Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells.
Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells.
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DOI：
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发表时间：
1999-04
影响因子：
4.4
通讯作者：
J. Cai;M. Wu;K. C. Nelson;P. Sternberg;D. P. Jones
中科院分区：
文献类型：
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作者：
J. Cai;M. Wu;K. C. Nelson;P. Sternberg;D. P. Jones
目的探讨氧化剂诱导培养的人视网膜色素上皮(HRPE)细胞死亡机制。方法用不同浓度的化学氧化剂叔丁基氢过氧化氢(TBH)处理培养的hRPE细胞不同时间。用末端脱氧核苷酸转移酶(TdT)介导的dUTP缺口末端标记法和流式细胞仪检测细胞的凋亡率。用罗丹明123染色和随后的流式细胞仪检测线粒体膜电位(mt-psi)。Western印迹法检测线粒体细胞色素c(Cytc)的释放及原天冬氨酸氨基转移酶3(Proaspase 3)和半胱氨酸天冬氨酸氨基转移酶(Caspase)底物的裂解。结果叔丁基氢过氧化氢可引起hRPE细胞凋亡的时间和剂量依赖性激活，表现为特征性的形态学改变；TUNEL阳性标记；磷脂酰丝氨酸(PS)暴露；Proaspase3、聚(ADP-核糖)聚合酶、层蛋白和微管蛋白的裂解。在半胱氨酸天冬氨酸氨基转移酶激活前，线粒体psi早期下降，线粒体cytc释放。结论：TBH可诱导hRPE细胞凋亡，其机制可能是通过触发线粒体通透性转换，导致线粒体膜间蛋白的肿胀和释放。
PURPOSE To determine the mechanism of oxidant-induced cell death in cultured human retinal pigment epithelium (hRPE). METHODS Cultured hRPE cells were treated with different concentrations of a chemical oxidant, t-butylhydroperoxide (tBH), for different periods of time. Apoptosis was determined with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Mitochondrial membrane potential (mtdelta psi) was measured by rhodamine 123 staining and subsequent flow cytometry. Release of mitochondrial cytochrome c (cyt c) and cleavage of procaspase 3 and caspase substrates were determined by western blot analysis. RESULTS t-Butylhydroperoxide caused time- and dose-dependent activation of apoptosis in hRPE, indicated by characteristic morphologic changes; TUNEL-positive labeling; phosphatidylserine (PS) exposure; and procaspase 3, poly(ADP-ribose)polymerase, lamin, and tubulin cleavage. An early decrease of mtdelta psi was observed before caspase activation, together with the release of mitochondrial cyt c. CONCLUSIONS Results indicate that tBH can induce apoptosis in hRPE, probably by triggering the mitochondrial permeability transition, which results in swelling and release of mitochondrial intermembrane proteins.