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)1×10-6mol/L维拉帕米(L-型钙离子通道阻断剂)和无钙离子细胞外液显著阻抑了1×10-6mol/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。
Objective To explore the effect and source of 15-KETE on the cytosolic [Ca2＋] 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 [Ca2＋]i measurement within 2h.Using laser-scanning confocal microscope technique,the effect of 15-KETE on cytosolic [Ca2＋] i in rat PASMCs was measured.Result （1） 15-KETE （1 × 10-8 ～ 1 ×10-6 mol/L） increased cytosolic [Ca2＋]i of rat PASMCs in a concentration-dependent manner.（2） The L-type Ca2＋ channel blocker verapamil （10-6 mol/L） and Ca2＋-free Krebs solution significantly inhibited the increase of cytosolic [Ca2 ＋] i induced by 15-KETE.Conclusion 15-KETE may increase cytosolic [Ca2 ＋] i of rat PASMCs,and the source of Ca2＋ was from intercellular solution.
出处
《实验动物科学》
2013年第5期28-32,共5页
Laboratory Animal Science
基金
黑龙江省科技攻关项目(No.PC09S03)
