山莨菪硷(654-2)预防大鼠实验性肺水肿的电镜观察

静脉注射肾上腺素可造成大鼠致死性肺水肿,这种水肿类似临床上的中枢性肺水肿,属于混合性肺水肿。既有肺毛细血管压力升高,又有肺毛细血管通透性增高。用中药山莨菪碱进行预防效果十分明显。电镜观察提供了这种预防效果的超微结构基础。水肿组内皮细胞及肺泡上皮细胞膨隆,水肿,部份细胞从基底膜脱落,基底膜裸露或断裂。间质腔及肺泡腔充满富含蛋白的水肿液。而预治组病变明显减轻,水肿液明显减少。内皮细胞及上皮细胞基本正常。间质、肺膈增宽,结缔组织增生。这是由于动物存活半个月,炎症被吸收有关。实验表明,山莨菪碱可以明显抑制血管通透性的增高。     

利用放射性同位素标记白蛋白的方法测定微血管通透性

同硫酸铵提取大鼠血清白蛋白，冰冻，干燥；经电泳，扫描测具纯度。按Ｇｒｅｅｎｗｏｏｄ的微量氯胺Ｔ标记法，参考Ｂｏｃｉ的常规血清蛋白标记程序，利用放射性同位素１２５Ｉ，室温下反应３ｍｉｎ，把放射性同位素１２５Ｉ标记在白蛋白酪氨酸的第７位碳原子上。大鼠尾静脉内注入１２５Ｉ白蛋白，放射强度为（７～９）＼Ｘ１０６Ｃｉ／ｍｉｎ。２ｈ后麻醉动物，剥离脏器，称重，测其放射性，计算通透性分值，并比较各脏器微血管通透分值的大小，有利于分析和判断微血管通透功能的改变。通过实际应用也显示了该法不同于其他方法的优点     

山莨菪碱预处置对大鼠ARDS血清及肺组织中细胞因子的影响

目的 观察山莨菪碱(654-2)预处置对大鼠油酸型急性呼吸窘迫综合征(ARDS)血清及肺组织中IL-6、IL-8、TNF-α及IL-10的影响,探讨654-2治疗ARDS的理论基础。方法 经舌下静脉注射油酸复制大鼠ARDS模型,用酶联免疫吸附试验测定血清及肺组织匀浆上清液中IL-6、IL-8、TNF-α和IL-10水平。结果 ARDS组大鼠血清IL-6、TNF-α、IL-10含量和肺组织匀浆上清液中IL-6、IL-10含量明显高于对照组(P<0.01或0.05)。654-2预处置组血清和肺组织中IL-10水平显著高于对照组(P<0.01或0.05)。654-2预处置组血清和肺组织中IL-6、TNF-α含量明显低于ARDS组(P<0.01或0.05),而IL-8和IL-10含量两者无显著性差异。结论 IL-6、IL-8、TNF-α及IL-10在大鼠油酸型ARDS炎症过程中可能起重要作用,654-2可能通过抑制IL-6、TNF-α过度分泌而减轻肺损伤。     

油酸型急性呼吸窘迫综合征血清及肺组织促炎症细胞因子变化及山莨菪碱干预的实验研究

目的:观察大鼠油酸型急性呼吸窘迫综合征(ARDS)不同时间血清及肺组织中白细胞介素-6(IL-6)、IL-8和肿瘤坏死因子(TNF-α)的水平变化以及山莨菪碱(654-2)对其影响。方法:经舌下静脉注射油酸复制大鼠ARDS模型,用酶联免疫吸附试验测定不同时间血清及肺组织匀浆上清液中IL-6、IL-8和TNF-α水平。结果:注射油酸4h组血清及肺组织中IL-6、IL-8和TNF-α水平明显高于对照组;注射油酸8h组上述细胞因子均明显低于4h组,但血清中IL-6、TNF-α和肺组织中IL-6仍高于对照组水平;注射油酸16h组血清IL-6、TNF-α水平明显低于8h组,但TNF-α水平仍高于对照组,肺组织中IL-6水平亦高于对照组。654-2干预组血清及肺组织中IL-6、TNF-α水平均明显低于未干预组。结论:IL-6、IL-8和TNF-α在大鼠油酸型ARDS炎症过程中可能起重要作用;654-2可能通过抑制IL-6、TNF-α过量分泌,减轻肺损伤,具有防治ARDS作用。     

Mesenchymal Stem Cell: Does it Work in an Experimental Model with Acute Respiratory Distress Syndrome?

We hypothesized that bone marrow-derived mesenchymal stem cells (BM-MSCs) would have a possible role in the treatment of acute respiratory distress syndrome (ARDS). ARDS disease model was developed in Wistar albino male rats by intratracheal instillation of physiological saline solution. Anesthezied and tracheotomized rats (n?=?8) with ARDS were pressure-controlled ventilated. Isolated and characterized rat (r-) BM-MSCs were labeled with GFP gene, and introduced in the lungs of the ARDS rat-model. After applying of MSCs, the life span of each rat was recorded. When rats died, their lung tissues were removed for histopathological examination. Also the tissue sections were analyzed for GFP labeled rBM-MSCs and stained for vimentin, CK19, proinflammatory (MPO, IL-1β, IL-6 and MIP-2) and anti-inflammatory [IL-1ra and prostaglandin E2 receptor (EP3)] cytokines. The histopathological signs of rat-model ARDS were similar to the acute phase of ARDS in humans. rBM-MSCs were observed to home in lung paranchyma. Although the infiltration of neutrophils slightly decreased in the interalveolar, peribronchial and perivascular area, a notable improvement was determined in the degree of hemorrhage, edema and hyaline membrane formation in rats treated with rBM-MSCs. Also decreased proinflammatory cytokines levels and increased the intensity of anti-inflammatory cytokines were established. Therefore MSCs could promote alveoar epithelial repair by mediating of cytokines from a proinflammatory to an anti-inflammatory response. As a novel therapeutic approach, mesenchymal stem cell treatment with intratracheal injection could be helpful in the management of critically ill patients with ARDS.     

大鼠脑缺血-再灌注损伤对脑微血管通透性的影响

目的 :探讨大鼠脑缺血 -再灌注后脑组织微血管通透性的变化规律 ,为进一步探讨脑缺血及再灌注损伤机制提供参考数据。方法 :应用荧光素钠 (分子量 3 86 ,Fl Na)和 FITC标记的右旋糖苷( FD4,分子量 40 0 0 )为荧光示踪剂 ,以单侧颈总动脉远心端及同侧颈静脉插管并连接二管造成颈总动脉向颈静脉的引流 ,同时用动脉夹夹闭对侧颈总动脉造成脑缺血模型。缺血 1h后松开动脉夹并中断引流造成再灌注模型。测量脑组织荧光强度反映脑微血管通透性的变化。结果 :单纯的脑缺血即可引起脑组织微血管通透性的增强 ,再灌注后对小分子量物质的通透性随再灌注时间的延长有增加的趋势 ,而对较大分子量物质的通透性基本维持在同一水平。结论 :即使是在再灌注损伤最严重时 ,脑微血管对较大分子量的物质通透仍不明显 ,说明血 -脑屏障的存在可以有效地防止有害物质通过微血管壁侵入脑组织 ,但一旦进入脑组织 ,将滞留于脑组织中不易清除。     

Activation of C5 by cobra venom factor is required in neutrophil-mediated lung injury in the rat.

Cobra venom factor (CVF)-induced systemic activation of the complement system in the rat has been shown to result in the development of acute lung microvascular injury and appearance in lungs and plasma of lipid peroxidation products. The pathogenesis of these events is dependent on complement and neutrophils and is sensitive to pretreatment of experimental animals with iron chelators or scavengers of hydroxyl radical. In order to further analyze the role of complement in the pathogenesis of acute lung injury in rats after systemic complement activation, two different CVFs have been employed in the present study. One was the previously used CVFn isolated from Naja n. naja venom, whereas the other factor, CVFh, was isolated from Naja h. haje venom. Both factors have been shown to activate the alternative complement pathway by forming a potent C3 convertase but differ with respect to their ability to bind and activate C5. CVFn but not CVFh activates C5 and distant complement components. When equal doses of C3-activating activity of CVFn or CVFh were injected intravenously into rats, CVFh-treated rats failed to develop acute lung injury, whereas CVFn-treated animals showed pronounced increases in lung vascular permeability. Similarly, in isolated blood perfused rat lungs neither the lung injury nor pulmonary hypertension caused by CVFn were found after injection of CVFh. In addition, CVFh-treated animals failed to show transient neutropenia or appearance in plasma of C5-derived chemotactic activity, although the extent of C3 conversion in vivo was identical to that seen in CVFn-treated rats. Morphologic examination of the lungs of the experimental animals revealed no signs of injury in CVFh-treated rats, whereas the lungs from CVFn-treated animals revealed interstitial and alveolar edema, as well as plugging of pulmonary capillaries with neutrophils, blebbing and/or destruction of vascular endothelial cells, fibrin deposition, and hemorrhage. These studies provide evidence that activation of the complement system involving C3 but not extending further in the complement sequence is not sufficient to bring about acute injury of the lung microvasculature and that the activation sequence must at least also involve C5.     

Acute generalized microvascular injury by activated complement and hypoxia: the basis of the adult respiratory distress syndrome and multiple organ failure?

It has been suggested that generalized endothelial damage and permeability changes, induced by prolonged activation of the complement system and ensuing release of lysosomal enzymes, prostaglandins and toxic oxygen products, underlie the genesis of the Adult Respiratory Distress Syndrome (ARDS) and Multiple Organ Failure (MOF). The effects in New Zealand white rabbits were investigated of a 4 h infusion of activated complement and its combination with a short hypoxic episode on respiratory function, leukocyte count, platelet count and morphology of the lungs, heart, liver, kidney and spleen. Prolonged activation of the complement system induced hyperventilation with respiratory alkalosis and hypocapnia, depletion of granulocytes (PMN), and a variable accumulation PMN in the capillaries of all organs examined, in combination with interstitial, and, in the liver, cellular oedema. Electron microscopy of the lungs revealed degranulation of PMN, endothelial swelling and widening of the alveolar septa. The combination of hypoxia and systemic complement activation appeared to aggravate this microvascular injury with the occurrence of protein rich alveolar oedema and haemorrhage in the lungs and accumulation of PMN debris containing macrophages in the spleen. The alterations in respiratory function and pulmonary morphology in these rabbits, imitate the clinical and morphological characteristics of the early phase of ARDS. The inflammatory reaction, found in all other organs examined, might represent the early phase of MOF. If so, ARDS and MOF -- clinically closely interconnected syndromes -- might be interpreted as manifestations of the same syndrome and as the clinical expression of an uncontrolled whole body inflammation.     

脂多糖对大鼠肺微血管内皮细胞[Ca~(2+)]_i和Gq蛋白的影响及山莨菪碱的干预

目的 :观察脂多糖对大鼠肺微血管内皮细胞 (RPMVECs) [Ca2 + ]i 和Gq蛋白的影响及山莨菪碱的干预作用。方法 :分离、培养并鉴定Wistar大鼠RPMVECs;应用Fura - 2 /AM法测定RPMVECs[Ca2 + ]i;流式细胞仪技术测定RPMVECsGq蛋白。结果 :①LPS作用于RPMVECs 30min和 90min后 ,[Ca2 + ]i 显著高于对照组 ;Gq蛋白显著低于对照组。②山莨菪碱可抑制LPS的上述作用。结论 :①LPS致RPMVECs[Ca2 + ]i 增加和Gq蛋白下降 ;②山莨菪碱通过抑制LPS诱导RPMVECs[Ca2 + ]i 增加和Gq蛋白下降的作用而保护其内皮屏障功能。     

No effects of large doses of catecholamines on vascular permeability in isolated blood-perfused dog lungs

Neurogenic pulmonary oedema (NPO) is believed to be induced by intense activation of the sympathetic nervous system, characterized by massive secretion of catecholamines into the blood stream. There is a possibility that NPO is partly the result of increased vascular permeability. However, the mechanism for an increase in pulmonary vascular permeability is not known. The present study was designed to test the hypothesis that large doses of catecholamines increase pulmonary microvascular permeability directly. Adrenaline or noradrenaline (100 and 300 pug) was injected as a bolus into isolated dog lungs perfused with heparinized autologous blood at constant pressure. Adrenaline or noradrenaline produced sustained lung weight loss although both catecholamines increased pulmonary capillary pressure, assessed by double occlusion pressure, by 2–5 mmHg above baseline. Vascular permeability, as measured by the capillary filtration coefficient and the isogravimetric capillary pressure, did not change significantly frombaseline at 30 and 60 min after catecholamine. Finally, the final-to-initial wet lung weight ratio of the catecholamine-treated lungs did not differ from that of saline-injected control lungs. Thus, we conclude that circulating catecholamines, even at supra-physiological doses, do not increase vascular permeability in isolated blood-perfused dog lungs.     

Ferritin and desferrioxamine attenuate xanthine oxidase-dependent leak in isolated perfused rat lungs

Iron, through its participation in reactions that generate reactive oxygen species, may contribute to the oxidative lung injury observed in patients with acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). A number of investigators have shown that the endogenous iron storage protein ferritin increases in the blood of patients with and at-risk for ALI and ARDS, but the significance of these increases are not known. In the present investigation, we measured lung tissue levels of thiobarbituric acid reactive substances (TBARS) and lung leak in isolated rat lungs perfused with xanthine oxidase (XO) and purine, an enzymatic system which generates reactive oxygen species. We found that adding ferritin (100 ng/mL) or desferrioxamine (DFO, 10 mM), an iron chelator, to the vascular perfusate solution decreased oxidant-induced leak in isolated rat lungs perfused with XO and purine. Addition of ferritin or DFO also decreased TBARS in isolated rat lungs perfused with XO and purine; neither ferritin nor DFO, however, decreased XO activity in vitro. Our results suggest that oxidative lung leak may be altered by the availability of reactive iron and that ferritin may contribute to protection against oxidative lung injury.     

Adult respiratory distress syndrome - an update

In adults, acute lung injury or adult respiratory distress syndrome (ARDS) may complicate a wide range of serious medical and surgical conditions, only some of which involve direct pulmonary insult. The characteristic histological feature of ARDS is an intense inflammatory process in the lungs, which may progress to fibrosis. The earliest physiological characteristic is an increase in the protein permeability across the endothelial and epithelial barriers of the lungs. This clinical syndrome is characterized by arterial hypoxaemia and bilateral radiographic infiltrates, which represent protein-rich oedema fluid. In addition there is a neutrophilic and macrophage infiltrate. Pulmonary endothelium is actively involved in the development of ARDS. It alters cell-cell adhesion as the initial step in leucocyte migration which, in turn, changes the permeability that allows protein-rich fluid to move into the interstitium. The quantity of this interstitial oedema may be sufficient to cause bulk flow through the epithelial barrier. There is probably independent epithelial injury. Finally, the endothelium can release and metabolize vasoactive and inflammatory substances, such as endothelins, nitric oxide and cytokines, etc. No single substance is responsible for acute lung injury, but rather a complex interplay exists between diverse pro- and anti-inflammatory mediators.     

糖尿病大鼠的骨膜反应及染料木素的保护作用

目的　探讨糖尿病大鼠骨膜反应的动态演变及染料木素(Gen)的保护作用。 方法　建立速发型链脲佐菌素（streptozotocin, STZ）糖尿病大鼠模型, 随机分为正常组(CON); 糖尿病组（DM）; Gen低剂量干预组（GenLow）; Gen高剂量干预组（GenHigh）(30 mg·kg-1·d-1)。每天1次, 灌胃给药（4.0 mg/kg）10周,每组20只。对各组大鼠骨膜作组织病理学分析及组织计量学测定; 利用Van Gieson胶原纤维染色法观察胶原纤维沉积变化; 墨汁灌注行边缘微血管密度测定。 结果　DM组骨膜厚度等均明显小于CON组(P<0.01); 微血管密度增大, 但渗透性大。GenHigh骨膜厚度明显减少（P<0.01）。 结论　糖尿病先后出现骨膜水肿、 增生、 退化等骨膜反应。Gen对糖尿病的骨膜反应具有改善保护作用。     

大鼠ARDS基因表达谱中信号传导基因的表达

 目的 对大鼠急性呼吸窘迫综合征(ARDS)基因表达谱中的信号传导基因进行研究与分析。方法 从正常和ARDS大鼠肺组织中提取总RNA,分离纯化mRNA,经反转录合成掺入生物素标记的cDNA探针,然后与基因芯片杂交,扫描芯片荧光信号图像,用Sam3.0进行统计处理,用博奥生物分子功能注释系统V4.0进行功能分析。随机选择3个差异表达基因 ,用荧光定量RT- PCR验证。结果 在ARDS大鼠肺组织中,信号传导相关基因上调的有2个,下调的有9个,涉及到的相关信号通路11个。结论 大鼠ARDS基因表达谱中涉及到许多信号传导基因和通路的差异表达。     

脂多糖对大鼠肺微血管内皮细胞[Ca2+]i和Gq蛋白的影响及山莨菪碱的干预

目的:观察脂多糖对大鼠肺微血管内皮细胞(RPMVECs)[Ca²⁺]_i和Gq蛋白的影响及山莨菪碱的干预作用。方法:分离、培养并鉴定Wistar大鼠RPMVECs;应用Fura-2/AM法测定RPMVECs[Ca²⁺]_i;流式细胞仪技术测定RPMVECsGq蛋白。结果:①LPS作用于RPMVECs30min和90min后,[Ca²⁺]_i显著高于对照组;Gq蛋白显著低于对照组。②山莨菪碱可抑制LPS的上述作用。结论:①LPS致RPMVECs[Ca²⁺]_i增加和Gq蛋白下降;②山莨菪碱通过抑制LPS诱导RPMVECs[Ca²⁺]_i增加和Gq蛋白下降的作用而保护其内皮屏障功能。     

Studies on cutaneous vascular permeability in the rat: Increases caused by histamine and histamine-like agents

The pharmacology of histamine-induced increases in microvascular permeability has been studied in rat skin. Histamine caused dose-dependent increases in microvascular permeability, assessed as increases in extravascular albumin accumulation. The responses to histamine were inhibited in a dose-dependent manner by pretreatment with mepyramine and were not changed by cimetidine. 2-(2-Aminoethyl)pyridine also increased microvascular permeability whereas impromidine did not. These results suggest that H₁-receptors and not H₂-receptors are involved in the permeability response to histamine in rat skin. In contrast, dimaprit increased microvascular permeability and responses to dimaprit exceeded the maximum response to histamine. The response to dimaprit proved to be independent of H₂ receptors and was consistent with an indirect response due to mast cell degranulation.     

山莨菪碱抗休克作用的细胞机制研究——对溶酶体稳定性的影响

山茛菪碱(654-2)可以稳定失血性休克大鼠的肝脏溶酶体、改善休克动物的预后,其效应与地塞米松相似。在体外实验中,山茛菪碱对分离出的休克大鼠肝溶酶体无直接作用,提示654-2在整体实验中显示出的保护肝脏溶酶体作用的机理与地塞米松不同。在离体肝灌流模型上,654-2对缺氧细胞有一定保护作用,提示山茛菪碱对溶酶体的稳定作用是其细胞保护的结果。     

大鼠软脑膜微血管通透性的计算机分析

本文应用微循环活体观察和荧光示踪技术 ,通过计算机数字图象分析对荧光素钠 (FiNa)在软脑膜微血管的通透过程进行了定量研究 ,以互成角度的两根血管为研究对象 ,建立了正常大鼠软脑膜和缺血大鼠软脑膜微血管对荧光素钠的通透方程 ,得到了不同缺血条件下的微血管通透速度方程及对通透速度评价的定量方法。通过对所得结果进一步分析 ,计算出两血管夹角与微血管通透性之间的关系 ,并代入不同缺血条件下大鼠软脑膜的通透曲线进行了验证。结果 :大鼠软脑膜微血管的通透方程成对数分布 ,通透速度成幂函数下降 ,一段时间后趋于稳定。以缺血 1h再灌注大鼠通透最为迅速 ,缺血 12h再灌注大鼠通透速度略快于正常大鼠。对于互成角度的两根血管 ,荧光物质的扩散速度与两血管间夹角存在一定关系。结论 :本方法能够较好地反映软脑膜微血管通透的实际情况 ,为进一步建立复杂结构血管的通透方程 ,以及定量评价微血管物质交换参数 ,提供了数学基础。     

油酸型呼吸窘迫综合征早期肺微血管通透性变化与前列腺素的关系

成人呼吸窘迫综合征以肺微血管通透性增加,肺水肿形成为特征。本实验发现用油酸复制的家兔呼吸窘迫综合征模型在30分钟内主要是油酸毒性直接作用引起的通透性增加,在这以后则以化学介质介导的间接损伤为主,而这种间接损伤与前列腺素类物质有关,特别是与血栓素和前列环素的比值有关。另外,实验还发现早期的直接损伤有部分在外源性前列环素作用下是可复性的。