摘要
目的建立子鼠肠上皮细胞(IEC)添加人工基底膜体外原代培养,模拟缺血缺氧损伤后胞内钙含量、膜脂流动性、IEC凋亡及采用钙通道阻断剂的影响。方法建立子鼠IEC分离与原代培养和缺血缺氧模拟环境,设立对照组(A组),缺氧组(B组),缺血组(C组),缺血缺氧组(D组)及加维拉珀米2mg/L的相应对照组。利用流式细胞仪(FCM)计算凋亡细胞率,激光共聚焦显微镜检测胞内钙含量;自旋标记-顺磁共振技术(ESR)检测膜脂流动性。结果B、C、D组IEC凋亡较A组明显增加(P<0.05);加用钙离子阻断剂后B1、C1、D1组较相应对照组减少(P<0.05)。缺血缺氧损伤导致胞内钙超载,尤以D组最显著,其次为B组,C组;加用钙通道阻断剂后细胞内钙含量下降,以D1组最明显(P<0.01),依次为B1组,C1组。损伤组膜蛋白构像改变、运动速度减慢,表现为强固定化与弱固定化组分谱线高度比值(hs/hw)明显增大及膜蛋白旋转相关时间(τc)显著延长,D组最为明显(P<0.01),其次为B组,C组;加用钙通道阻断剂后hs/hw和,τc有不同程度的恢复,与损伤组比较差异有统计学意义(P<0.05)。结论缺血缺氧损伤后,膜脂流动性发生变化及胞内钙超载和IEC凋亡一致;钙通道阻断剂可降低胞内钙含量,减轻膜蛋白的构像改变,减少细胞凋亡。
Objective To investigate the relationship between the apoptosis and intracellular calcium amount and the change of cellular membrane fluidity, and the influence of calcium antagonist. Methods Primary culture of rat intestinal epithelial cells （IECs） with construction of artificial basal membrane and ischeia and anoxia environment was established. IECs were divided into 8 groups： group A, control group; B,anoxia group; C,ischemia group; D,ischemia and anoxia group; A1 ,A ＋ Verapamil （2 mg/ L） ; B1 ,B ＋ Verapamil （2 mg/L） ; C1 ：C ＋ Verapamil （2mg/L） ; D1 ：D ＋ Verapamil （2 mg/L）. Apotosis rate （AR） was detected by FCM, intracellular calcium overload by LSCM, and cellular membrane fluidity by spin labeling-ESR spectra. Results The AR of IECs in groups B, C and D was much higher than that in group A （P〈0.01 ,P 〈0.05）.The AR in groups B1,C1 and D1 that were treated with calcium channel antagonist were lower than that in groups B, C and D. Ischemia and anoxia injury led to intracellular calcium overload,especially in group D （P 〈 0.01 ）. Calcium channel antagonist could markedly decrease the amount of intracellular calcium （P 〈 0.05 ）. The hs/hw ratio in groups B, C, D was increased significandy as compared with group A,so did the rotational correlation time （τc）. The increase in group D was the most significant （ P 〈 0.01 ） , and that in group B was more significant than in group C. Calcium channel antagonist could markedly decrease the hs/hw ratio and τc（ P 〈 0.05 ）. Conclusion Under the injury of anoxia and ischemia, the AR of IEC is increased, and calcium overload and cellular membrane fluidity markedly change. Calcium channel antagonist can decrease AR and intracellular calcium and improve the cellular membrane fluidity.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2007年第5期567-569,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(30271286
30471687)
上海市科委青年科技启明星跟踪计划项目(02QMB1406)
关键词
钙
肠
上皮细胞
缺血
缺氧
Calcium
Intestinal
Epithelial cells
Ischemia
Anoxia
