Prostacyclin production in vitro by rabbit aortic endothelium: correction for unstirred diffusional layers.
Prostacyclin production in vitro by rabbit aortic endothelium: correction for unstirred diffusional layers.
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DOI：
10.1182/blood.v66.5.1047.bloodjournal6651047
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发表时间：
1985-11
期刊：
影响因子：
20.3
通讯作者：
E. Grabowski;G. Naus;B. Weksler
中科院分区：
文献类型：
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作者：
E. Grabowski;G. Naus;B. Weksler
紧邻内皮表面的流体层中的混合程度是体外培养的内皮细胞或完整血管内皮产生前列环素（PGI 2）的主要变量。缺乏充分的混合会导致低估真实的生产，因为在缓冲液收集时，紧邻内皮的PGI 2只能很好地采样。因此，切除38只新西兰白色家兔的胸主动脉，纵向打开，并将内皮侧最上方固定在缓冲液填充室中，该缓冲液填充室从研究中排除切割的组织边缘。通过放射免疫测定法（RIA）测定6-酮-PGF 1 α在未搅拌和磁力搅拌条件下的PGI 2生成。对于用罂粟碱和肝素联合预处理的动物（见下文），未刺激和花生四烯酸盐刺激的6-酮-PGF 1 α随搅拌速率分别向2.9和28.5 ng/cm 2/min的限度增加。例如，在650 rpm下测量的未刺激和刺激的6-酮PGF 1 α分别比0 rpm下的值大3.5倍（2 P小于0.01）和3.7倍（2 P小于0.001）。然而，血管切除的过程产生了另一个变量：由继发性血管收缩和凝血酶生成等因素引起的内皮损伤程度。通过在处死动物前使用平滑肌松弛剂和肝素，可以分别最大限度地减少血管收缩和凝血酶生成。因此，在处死前，根据静脉内（IV）处理对家兔进行分组，分别给予生理盐水、罂粟碱（4 mg/kg）、肝素（200 U/kg）或罂粟碱和肝素的组合（相同剂量）。与生理盐水、单独罂粟碱或单独肝素预处理相比，罂粟碱和生理盐水联合预处理导致刺激的6-keto-PGF 1 α增加1.6- 2.8倍。通过透射电子显微镜观察，用生理盐水预处理的动物的内皮细胞显示超微结构变化，包括细胞质破坏，大多数内皮细胞与内皮下分离而不分离，以及局部区域的剥脱。与此相反，内皮细胞的超微结构完整性保存在罂粟碱和肝素联合预处理的动物的动脉瘤。这些结果支持这一假设，即未搅拌的扩散层导致，在体外，低估PGI 2的生产，特别是当血管被保护免受切除损伤。（400字处截断摘要）
The degree of mixing in fluid layers immediately adjacent to the endothelial surface is a major variable in assessment of prostacyclin (PGI2) production by cultured endothelial cells or intact vessel endothelium in vitro. Lack of adequate mixing should lead to underestimation of true production because PGI2 immediately adjacent to endothelium would be only poorly sampled upon buffer collection. Thoracic aortas from 38 New Zealand white rabbits were therefore excised, opened longitudinally, and mounted endothelial side uppermost in a buffer-filled chamber which excluded cut tissue edges from study. Production of PGI2 under unstirred and magnetically stirred conditions was measured by radioimmunoassay (RIA) for 6-keto-PGF1 alpha. For animals pretreated with the combination of papaverine and heparin (see below), unstimulated and arachidonate-stimulated 6-keto-PGF1 alpha increased with stirring rate toward limits of 2.9 and 28.5 ng/cm2/min, respectively. Unstimulated and stimulated 6-keto PGF1 alpha measured at 650 rpm, for example, were greater than their values at 0 rpm by factors of 3.5 (2P less than .01) and 3.7 (2P less than .001), respectively. The process of vessel excision, however, produces another variable: degree of injury to endothelium caused by such factors as secondary vessel contraction and thrombin generation. Vessel contraction and thrombin generation can be minimized, respectively, by the use of a smooth muscle relaxant and heparin administered prior to killing of the animals. The rabbits were, therefore, grouped according to intravenous (IV) treatment, prior to killing, with saline, papaverine (4 mg/kg), heparin (200 U/kg) or the combination of papaverine and heparin (same doses). As compared with pretreatment with saline, papaverine alone, or heparin alone, pretreatment with the combination of papaverine and saline led to increases in stimulated 6-keto-PGF1 alpha of 1.6- to 2.8-fold. By transmission electron microscopy, endothelium from animals pretreated with saline showed ultrastructural changes, including disruption of cytoplasm, separation without detachment of most endothelial cells from subendothelium, and focal areas of denudation. In contrast, ultrastructural integrity of endothelium was preserved in aortas of animals pretreated with combined papaverine and heparin. These results support the hypothesis that unstirred diffusional layers lead, in vitro, to underestimation of PGI2 production, especially when vessels are protected from excisional injury.(ABSTRACT TRUNCATED AT 400 WORDS)