H_2O_2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 H_2O_2-induced apoptosis of SD rat retinal cells

作　　者：冯燕[1,2] 张波[1,3] 王少兰[1] 王宝英[1] 李红波[1] 杜芳英[1] 俞小瑞[1] 

机构地区：[1]西安交通大学医学部遗传与分子生物学系,陕西西安710061 [2]西安交通大学附属红会医院骨质疏松科,陕西西安710054 [3]西安交通大学附属3201医院骨科,陕西汉中723000

出　　处：《西安交通大学学报（医学版）》2015年第5期580-586,共7页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.81271013);国家教育部高等学校博士点专项科研基金资助项目(No.20120201110051)~~

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-3AM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。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＋]ion 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＋]iby 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＋]isourced 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 from0 h to 24 h after 100μmol/L H2O2 stressed,but[Ca2＋]iincreased at 2 h,sustained to 12 h,and then recovered gradually.Increased[Ca2＋]icaused by 100μmol/L H2O2 treatment for 2 h significantly attenuated by 0.5-5 mmol/L EGTA,which was dose-dependent.Conclusion[Ca2＋]iincrease occurred at the early stage of primary cultured SD rat retinal cells apoptosis induced by H2O2.The increase of[Ca2＋]isourced partly from extracellular Ca2＋influx.

关 键 词：[CA2+]I H2O2 SD大鼠 视网膜细胞 凋亡 

分 类 号：R774.1[医药卫生—眼科]