Density-dependent resistance to apoptosis in retinal cells.
Density-dependent resistance to apoptosis in retinal cells.
复制标题
视网膜细胞对细胞凋亡的密度依赖性抵抗。
DOI：
10.1076/ceyr.19.5.377.5293
复制
发表时间：
1999
影响因子：
2
通讯作者：
Wax,MB
中科院分区：
文献类型：
--
作者：
Tezel,GM;Seigel,GM;Wax,MB
目的研究细胞密度对视网膜细胞存活的影响。方法采用模拟缺血、兴奋毒性、抗热休克蛋白27 （hsp27）抗体等多种刺激方法，分别以不同密度或不同密度培养的视网膜细胞诱导光性细胞死亡。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记技术和流式细胞术对凋亡细胞进行定量分析。免疫细胞化学检测细胞骨架，phalloidin和DNase i对肌动蛋白进行特异性染色。western blotting检测细胞骨架蛋白、bcl-2家族蛋白和hsp27蛋白的变化。结果TUNEL技术和流式细胞术分析表明，在低密度培养下，细胞在凋亡刺激下的凋亡率较高。细胞骨架形态学检查和western blotting均显示，在各种应激源孵育后，与高密度培养相比，低密度培养中肌动蛋白和微管蛋白的降解更为突出。在基础条件下，低密度培养中bcl-2和bcl-xL的表达量高于高密度培养，bax的表达量低于高密度培养。与应激源孵育后，bcl-2和bcl-xL在低密度和高密度培养中表达降低，bax表达升高。然而，我们观察到，在存在或不存在凋亡刺激的情况下，高密度培养中hsp27的表达高于低密度培养。结论视网膜细胞在高密度培养中具有较强的抗凋亡能力，不受诱导因子的影响。这可能部分是由于内源性hsp27在更高密度的细胞中具有保护活性，这有助于在细胞凋亡刺激下保持细胞骨架的完整性。
PURPOSETo study effects of cell density on retinal cell survival.METHODSApoptotic cell death was induced in cultured retinal cells seeded at higher or lower density by various stimuli including simulated ischemia, excitotoxicity and antibody against heat shock protein 27 (hsp27). Quantitative analysis of apoptotic cells was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique and flow cytometry. Cytoskeleton was examined using immunocytochemistry and specific staining of actin by phalloidin and DNase I. In addition, alterations in the cytoskeletal proteins, bcl-2 family of proteins and hsp27 were studied using western blotting.RESULTSIncubation of the cells under apoptotic stimuli caused higher rates of apoptosis in lower density cultures as determined by TUNEL technique and flow cytometric analysis. Both morphologic examination of cytoskeleton and western blotting revealed that after incubation with various stressors, degradation of actin and tubulin was more prominent in lower density cultures compared to higher density cultures. The expression of bcl-2 and bcl-xL was higher and the expression of bax was lower in lower density cultures compared to higher density cultures at basal condition. After incubation with stressors, bcl-2 and bcl-xL expressions decreased and bax expression increased in both lower and higher density cultures. However, we observed that the expression of hsp27 was higher in higher density cultures than in lower density cultures in the presence or absence of apoptotic stimuli.CONCLUSIONSThese findings demonstrate that retinal cells are more resistant to apoptosis in higher density cultures, independent of the inducer. This might be partly due to protective activity of endogenous hsp27 in the cells at higher density, which contributes to cytoskeletal integrity in response to apoptotic stimuli.