体外维拉帕米诱导视网膜色素上皮细胞凋亡过程中胞内钙离子稳态及半胱天冬酶-3基因表达
[Ca(2+)]i homeostasis and caspase-3 gene expression in verapamil-induced retinal pigment epithelium cells apoptosis in vitro.
作者信息
Pang Dong-Bo, Hong Jing
机构信息
Department of Ophthalmology, the First Hospital Affiliated to Liaoning Medical University, Jinzhou 121001, Liaoning Province, China.
出版信息
Int J Ophthalmol. 2010;3(4):288-90. doi: 10.3980/j.issn.2222-3959.2010.04.02. Epub 2010 Dec 18.
AIM
To study caspase-3 gene expression and [Ca(2+)]i homeostasis in verapamil (Ver)-induced human retinal pigment epithelium (RPE) cells apoptosis.
METHODS
Ver 80mg/L was applied in cultured human RPE cells for 12, 24 and 48 hours to induce RPE cells apoptosis. The expression of apoptotic effector gene caspase-3 was assessed by reverse transcription polymerase chain reaction (RT-PCR). Single cell was measured using fluorescence indicator Fura-3/AM with MetaFluo4.5/coolsnapfx/IX70 intracellular Ca(2+) fluorescence imaging system.
RESULTS
High levels of expression of caspase-3 mRNA were observed in normal RPE cells and it significantly increased after co-cultured with Ver. The fluorescence in resting RPE cells was strong and distributed throughout the cells. The nucleus appeared more fluorescent than the cytoplasm. Calcium fluorescence of RPE cells attenuated after co-cultured with Ver.
CONCLUSION
Up-regulation of caspase-3 gene expression and disturbance of [Ca(2+)]i homeostasis might play pivotal roles in Ver-induced RPE cells apoptosis.
目的
研究维拉帕米（Ver）诱导人视网膜色素上皮（RPE）细胞凋亡过程中半胱天冬酶-3基因表达及细胞内钙离子（[Ca(2+)]i）稳态。
方法
将80mg/L的Ver应用于培养的人RPE细胞12、24和48小时以诱导RPE细胞凋亡。通过逆转录聚合酶链反应（RT-PCR）评估凋亡效应基因半胱天冬酶-3的表达。使用荧光指示剂Fura-3/AM与MetaFluo4.5/coolsnapfx/IX70细胞内Ca(2+)荧光成像系统测量单细胞。
结果
在正常RPE细胞中观察到半胱天冬酶-3 mRNA的高水平表达，与Ver共培养后其显著增加。静息RPE细胞中的荧光较强且分布于整个细胞。细胞核比细胞质荧光更强。与Ver共培养后RPE细胞的钙荧光减弱。
结论
半胱天冬酶-3基因表达上调及[Ca(2+)]i稳态紊乱可能在Ver诱导的RPE细胞凋亡中起关键作用。
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