【作者】 胡潇；

【导师】 梁高林；

【作者基本信息】 中国科学技术大学 ， 分析化学， 2021， 硕士

【摘要】 癌症是世界上最主要的死亡原因之一。癌症的早期精确诊断对于降低癌症导致的死亡率至关重要。临床上对癌症的诊断主要依靠细胞和组织形态学分析,以及癌症相关生物标志物的检测(例如,与癌症各个时期相关的酶、蛋白质和代谢物)。但是,在癌症的早期阶段,这些检查指标表达水平较低,会影响癌症检测的准确性和可靠性。因此,有必要开发出新的方法,以提高癌症早期诊断的准确性。在上述方法中,测定癌症相关生物标志物的水平在癌症早期诊断中应用广泛。而弗林蛋白酶是一种肿瘤过度表达的前蛋白转化酶,可以作为区分肿瘤细胞与正常细胞的重要指标。弗林蛋白酶的成像则可以直观地用于癌症的早期检测。在过去的十年中,已有多种成像方式被成功用于弗林蛋白酶的成像或检测,包括正电子发射断层扫描成像(PET)、磁共振成像(MRI)和分子荧光成像等。在几种成像模式中,分子荧光成像因具备分辨率高、选择性高、仪器简单和无创性等特点,在弗林蛋白酶的检测中具有优势。已报道的用于弗林蛋白酶检测的荧光探针通常使用Arg-Val-Arg-Arg(RVRR)肽序列作为弗林蛋白酶的特异性底物。尽管探针包含的特异性底物对于弗林蛋白酶具有高亲和力,可以粗略地指示弗林蛋白酶的位置(即高尔基体),但它们并未配备其他靶向高尔基体的官能团,难以实现对弗林蛋白酶的精准成像。此外,先前的工作通常仅使用单色荧光。在此,我们开发了一种靶向高尔基体的双色荧光“点亮”探针Q-RVRR-DCM,用于弗林蛋白酶的空间精准成像。利用荧光共振能量转移和分子内电荷转移的原理,探针一开始几乎不发荧光。经弗林蛋白酶酶切后,Q-RVRR-DCM转化为Q-RVRR和DCM-NH2,在420和640 nm处将双色荧光“点亮”,且两个荧光通道没有光谱串扰。在过度表达弗林蛋白酶的HCT-1 16细胞中,Q-RVRR-DCM不仅显示弗林蛋白酶特异性的双色“点亮”荧光,而且与单色“点亮”荧光探针RVRR-DCM相比,还与高尔基体绿色荧光探针的信号具有优越的共定位性。我们设想,凭借靶向高尔基和双色“点亮”荧光的优异特性,实现空间精准成像,我们的弗林蛋白酶探针Q-RVRR-DCM可以在不久的将来用于癌症的精准早期诊断。更多还原

【Abstract】 Cancer is one of the leading causes of death worldwide.Early diagnosis of cancer is important for reducing the mortality rate of cancer.In clinic,existing methods for cancer diagnosis mainly rely on the morphological analysis of tissues(histopathology),cells(cytology),or detection of cancer-related biomarkers(e.g.,enzymes,proteins,or metabolites associating with the stages of cancer).However,at early stage of cancer,low abundance of these examination indicators challenges the accuracy and reliability of cancer detection.Therefore,it is of great urgency and interest to develop new methods to improve the detection accuracy for the early cancer diagnosis.Among the methods abovementioned,measuring the levels of cancer-related biomarkers is one promising approach for the early diagnosis of cancer.Furin,known as a tumor-overexpressing protein convertase mainly located in the trans-Golgi apparatus,can serve as an essential indicator for distinguishing tumor cells from normal cells.Furin imaging could be used intuitively for the early detection of cancer.In the past decade,several imaging modalities have been used for furin imaging(or detection),including positron emission tomography(PET),magnetic resonance imaging(MRI),and molecular fluorescence imaging.Among these imaging modals,molecular fluorescence imaging is advantageous in furin detection due to its high resolution,high selectivity,simple apparatus,and noninvasive capabilities.The reported fluorescence probes for furin detection commonly used the Arg-Val-Arg-Arg(RVRR)peptide sequence as the specific substrate for furin cleavage.Although these probes are able to roughly indicate the location of furin(i.e.,Golgi apparatus)by using their high affinity to furin and fluorescence "Turn-On",they are not equipped with another organelle-targeting warhead for additionally precise furin imaging.Moreover,previous works usually used merely monochromatic fluorescence for furin detection.Herein,we report a Golgi-targeting and dual color"Turn-On" probe Q-RVRR-DCM for imaging furin with high spatial precision.By integrating the principles of Forster resonance energy transfer and intramolecular charge transfer,the probe was designed non-fluorescent.Upon furin cleavage,Q-RVRR-DCM was converted into Q-RVRR and DCM-NH2,turning the dual fluorescence color "On" at 420 and 640 nm without spectral crosstalk.In furin-overexpressing HCT-116 cells,Q-RVRR-DCM showed not only furin-specific,dual color "Turn-On" fluorescence but also superior co-localization with Golgi tracker than that single color "Turn-On" probe RVRR-DCM.We envision that,with the excellent properties of Golgi-targeting and dual fluorescence color "Turn-On",our furin probe Q-RVRR-DCM could be applied for accurate early diagnosis of cancer in the near future.更多还原