摘要
目的:观察不同浓度的氯化钴(CoCl2)对H9C2心肌细胞的影响及其诱导化学性缺氧的最佳浓度和时间。方法:用100、300、600、900、1200μmol/L的CoCI2处理H9C2心肌细胞,建立化学性缺氧诱导心肌细胞损伤的实验模型,分别于12、18、24、36、48h用Hoechst33342细胞核染色法检测心肌细胞凋亡:CCK-8试剂盒检测心肌细胞存活率;确定最佳诱导浓度600μmol/L后,分别于0、3、6、9、12、18、24、36、48h采用蛋白印迹法检测缺氧诱导因子1α(HIF-1α)蛋白的表达;确定最佳诱导时间后,用蛋白印迹法检测100、300、600、900、1200μmol/LCoCl2处理H9C2心肌细胞12h后HIF-1α蛋白的表达情况。结果:在600~1200μmo1/L浓度范围内,CoCl2呈剂量依赖性地抑制H9C2心肌细胞的存活。600μmol/LCoCl2诱导12h后H9C2心肌细胞产生明显凋亡.诱导12~48h范围内.12h时H9C2心肌细胞表达的HIF-1α蛋白最高:100~1200μmoI/LCoCl2诱导H9C2心肌细胞12h.其中600μmol/LCoCl2诱导时H9C2心肌细胞表达的HIF-α蛋白最高。结论:CoCl2诱导H9C2心肌细胞化学性缺氧的最佳浓度为600μmol/L.此时最佳时间为12h。
Objective: To investigate the effect of different concentrations of Cobalt chloride (COC12) on myocardial H9C2 cells and the optimum concentration and time to induce chemical hypoxia in H9C2 cells. Methods: Cobalt chloride was used to induce the chemical hypoxia injury model in H9C2 cells at different concentrations from 100 to 1 200 μmol/L. Apoptotic changes in H9C2 cells were observed by using Hoechst 33342 staining and photofluorography at 12, 18, 24, 36, 48 h; myocardial cell viability was detected by the CCK-8 assay kit; HIF-lα (hypoxia-indueible factor 1alpha) protein was detected by Western blot at the optimum concentration (600 μmol/L) and different time interval from 0-48 h . Cobalt chloride induced HIF-lα protein was detected by Western blot to define the optimum time at different concentrations of CoC12 from 100-1 200 μmol/L. Results: At the concentration from 600 to 1 200 μmol/L, COC12 dose-dependently inhibited the cell viability in H9C2 cells. Twelve hours COC12 treatment significantly induced H9C2 cell apoptosis at the concentration of 600 μmol/L. HIF-lα protein expression was the highest at 12 h with the concentration of 600 μmol/L. Conclusions :The optimum concentration of COC12 for inducing chemical hypoxia in H9C2 cells is 600 μmol/L while duration is 12 hours.
出处
《诊断学理论与实践》
2013年第5期532-536,共5页
Journal of Diagnostics Concepts & Practice
基金
上海市科技人才计划项目(11XD1403700)
瑞金医院学科带头人攀登计划