H2O2诱导SD大鼠视网膜细胞凋亡过程中细胞内钙离子浓度的变化
Alteration of intracellular calcium concentration in the process of H2O2-induced apoptosis of SD rat retinal cells

摘要：目的 观察H2O2诱导原代培养SD大鼠视网膜细胞凋亡过程中细胞内Ca2+浓度([Ca2+]i)的变化及其来源。方法 取1～3d内新生SD大鼠视网膜进行原代细胞培养，以100μmol/L H2O2作用0、2、4、8、12、24h，采用MTT法进行细胞活力检测，应用Hoechst 33342染色法进行细胞凋亡检测，以Fluo-3 AM为细胞内Ca2+探针并利用荧光激活细胞分类技术(FACS)进行[Ca2+]i检测，确定H2O2诱导细胞损伤及凋亡过程中[Ca2+]i的变化；应用细胞外Ca2+螯合剂EGTA初步检测H2O2诱导的[Ca2+]i变化是否源于细胞外Ca2+内流。结果 100μmol/L H2O2可诱导原代培养的SD大鼠视网膜细胞发生凋亡，且凋亡数目呈时间依赖性递增；100μmol/L H2O2作用2～24h均可显著降低细胞活力，而[Ca2+]i的升高出现在H2O2作用2h且维持至12h，然后逐渐下降，至24h恢复至正常水平；0.5～5mmol/L EGTA可显著削弱由100μmol/L H2O2作用2h导致的[Ca2+]i增加。结论 在H2O2诱导原代培养的SD大鼠视网膜细胞发生凋亡过程中，[Ca2+]i的升高发生在凋亡的早期阶段，而非细胞凋亡全过程中；H2O2的损伤作用所导致的[Ca2+]i升高部分来源于细胞外Ca2+的内流。
ABSTRACT: Objective To observe the alteration of intracellular Ca2+ concentration ([Ca2+]i) in the process of H2O2-induced apoptosis of retinal cells and to detect the source of [Ca2+]i on the in vitro model of primary cultured SD rat retinal cells. Methods In this study, we used primary cultured retinal cells as experimental model, detected the [Ca2+]i by FACS technique with Fluo-3 AM as a Ca2+ indicator. Cell viability was measured by MTT. Apoptosis was assayed by Hoechst 33342 staining. Besides, we used extracellular Ca2+ chelator EGTA to preliminarily determine whether the alteration of [Ca2+]i sourced from Ca2+ influx or not. Results H2O2 of 100μmol/L could induce the apoptosis of primary cultured SD rat retinal cells in a time-dependent manner. Cell viability of primary cultured retinal cells was reduced time dependently from 0h to 24h after 100μmol/L H2O2 stressed, but [Ca2+]i increased at 2h, sustained to 12h, and then recovered gradually. Increased [Ca2+]i caused by 100μmol/L H2O2 treatment for 2h significantly attenuated by 0.5-5mmol/L EGTA, which was dose-dependent. Conclusion [Ca2+]i increase occurred at the early stage of primary cultured SD rat retinal cells apoptosis induced by H2O2. The increase of [Ca2+]i？？sourced partly from extracellular Ca2+ influx