Abstract: Abstract: Objective To explore the effect and source of 15-KETE on the cytosolic ［Ca^{2 +}］i in rat pulmonary arterial smooth cells( PASMCs) ． Method PASMCs obtained with enzyme ( collagenase I plus elastase) dispersing method were cultured in the first passage in vitro． Cells with concentration of 2 × 10 ^{－ 5} cells /mL were seeded onto a glass coverslip in 6 well plate，and cultured for 24 h in the incubator at 37℃． After cell adherence，the glass coverslip was taken out from 6 well plate，placed in special-made chamber，and washed with D-Hanks solution for 3 times． Cells were loaded with Fluo-3 /AM ( final concentration 10μmol /L) ，followed in the incubator at 37℃ for 30 min，then washed with D-Hanks solution for 3 times，extracellular dye was washed out，the Fluo-3 /AM loaded cells were available for ［Ca^{2 +}］i measurement within 2h． Using laser-scanning confocal microscope technique，the effect of 15-KETE on cytosolic ［Ca^{2 +}］i in rat PASMCs was measured． Ｒesult ( 1) 15-KETE ( 1 × 10 － 8 ～ 1 × 10 － 6 mol /L) increased cytosolic ［Ca^{2 +}］i of rat PASMCs in a concentration-dependent manner． ( 2) The L-type Ca^{2 +} channel blocker verapamil ( 10 － 6 mol /L) and Ca^{2 +} -free Krebs solution significantly inhibited the increase of cytosolic ［Ca^{2 +}］i induced by 15-KETE． Conclusion 15-KETE may increase cytosolic ［Ca^{2 +}］i of rat PASMCs， and the source of Ca^{2 +} was from intercellular solution．

Key words: 15-ketoeicosatetretraenoic acid, pulmonary arterial smooth cells, ［Ca^{2 +}］i

摘要： 摘要: 目的探讨15-KETE 对大鼠肺动脉平滑肌细胞内Ca2 + 的作用及其来源。方法以酶法( 胶原酶Ⅰ型和弹性 酶) 分离培养原代大鼠肺动脉平滑肌细胞，将细胞稀释所需密度( 2 × 105 个/mL) ，加样于6 孔板中的盖玻片上，放 入37℃孵箱中培养12 h，细胞贴壁后，取出6 孔板中的盖玻片，放入特制的小槽内，D-Hanks 液冲洗细胞3 次，加入 1 × 10 － 5mol /L 的Flou-3 /AM，置37℃孵箱中避光孵育约30 min，用D-Hanks 液洗去细胞外残留染料，应用激光扫描 共聚焦显微镜技术，测定了15-KETE 对大鼠肺动脉平滑肌细胞游离钙离子浓度的影响。结果1) 15-KETE ( 1 × 10 － 8—1 × 10 － 6 ) mol /L 可依赖性引起肺动脉平滑肌细胞内钙离子浓度( ［Ca2 +］i) 增加; 2) 1 × 10 － 6 mol /L 维拉米( L-型钙离子通道阻断剂) 和无钙离子细胞外液显著阻抑了1 × 10 － 6 mol /L 15-KETE 引起肺动脉平滑肌［Ca2 +］i 增 加。结论15-KETE 可引起大鼠肺动脉平滑肌［Ca2 +］i 增加，并且此钙来源于细胞外液钙离子。

关键词: 15-酮基二十碳四烯酸, 肺动脉平滑肌细胞, 细胞内Ca2 + 浓度