摘要/Abstract
弗林蛋白酶是前体蛋白转化酶家族中最具特色的酶之一，具有重要的生物学功能，其表达量水平与许多疾病有密切的关系，如癌症的发生和发展与弗林蛋白酶表达水平有着密切关联.目前文献中报道了一些单光子荧光探针用于弗林蛋白酶的检测，但这些探针不能应用于深层组织成像，且弗林蛋白酶在肿瘤发展过程的作用仍没有得到很好地研究.针对这些问题，本工作构建了一种新型双光子荧光探针Nap-F用于细胞和肿瘤组织内弗林蛋白酶的检测与双光子成像.Nap-F是由经典双光子荧光染料1，8-萘酰亚胺、弗林蛋白酶特异性多肽序列RVRR和自消除连接体整合而成.实验结果表明Nap-F对弗林蛋白酶具有很好的特异性，能够定量检测弗林蛋白酶的活性.在飞秒激光820 nm激发下，Nap-F能有效降低生物背景，并提高组织穿透深度，适用于细胞和组织的双光子成像.Nap-F成功地实现了几种活细胞中弗林蛋白酶的双光子成像，揭示了癌细胞和表达缺陷细胞中弗林蛋白酶含量的差异.更重要的是，我们将该探针用于CoCl2固定HIF-1构建的肿瘤细胞缺氧模型成像，实验结果表明弗林蛋白酶的表达与肿瘤细胞缺氧程度存在正相关性.
关键词: 双光子, 荧光探针, 弗林蛋白酶, 癌症, 缺氧
Furin, the most characteristic member of the proprotein convertase (PCs), has important biological functions. The expression level of furin is related to many diseases, for example, the occurrence and development of cancer is closely related to the expression level of furin. Although several small-molecule fluorescent probes for furin have been developed, which were designed based on near-infrared dye or one-photon dye. These probes exhibit low Stocks' shift or shallow penetration depth, which leading to self-quenching and strong interference. Two-photon fluorescent probes, which utilize two near-infrared photons as the excitation source, can overcome these problems. Herein, a furin-activatable two-photon fluorescent probe (Nap-F) was developed firstly that allowed for detection and imaging of furin in live cells and tumor tissues. Nap-F consists of a classical two-photon fluorophore (1,8-naphthalimide), a furin-particular polypeptide sequence RVRR and a self-eliminating linker. Nap-F is water-soluble and in a fluorescence-off state itself due to the inhibited intramolecular charge transfer (ICT). In the absence of furin, no noticeable fluorescence enhancement was detected, even over 3 days in buffer solution, indicating its good stability. Upon the conversion by furin, it displayed a dramatically fluorescence enhancement at 545 nm, and exhibits high specificity and sensitivity to furin. Nap-F was applied for visualizing the difference in the expression level of furin in various cells, demonstrating its capacity of distinguishing some cancer cells from normal cells. Furthermore, Nap-F was utilized to visualize the variation of furin expression level efficiently after immobilization of hypoxia-inducible factor-1 (HIF-1) by CoCl2, with the results indicating that there is a positive correlation between the expression level of furin and the degree of hypoxia in tumor cells. Owing to the excellent property of Nap-F, the probe was also successful utilized to imaging furin activity in tumor tissues. Thus, Nap-F is able to serve as a potential tool for better exploring the intrinsic link between hypoxic physiological environment and cellular carcinogenesis and detecting cancer in preclinical applications.
Key words: two-photon, fluorescent probe, furin, cancer, hypoxia
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