti-VEGF receptor (VEGFR) antibodies against VEGFR-1 and VEGFR-2 suppressed proliferation of acid exposed A549 cells but had no effect on control cells. CONCLUSIONS: Exogenous VEGF interacts with VEGFR-1 and VEGFR-2 on the surface and regulates the proliferation of injured alveolar lining epithelial cells in an autocrine or paracrine fashion.     

Proliferation of alveolar type II epithelial cells in residual lungs following thoracotomy

We have found that the number of alveolar type II epithelial cells was markedly increased in the alveolar walls along with the visceral pleura of an excised residual lung in patients who had undergone an exploratory thoracotomy with partial lobectomy seven or nine days prior to the second thoracotomy with resection of the residual lung. Such increase in proliferation of alveolar type II epithelial cells was not found in the excised lung not exposed to a prior thoracotomy, and not in the excised lung exposed to a prior thoracotomy more than 1 year before the second thoracotomy. These findings suggested that remodelling of the alveolar epithelial cells occurred in the residual lung in the early period following thoracotomy.     

Injury to murine airway epithelial cells by pollen enzymes

BACKGROUND—Pollens are importanttriggers for asthma but the mechanism of sensitisation to theirproteins remains poorly understood. The intrinsic protease activity ofsome allergens may contribute to sensitisation by disrupting theintegrity of the airway epithelial barrier. Pollens release a varietyof enzymes, including proteases, upon hydration. The hypothesis thatsuch enzymes might be able to damage airway epithelial cells wastherefore tested.
METHODS—Diffusates from pollens ofLolium perenne (ryegrass), Poa pratensis(Kentucky bluegrass), Acacia longifolia (Sydney golden wattle), or Casuarina distyla (she-oak) were incubatedwith mouse tracheal epithelial cells in culture and cellular detachmentwas quantified using a methylene blue dye binding assay.
RESULTS—Diffusates prepared using100 mg/ml of pollen caused detachment of 30-90% of airway epithelialcells in separate experiments. Within each experiment comparabledetachment was observed with all diffusates tested, although totalprotein in the diffusates varied markedly between species. Viability ofthe cells recovered after exposure to Acacia diffusate washigher than after detachment by exposure to Loliumdiffusate. Cellular detachment by all of the diffusates could be almostcompletely inhibited by addition of 10% serum. Aprotinin, an inhibitorof serine proteases, partially blocked activity in diffusates ofLolium pollen but not of Acacia pollen. Incontrast, α₁-protease inhibitor and secretory leucocyte protease inhibitor (SLPI) were not able to block the activity of eitherdiffusate at concentrations which inhibited cellular detachment by trypsin.
CONCLUSIONS—Proteases released bypollens are able to cause detachment of airway epithelial cells fromtheir substratum in vitro and may not be effectively inhibited byendogenous antiproteases.

     

瘢痕疙瘩的细胞信号转导通路的研究进展

瘢痕疙瘩(Keloids,KD)是一种继发于创伤的结缔组织过度增生性皮肤病,易感人群好发,通常不会自发消退,手术切除后极易复发,是目前整形外科亟待解决的临床难题。KD的发病机制十分复杂,是多种因素共同作用的结果,如细胞因子、细胞信号传导通路、胶原代谢、基因表达和细胞的增殖与凋亡等。本文对近几年有关KD细胞信号传导通路和特异性蛋白的研究进展进行综述。