大鼠肺动脉平滑肌细胞培养及对不同类型激动剂刺激产生的反应

Detection of intracellular calcium concentration on pulmonary artery smooth muscle cells on rats

目的分离和培养SD大鼠肺动脉平滑肌细胞（PASMCc），并检测其功能状态。方法显微分离肺内小动脉，并在含胶原酶（1750 U·mL^-1）和木瓜蛋白酶（9．5U·mL^-1）的低钙HBSS溶液中酶解和培养PASMCs，采用动态细胞荧光成像技术检测PASMCs胞浆游离Ca^2＋浓度（[Ca^2＋]i）的变化。结果在18～24h内可获得PASMCs，并可观察到环匹阿尼酸和5-HT可引起PASMCs[Ca^2＋]i的升高效应。结论大鼠PASMCs一步酶消化法，方法简便实用。所分离的PASMCs细胞形态和功能正常，适用于动态细胞荧光成像技术检测实验及PASMCs信号转导功能的研究。

OBJECTIVE To detect intracellular calcium concentration （ [Ca^2＋ ] i） in fresh culturing pulmonary artery smooth muscle cells（PASMCs） on rats. METHDOS The pulmonary arteries of rats were isolated on microscope and were digested with low-Ca^2＋ HBSS＇ s solution（37℃ ）, containing collagenase I （1750U· mL^-1） and papain （9.5U· mL^-1）. [Ca^2＋]i of PASMCs were measured using real-time cellular fluorescence imaging technique. RESULTS Enough PASMCs for experiments can been obtained within 18--24h, and the enhancement [Ca^2 ＋]i of PASMCs can been induced by both 10μmol·L^-1 cyelopiazonic acid and 10μmol·L^-1 5-HT. CONCLUSION This isolated and cultured method is convenient and useful. The PASMCs that the structure and function was normal could be applied to real-time cellular flourescence imaging technique experiment and the research of PASMCs signal transduction function.