【摘要】 目的:分离大肺血管内皮细胞,建立研究ACE表达调控的体外模型。方法:分离大鼠肺血管内皮细胞采用活体胶原酶灌注法;用胰酶不完全消化和无血清培养相结合的方法得到纯化的内皮细胞;用定量PCR技术检测血管紧张素转换酶(ACE)mRNA水平的变化,速率法测定ACE酶活性。结果:传代细胞第四代后细胞增殖速度趋于稳定,内皮细胞纯度达到95%以上,ACE的活性及mRNA的表达也处于稳定期。表皮生长因子(EGF)可以使ACEmRNA表达受到抑制(P=0.025)。冻存后存代复苏率(0.816±0.085)和贴壁率(0.758±0.079)均较高,生长良好。结论:本法分离到的大鼠肺血管内皮细胞,纯度高,ACE活性及其mRNA表达水平在第四代后稳定,以作为进一步研究ACE调控的良好的体外模型更多还原

【Abstract】 Objective:To establish the model of regulation of ACE expression in vitro using isolated using rats pulmonary vessel endothelial cells (RPVEC).Methods:The RPVEC was isolated by collagenase infusion in vivo and the purified endothelial cells were obtained by typsin incomplete digestion combined with cuhure in serum free medium.The level of ACE mRNA was measured by quantitative PCR and the activity of angiotensin converting enzyme (ACE) was determined by kinetics PCR.Results:The stabilization of RPVEC was obtained after four or five subcultures and the purity of RPVEC was over 95%.The actiyity of ACE and the level of ACE mRNA expression were stabilized as well.The epidermal growth factor (EGF) might inhibit the expression of ACE mRNA ( P =0.025).The rate of resuscitation (0.816±0.085) and the rate of reattachment (0.758±0.079) after refrigeration were relatively high and the cells grew well.Conclusions:The RPVEC have high purity and activity.The activity of ACE and the level of ACE mRNA were stabilized after subcultures.The RPVEC can be used as models in vitro for further study of ACE regulation.更多还原