Pfister Sandra L, Hughes Miranda J, Rosolowsky Mark, Campbell William B
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee 53226, USA.
Prostaglandins Other Lipid Mediat. 2002 Sep;70(1-2):39-49. doi: 10.1016/s0090-6980(02)00009-6.
Previous studies suggested that cultured human endothelial cells metabolize arachidonic acid to thromboxane A2. When primary cultures of human umbilical vein endothelial cells were incubated with 14C-arachidonic acid and the 14C-metabolites resolved by reverse phase high pressure liquid chromatography, radioactive products were observed that comigrated with 6-keto-prostaglandin F1alpha and thromboxane B2, the degradation products of prostacyclin and thromboxane A2, respectively. Since platelets synthesize thromboxane A2, the present study examined the hypothesis that adherent platelets may contaminate the primary cultures of human umbilical vein endothelial cells and be responsible for thromboxane B2 production. Confluent primary cultures or passaged cells were stimulated with histamine (10(-5) M). Incubation buffer was analyzed by specific radioimmunoassays for 6-keto-prostaglandin F1alpha and thromboxane B2. The production of thromboxane B2 decreased in the passaged cells (207 +/- 44 pg/ml versus 65 +/- 12 pg/ml; primary versus passaged cells). A moderate decrease in the yield of 6-keto-prostaglandin F1alpha was measured in the passaged cells compared to the primary cultures (3159 +/- 356 pg/ml versus 1678 +/- 224 pg/ml, primary versus passaged cells). If the primary cultures were incubated with human platelet-rich plasma for 30 min prior to stimulation with histamine, the amount of thromboxane B2 increased approximately 10-fold. In an additional experiment, sub-confluent primary cells were incubated with platelet-rich plasma for 30 min, washed to remove non-adherent platelets, and allowed to reach confluency. Confluent cells were then passaged and stimulated with histamine. The amount of thromboxane B2 was not significantly different from that obtained with passaged cells that had not been incubated with platelet-rich plasma during the primary culture (83 +/- 15 pg/ml versus 65 +/- 12 pg/ml, respectively). If the cyclooxygenase inhibitor indomethacin was included in the incubations, the amounts of both thromboxane B2 and 6-keto-prostaglandin F1alpha decreased. In contrast, the thromboxane A2 synthase inhibitor dazoxiben blocked thromboxane production and had no effect on the amount of 6-keto-prostaglandin F1alpha. Light microscopy revealed the presence of adherent platelets in primary cultures with and without platelet-rich plasma but no platelets were observed in any group of passaged cells. Histofluorescence for platelet serotonin indicated the presence of platelets only in primary cultures of human umbilical vein endothelial cells or in cultures pre-incubated with platelet-rich plasma. These studies suggest that primary cultures of human umbilical vein endothelial cells contain adherent platelets that contribute to thromboxane synthesis.
先前的研究表明，培养的人内皮细胞可将花生四烯酸代谢为血栓素A2。当用人脐静脉内皮细胞的原代培养物与14C-花生四烯酸一起孵育，并通过反相高压液相色谱法分离14C-代谢产物时，观察到放射性产物，其迁移位置与6-酮-前列腺素F1α和血栓素B2相同，分别是前列环素和血栓素A2的降解产物。由于血小板可合成血栓素A2，因此本研究检验了以下假设：贴壁血小板可能会污染人脐静脉内皮细胞的原代培养物，并导致血栓素B2的产生。用组胺（10(-5)M）刺激汇合的原代培养物或传代细胞。通过特异性放射免疫测定法分析孵育缓冲液中的6-酮-前列腺素F1α和血栓素B2。传代细胞中血栓素B2的产生量减少（原代细胞与传代细胞分别为207±44 pg/ml对65±12 pg/ml）。与原代培养物相比，传代细胞中6-酮-前列腺素F1α的产量适度下降（原代细胞与传代细胞分别为3159±356 pg/ml对1678±224 pg/ml）。如果原代培养物在用组胺刺激前与人富含血小板血浆孵育30分钟，则血栓素B2的量增加约10倍。在另一项实验中，将亚汇合的原代细胞与人富含血小板血浆孵育30分钟，洗涤以去除未贴壁的血小板，然后使其达到汇合状态。然后将汇合细胞传代并用组胺刺激。血栓素B2的量与原代培养期间未与人富含血小板血浆孵育的传代细胞所获得的量无显著差异（分别为83±15 pg/ml对65±12 pg/ml）。如果在孵育中加入环氧化酶抑制剂吲哚美辛，则血栓素B2和6-酮-前列腺素F1α的量均减少。相反，血栓素A2合酶抑制剂达唑氧苯可阻断血栓素的产生，并且对6-酮-前列腺素F1α的量没有影响。光学显微镜检查显示，无论有无富含血小板血浆，原代培养物中均存在贴壁血小板，但在任何传代细胞组中均未观察到血小板。血小板5-羟色胺的组织荧光表明，仅在人脐静脉内皮细胞的原代培养物中或在与富含血小板血浆预孵育的培养物中存在血小板。这些研究表明，人脐静脉内皮细胞的原代培养物中含有有助于血栓素合成的贴壁血小板。
