Multimodal in vivo imaging and blood monitoring of intrinsic and extrinsic apoptosis.
Multimodal in vivo imaging and blood monitoring of intrinsic and extrinsic apoptosis.
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DOI：
10.1038/mt.2011.17
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发表时间：
2011-06-01
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影响因子：
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通讯作者：
Tannous, Bakhos A
中科院分区：
文献类型：
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作者：
Niers, Johanna M;Kerami, Mariam;Tannous, Bakhos A
细胞凋亡的非侵入性检测和体内成像在包括癌症在内的许多不同领域的治疗方法的发展中起着关键作用。我们通过将绿色荧光蛋白（GFP）融合到天然分泌的Gaussia荧光素酶的N-末端，所述Gaussia荧光素酶被由天冬氨酸（D）、谷氨酸（E）、缬氨酸（V）和天冬氨酸（D）组成的半胱天冬酶-3切割肽或DEVD分开，从而开发了凋亡生物传感器。我们发现这种融合以非活性形式保留在细胞的细胞质中。在细胞凋亡时，DEVD肽响应于半胱天冬酶-3活化而裂解，释放ssGluc，其现在可以进入分泌途径，在那里其被正确折叠并从细胞释放，并且可以随时间在培养物的条件培养基中或离体活动物的血液中检测到。由于Gluc是通过内质网（ER），高尔基体和囊泡通过常规途径从细胞中分泌的，我们表明，在这些隔室中的Gluc的存在下，细胞凋亡的反应可以在体内可视化使用生物发光成像。该报告基因为细胞凋亡的成像和实时监测提供了有价值的工具，并且与培养细胞和动物模型中的高通量功能筛选应用兼容。
Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form. Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc, which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time. Because Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum (ER), Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging. This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models.