【摘要】 目的利用低氧/厌氧工作站,结合不同的缺氧条件,探讨建立H9c2细胞缺氧复氧(hypoxia/reoxygenation,H/R)模型的最佳方法。方法缺氧时将H9c2细胞置于低氧/厌氧工作站中,分别以完全培养基,无糖培养基和酸性缺氧液培养1、2、4、6、8 h,缺氧后换用完全培养基于培养箱中按常规条件培养1 h。流式细胞仪测定细胞内活性氧(reactive oxygen species,ROS)含量,噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞生存率,倒置显微镜下观察H9c2细胞形态变化。结果与对照组比较,缺氧时以完全培养基和无糖培养基处理的H9c2细胞H/R后,细胞内ROS含量和细胞存活率随缺氧时间延长无明显改变,且倒置显微镜下未观察到明显的形态变化。缺氧时以酸性缺氧液处理的H9c2细胞,随H/R时程的延长,细胞内ROS含量不断增加(P<0.01),且细胞存活率逐渐降低(P<0.01),倒置显微镜下观察到细胞H∶1 h/R∶1 h后开始出现部分细胞皱缩,少量坏死细胞漂浮,随时间延长损伤加重;缺氧8 h后,细胞皱缩成圆形,大量坏死细胞漂浮,复氧1 h后可见细胞膜表面不平滑,复氧液中仍有少量坏死细胞漂浮。结论采用低氧/厌氧工作站,并结合酸性缺氧液,可成功建立重现性非常好的H9c2细胞H/R模型。更多还原

【Abstract】 Objective To investigate optimal method of establishing hypoxia/reoxygenation( H/R) model of H9c2 cell by using hypoxia/anoxic workstation under different conditions in hypoxia. Methods H9c2 cell was placed into hypoxia / anoxic workstation and simultaneously cultured with complete medium,glucose-free DMEM and acidic hypoxic solution for 1,2,4,6 and 8 h respectively,and then reoxygenated with complete medium for1 h in normoxic incubator. The level of ROS was measured by flow cytometry. The cell viability was detected by MTT assay. The cellular morphology was observed by inverted microscope. Results With the extension of cell hypoxia time,there were no significant differences in the ROS level and cell viability in complete medium- and glucose-free DMEM-treated H / R groups compared with control group( P < 0. 05). There was no obvious morphologic change observed with inverted microscope,either. Nevertheless,when H9c2 cells were treated with acidic hypoxic solution in hypoxia,the ROS level continuously increased and the cell viability decreased with the extension of cell hypoxia time( P < 0. 01). Since H ∶1 h / R ∶1 h,some of the cells shrunk and a few necrotic cells floated in the media under the inverted microscope,and the damage was aggravated with the extension of hypoxia time.After the cells were exposed in hypoxia for 8 h,they wrinkled to be round and a large number of floating necrotic cells were observed. When the cells were reoxygenated for 1 h,the cytomembrane was not smooth and there were still a few necrotic cells floating in culture dish. Conclusion The H9c2 cell H / R model with good repeatability can be established successfully by using hypoxia / anoxic workstation combining with acidic hypoxic solution.更多还原