模拟缺血/再灌注诱导人离体胃黏膜上皮细胞的凋亡

目的模拟缺血/再灌注诱导人离体的胃黏膜上皮细胞损伤模型,观察再灌注不同时间点胃黏膜上皮细胞凋亡情况并分析其规律。方法采用人胃黏膜上皮细胞株进行体外培养并传代,细胞以缺氧（5%CO2、1%O2、94%N2）＋无糖KR液模拟缺血、缺氧,复氧、复糖模拟再灌注诱导胃黏膜上皮细胞凋亡,用流式细胞仪、Ho-echst33258染色法观察正常对照组及模拟损伤组于模拟缺血1 h,再灌注3、6、12 h细胞凋亡的情况及变化规律。结果①胃黏膜上皮细胞12～24 h贴壁,24 h后有少量上皮样细胞从周边爬出,48 h后细胞呈指数样生长,72 h后细胞已基本能达到融合。②模拟胃缺血/再灌注后细胞凋亡率于缺血1 h、再灌注6 h时出现高峰,且与正常对照组相比差异有统计学意义（P〈0.01）。经Hoechst33258染色,荧光显微镜下观察可见细胞于缺血1 h、再灌注6h时产生典型的凋亡形态学变化如核浓染、核碎裂、边集等。结论成功模拟了缺血/再灌注诱导人离体的胃黏膜上皮细胞损伤模型;该损伤模型可引起胃黏膜上皮细胞的凋亡,且在缺血1 h、再灌注6 h时为高峰。

Simulation of ischemia/reperfusion-induced apoptosis of human gastric mucosal epithelial cells cultured in vitro

Objective To simulate the ischemia/reperfusion-induced injury to the epithelial cells of gastric mucosa and to investigate the cellular apoptosis at different time points in order to explore its regularity.Methods Human gastric epithelial cell line was cultured and subculture in vitro.The cells were subjected to hypoxia（5% CO2,1% O2 and 94% N2） ＋ glucose-free KR bicarbonate buffer and succedent reperfusion（5%CO2 and 95% air） in DMEM/F12 to simulate conditions of ischemia and hypoxia,reoxygenation and glucose reintroduction.The morphological changes and the percentage of apoptotic cells were evaluated by Hoechast33258 staining,flow cytometry（FCM） at reperfusive 3,6 and 12 h secondary to 1 h simulated ischemia culture.Results ①Observation under the inverted microscope revealed the gastric mucosa epithelial cell adherence after 12 to 24 h;and at 24th h,there was outgrowth of epithelial cells;at 48th h the cells were in exponential growth;and at 72nd h the cultured cells reached nearly confluence,displaying the typical cobblestone appearance.②FCM analysis revealed that the percentage of cell apoptosis peaked at simulated ischemic 1 h and reperfusive 6 h,with significant difference（P0.01） compared with control group.Hoechst33258 staining and fluorescent microscopy revealed typical morphological changes of apoptotic cells including hyperchromatic nuclei,karyorrhexis,margination,etc.at ischemic 1 h and reperfusive 6 h.Conclusion The ischemia/reperfusion-induced apoptosis of human gastric mucosal epithelial cells cultured in vitro was successfully simulated.This injury model is able to induce apoptosis of gastric mucosal epithelial cells with a peak at the time point of reperfusive 6th h secondary to ischemic 1st h.