利用低氧／厌氧工作站建立H9c2细胞缺氧复氧模型的探讨

利用低氧／厌氧工作站建立H9c2细胞缺氧复氧模型的探讨储倩雯;李伟秋;鲁诗史;黄丹梅;张艳美

【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砄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8h后，细胞皱缩成圆形，大量坏死细胞漂浮，复氧1h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H9 c2细胞H／R模型。%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 for 1 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.

【期刊名称】《中国生化药物杂志》

【年(卷),期】2015(000)011

【总页数】4页(P11-14)

【关键词】低氧/厌氧工作站;H9 c2细胞;缺氧复氧模型

【作者】储倩雯;李伟秋;鲁诗史;黄丹梅;张艳美

【作者单位】汕头大学医学院药理教研室，广东汕头 515041;汕头大学医学院分析细胞实验室，广东汕头 515041;汕头大学医学院第一附属医院药剂科，广东汕头 515041;汕头大学医学院药理教研室，广东汕头 515041;汕头大学医学院药理教研室，广东汕头 515041

【正文语种】中文

【中图分类】R363

心肌缺血再灌注(ischemia/reperfusion， I/R)损伤是心脏缺血性疾病和心脏外科手术后常见的一种病理生理现象。体外心肌细胞缺氧复氧

(hypoxia/reoxygenation， H/R)模型一直被广泛应用于心脏I/R损伤的研究，对于探讨I/R损伤的分子机制具有重要的意义。但国内外建立的H/R细胞模型没有统一的标准，目前常用的方法主要有：混合气体培养法、模拟缺氧/再灌注液培养法、氮气饱和培养基法、液体石蜡覆盖法等单纯物理性模型[1]。而化学模型建立方法主要是利用氧清除剂二亚硫酸钠和氯化钴[2-3]。这些方法都存在一些缺点，如操作复杂，无法准确地控制缺氧过程的氧分压，缺氧条件的重复性差等。探寻一个稳定，可靠，便于操作的H/R损伤的细胞模型，将为研究心脏I/R损伤提供一个可靠的平台。来源于大鼠胚胎期心室的H9c2细胞株被用于本实验中，该细胞株保持着心肌细胞的多种特性，被广泛应用于心脏疾病的体外研究中。本研究利用新购置的低氧/厌氧工作站，结合不同的缺氧培养条件，以心肌I/R最重要的病理生理改变之一—活性氧(reactive oxygen species，ROS)[4]作为评判模型成功与否的标准，探讨建立H9c2细胞H/R模型的最佳方法。