摘要
目的:利用高内涵筛选(high-content screening,HCS)、流式细胞术(flow cytometry,FCM)、激光扫描细胞分析术(laser scanning cytometry,LSC)等细胞组学技术对心肌细胞凋亡特征进行研究。方法:缺氧缓冲液处理H9C2心肌细胞,建立体外心肌细胞凋亡模型,以常规DMEM培养为对照组,处理6h后收取细胞行Annexin V-FITC、PI染色,分别利用HCS和FCM检测,分析心肌细胞早、晚期凋亡以及坏死的比例。利用小鼠心脏左前降支结扎,建立心肌梗死在体模型,术后28d收取心脏,进行TUNEL染色,利用HCS和LSC分析心脏组织健存区、交界区和梗死区心肌细胞的凋亡。结果:缺氧缓冲液处理H9C2心肌细胞6h后,HCS和FCM方法均显示早期凋亡细胞缺氧组与DMEM组,差异有统计学意义,分别为[(17.69±2.85)vs.(5.31±0.86)%,P<0.01]和[(12.18±0.61)vs.(3.08±0.15)%,P<0.01],晚期凋亡与坏死细胞比例变化不明显,表明缺氧缓冲液处理6h主要诱导心肌细胞早期凋亡。小鼠心肌梗死28d后,HCS和LSC方法均显示心脏梗死区和交界区有凋亡细胞,梗死区更明显,整体凋亡率分别为6.08%和4.35%,健存区无细胞凋亡。结论:HCS和FCM方法能够可靠、稳定和量化评价心肌细胞凋亡,准确区分细胞早、晚期凋亡和坏死,两种方法结果一致;HCS和LSC方法还可分析组织水平的细胞凋亡;而LSC优势在于可明确凋亡细胞的组织定位。
Objective：To study of myocardial cell apoptosis cytomics technology was used, including high-content screening （ HCS）, flow cytometry （FCM）, laser scanning cytometry （LSC）. Methods： To estab- lish the model of myocardial cell apoptosis in vitro, H9C2 cell line was treated with hypoxia buffer for 6 hours. The DMEM treatment was as control group. The cells were stained with Annexin V-FITC and PI, and then ana- lyzed for the ratio of early and late apoptosis and necrosis using HCS and FCM. Myocardial infraction was per- formed in mice by ligation of left anterior descending branch. After 28 days, heart sections were performed with TUNEL assay. The myocardium apoptosis was evaluated with HCS and LSC in the normal, border and infracted area of the hearts. Results ： After 6 hours of hypoxia buffer treatment, HCS method displayed the ratio of early apoptotic cells （ Anenxin V ＋ PI - ） significantly higher in hypoxia group than DMEM group [ （ 17.69 ± 2. 85 ） vs. （ 5.31 ± 0. 86） %, P 〈 0. 01 ] , but there was no difference in the ratio of late apoptotic （ Anenxin V ＋ PI ＋ ） and necrotic （ Anenxin V-PI ＋ ） cell between the two groups. Moreover, FCM methods revealed the same trend in cell apoptosis. The percentage of early apoptosis significantly increased in hypoxia group compared in DMEM group [ （ 12. 18 -±0. 61） vs. （3.08 ±0. 15）%, P 〈0. 01]. There was no difference in the ratio of late apoptotic and necrotic cells. After 28 days of myocardial infarction, both HCS and LSC methods showed thatthere were apoptotic cells in the border and infracted zone of the hearts. The percentage of the apoptotic cells were 6. 08% （HCS） and 4. 35% （LSC） respectively. Conclusion： HCS and FCM methods are reliable, sta- ble and consistent in assessment of myocardial cell apoptosis. Two methods are able to quantify and distinguish the process of the apoptosis. LSC and HCS methods are applicable at the tissue sections. LSC may clearly local- ized apoptotic cells in tissues.
出处
《心肺血管病杂志》
CAS
2013年第6期768-772,共5页
Journal of Cardiovascular and Pulmonary Diseases
基金
国家自然科学基金资助项目(No:81230006
81000069)
北京市自然科学基金(No:2010B031
7132043)
