Bis(zinc-dipicolylamine), Zn-DPA, a new marker for apoptosis.
Bis(zinc-dipicolylamine), Zn-DPA, a new marker for apoptosis.
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DOI：
10.1167/iovs.13-13346
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发表时间：
2014-07
影响因子：
4.4
通讯作者：
Jacky M K Kwong;Celia Hoang;Reshil T Dukes;R. Yee;B. Gray;K. Pak;J. Caprioli
中科院分区：
文献类型：
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作者：
Jacky M K Kwong;Celia Hoang;Reshil T Dukes;R. Yee;B. Gray;K. Pak;J. Caprioli
目的研究双吡啶甲苯胺锌（Zn（II）-dipicolylamine）（Zn-DPA）标记的荧光分子探针在N-甲基-D-天冬氨酸（NMDA）诱导的大鼠视网膜神经节细胞（RGC）变性模型中对凋亡细胞的标记作用。方法成年Wistar大鼠玻璃体内注射3 μL 40 mM中性NMDA，分别于1、2、4、24和48 h处死。在安乐死前1小时，玻璃体内注射3 μL与荧光素缀合的Zn-DPA（Zn-DPA 480）。用荧光金（FG）标记RGC，TUNEL法检测RGC，用β-tubulin和vimentin免疫组化检测RGC。结果注射NMDA后1 ~ 24 h，视网膜内可见Zn-DPA 480荧光标记，注射后48 h标记减弱。在注射后4和24小时，RGC层中的大多数Zn-DPA 480阳性细胞被FG和III β-tubulin标记。TUNEL阳性细胞数从4小时增加到24小时。在24小时，95.7%的Zn-DPA 480阳性细胞为TUNEL阳性，而95.1%的TUNEL阳性细胞为Zn-DPA 480阳性。注射NMDA后1h和2 h Zn-DPA 480阳性细胞数显著高于TUNEL阳性细胞数。结论：我们的研究结果表明，荧光Zn-DPA 480的玻璃体内注射标记视网膜神经元凋亡，暴露的磷脂酰丝氨酸的识别出现早于DNA片段的检测，这表明Zn-DPA作为成像探针跟踪变性视网膜神经元的潜力。
PURPOSE To characterize the labeling of apoptotic cells with a molecular probe of bis(zinc(II)-dipicolylamine) (Zn-DPA) conjugated with a fluorescent reporter in a rat model of retinal ganglion cell (RGC) degeneration induced by N-methyl-D-aspartate (NMDA). METHODS Adult Wistar rats were given unilateral intravitreal injections of 3 μL 40 mM neutralized NMDA and euthanized at 1, 2, 4, 24, and 48 hours. One hour before euthanasia, 3 μL Zn-DPA conjugated with fluorescein (Zn-DPA 480) was intravitreally injected. Prelabeling of RGC with retrograde fluorogold (FG), TUNEL, and immunohistochemistry with III β-tubulin and vimentin were performed. RESULTS Fluorescence labeling of Zn-DPA 480 was observed in the retinas from 1 hour up to 24 hours after NMDA injection, whereas the labeling was reduced at 48 hours postinjection. At both 4 and 24 hours postinjection, most Zn-DPA 480-positive cells in the RGC layer were labeled by FG and III β-tubulin. The number of TUNEL-positive cells increased from 4 to 24 hours. At 24 hours, 95.7% of Zn-DPA 480-positive cells were TUNEL positive, whereas 95.1% of TUNEL-positive cells were Zn-DPA 480 positive. The numbers of Zn-DPA 480-positive cells at 1 and 2 hours after NMDA injection were significantly higher than TUNEL. CONCLUSIONS Our findings demonstrate that intravitreal injection of fluorescent Zn-DPA 480 labels retinal neurons undergoing apoptosis and that recognition of exposed phosphatidylserine appears earlier than detection of DNA fragmentation, indicating the potential of Zn-DPA as an imaging probe for tracking degenerating retinal neurons.