滨蒿内酯对原代和传代培养豚鼠气道平滑肌细胞内钙的影响

Effect of scoparone on intracellular calcium concentration of guinea pig airway smooth muscle cells in primary culture and subculture

目的探讨滨蒿内酯(scoparone,Scop)对原代及短期传代培养的豚鼠气道平滑肌细胞(airway smooth musclecells,ASMCs)内钙的影响,同时,比较原代与传代ASMCs在形态、生长曲线及内钙释放受Scop及caffeine影响的异同。方法细胞计数法绘制原代及传代培养ASMCs的生长曲线,应用Fluo-3/AM为细胞内Ca2+示踪剂,通过倒置荧光纤维镜观察和记录原代及短期传代培养的ASMCs的细胞形态及其细胞内钙离子浓度([Ca2+]i)的改变。结果原代和传代培养ASMCs的倍增时间分别为(31.89±1.24)h和(22.91±6.82)h,传代培养ASMCs的倍增时间明显缩短(P<0.05),传代培养的ASMCs相对原代培养ASMCs体积增大。在细胞外液无钙条件下,不同浓度的Scop(10-6、10-5、10-4mol.L-1)可降低静息状态下培养的ASMCs的[Ca2+]i,并与给药浓度有关,原代与传代培养的ASMCs比较,对不同浓度的Scop的降钙反应无明显异同(P>0.05);不同浓度咖啡因(caffeine,10-4、10-3、10-2mol.L-1)在10-4mol.L-1Scop存在下,可升高ASMCs的[Ca2+]i,传代培养的ASMCs[Ca2+]i较原代对caffeine的反应下降(P<0.01)。结论Scop可降低培养的ASMCs的[Ca2+]i,并且不受细胞传代影响。短期传代培养的ASMCs相对于原代细胞,形态及内钙释放通道特性发生了改变。

Aim To determine the effects of scop- arone （Scop） on （[Ca^2＋ ]i） of guinea pig airway smooth muscle cells （ASMCs） in primary culture and subculture, and to compare the differences between primary and subcultured ASMCs in cell shape, growth curve, and the reactions to caffeine and Scop. Methods Cell growth curves of ASMCs in primary culture and subculture were obtained by cell counting. ASMCs in primary culture and subculture were loaded with the fluorescent calcium indicator Fluo-3/AM and changes of [ Ca^2＋] i were monitored using an inverted fluorescence microscope system. Results The doubling time of ASMCs in primary and subcultured were （31.89 ± 1.24） h and （22.91±6.82） h respectively. The shape of ASMCs in subculture became large and flat, and the doubling time of subcuhured ASMCs was shorter than that of the primary cultures （P 〈 0.05）. In extracellular Ca^2＋-free condition, the resting [ Ca^2＋ ]i of ASMCs could be reduced by Scop （ 10^-6, 10^-5, 10^-4 mol · L^-1） in a concentration-dependent manner and there was no significant difference between the primary and subcuhured ASMCs （P 〉 0.05 ）. Caffeine （ 10-4,10 -3,10-2 mol · L^-1 ） raised the [ Ca^2＋ ] i of ASMCs in the presence of 10^-4 mol · L^-1 Scop and the reaction to eaffeine was lower in subcuhured ASMCs than in primary ASMCs （ P 〈 0.01 ）. Conclusion The effect of redueed [ Ca^2＋ ]i levels of Seop in cultured ASMCs remains unchanged after further cell passages. The eell shape and character of calcium releasing pathway of ASMCs in subeuhured have altered compared with the primary cultures.