具有自发级联扩增功能的开关型荧光探针开发及其在循环肿瘤细胞检测中的应用

Circulating tumor cells (CTCs) are the leading causes of tumor metastasis. The presence of CTCs is highly associate with tumor development and progression. It is now considered as an important indication for tumor diagnosis and prognosis monitoring. Generally, the current CTCs diagnosis is based on polymerase chain reaction or fluorescent antibody labeling. These methods have good sensitivity and specificity, but expensive, specialized equipments and highly trained personnel are required. The aim of this study is to develop a self-immolative dendritic probe with off-on fluorescent ability for tumor cell sensoring and establish a convenient method for cancer cells detection. This fluorescent probe is stabilized by a substrate of Cathepsin B to prevent self-immolative decomposition. It is quenched in dendritic form, but can produce a lot of fluorescent monomers after de-protection specifically by Cathepsin B in cancer cells. The increased degraded products can elevate the intra-cellular osmotic potential. The cell bursts due to swelling which is accelerated by low osmotic potential reaction buffer. The released Cathepsin B can start very quick degradation of probes and increase the concentration of fluorescent monomers. It is easy to realize full automatic detection without labeling or washing. Due to its convince and low-cost, this assay has great value in clinical applications.

循环肿瘤细胞是肿瘤转移最主要的原因之一，因与肿瘤的发生、发展密切相关，目前已被作为肿瘤早期诊断、疗效评估及复发检查的关键指标。现有循环肿瘤细胞检测主要基于核酸扩增或荧光抗体识别，因此操作较复杂、检测耗时长、费用高、不易实现全自动化。针对现有技术的不足，我们拟开发一种靶向开关型荧光探针用于循环肿瘤细胞的体外检测。该探针为叶酸标记的具有自发级联降解功能的荧光分子多聚体，控制自身降解的分子开关被组织蛋白酶B底物构成的保护基团封闭。探针在聚合状态时被淬灭，当靶向进入肿瘤细胞内经酶切去除除保护基团后引发探针自发级联降解，产生数量众多且能被激发光激发的荧光分子单体。探针降解产物浓度的提高引起胞内渗透压升高，在低渗缓冲液作用下细胞破裂释放组织蛋白酶B到缓冲液中，进一步引发探针的快速降解发光。该方法无须标记和洗脱，检测方法简单，易于实现全自动化操作，具有较高的临床应用价值和产业化前景。

因手术切除完整程度与术后复发及生存期密切相关，所以用于术中肿瘤显影的探针开发具有较大的需求。本项目开发了一种叶酸-PEG修饰的包载开关型荧光探针的纳米CaCO3用于肿瘤细胞的体内外检测。开关型荧光探针（JF2）的合成是由组织蛋白酶B底物（苯丙氨酸-赖氨酸）为保护基，以对氨基苯甲醇为自分解连接链（Self-immolative linker），末端连接一个开关型荧光分子（JF1）及一个叶酸分子。采用乳液法将荧光探针JF2包埋在纳米CaCO3中，并在纳米CaCO3表面进行叶酸标记的PEG修饰。体外通过多种肿瘤细胞系进一步优化体系，建立体外肿瘤细胞的快速检测方法。我们此外还构建了小鼠肿瘤模型，建立体内肿瘤显影方法，成功在体外以及小鼠肿瘤模型中实现肿瘤细胞的显影。该材料在体内外显示了良好的PH和蛋白酶B依赖性，且极大降低了背景信号，在中性PH能保持稳定，在酸性条件下快速降解释放，实现在5分钟内对小鼠瘤体的显影，且无急性毒性的发生，具有较高的临床应用价值和产业化前景。