氯化钴诱导心肌细胞化学性缺氧HIF-1α表达的研究

Study on expression of HIF-1α in myocardial cells with CoCl2 induced chemical hypoxia

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.