基于肽识别的肿瘤细胞早期凋亡传感分析新方法研究

The disorder of apoptosis plays a key role in the uncontrolled proliferation and fast growing of cancer cells. To effectively interfere and regulate such behavior of cancer cells, it is of great importance to develop analytical approaches for probing the early apoptosis in cancer cells. In this proposal, cancer-related proteins and molecular events involved in apoptosis are chosen as the targets. Peptides with designable structures, various optional building blocks and high biocompatibility were chosen for the design and synthesis of novel bioprobes. Based on the high affinity of peptides, novel methods can be developed for sensing early apoptosis of cancer cells. By targeting one or multiple targets, peptide-based bioprobes for selective and sensitive real-imaging analysis of cancer-cell apoptosis can be functionally evolved. Such peptide probes will be applied for probing the apoptotic responses from cancer cells to endogenous stimulations such as changes microenvironment, drugs or oxidative stress. The potential application of peptide probes for analysis and screening of the sensitivity of cancer cells to apoptosis will be also investigated. These studies can provide potential tools and methods for the evaluation of progression status, multidrug resistance and individualized therapy of tumors.

细胞凋亡程序的破坏与紊乱是肿瘤细胞增殖失控和恶性生长的关键原因。肿瘤细胞早期凋亡信号的探测分析，对于干预与调控手段的有效实施具有重要指导意义。本项目拟从肿瘤相关关键蛋白质及其抗细胞凋亡的特征分子活动出发，利用多肽结构的可设计性、构建单元的多样性和高度的生物相容性，设计并合成肿瘤细胞凋亡特异性响应的新型多肽探针，发展基于亲和肽识别的肿瘤细胞早期凋亡传感检测新方法；建立针对单一或多靶标的、高选择性、高灵敏度的肿瘤细胞凋亡成像分析新方法。利用多肽探针探测外源性刺激，如微环境因素、药物或氧化压力等对肿瘤细胞凋亡进程的诱导和干扰作用，探索基于多肽探针的肿瘤细胞凋亡敏感性传感筛查与活体分析新应用，为肿瘤细胞的恶性度评价、多药耐药性以及个性化治疗提供新的思路和方法。

细胞凋亡程序的破坏与紊乱是肿瘤细胞增殖失控和恶性生长的关键原因。肿瘤细胞早期凋亡信号的探测分析，对于干预与调控手段的有效实施具有重要指导意义。本研究选择细胞凋亡关键蛋白质天冬氨酸蛋白水解酶-3（Caspase-3）为靶蛋白，利用多肽结构的可设计性和高度的生物相容性，筛选RKKRRORRR作为细胞穿膜基团，DEVD作为Caspase-3底物识别基团，长波长荧光团甲酚紫CV作为检测报告基团，设计并构建了新型比例型多肽荧光探针。Caspase-3的含量与细胞凋亡正相关，通过对Caspase-3的定量成像分析，实现了对肿瘤细胞凋亡的评价，发展了基于亲和肽识别的肿瘤细胞早期凋亡传感新方法，其检测灵敏度为5.3 pmol/L。外源性星形孢菌素（STS）的加入，可引起细胞荧光比例通道信号升高，表明STS可以快速诱导细胞凋亡，且随细胞凋亡程度的加深，其信号不断增强。该多肽探针有望用于细胞凋亡相关疾病的检测与诊疗评价。以凋亡相关蛋白质细胞色素C为识别对象，以细胞色素C碳端九肽作为模板分子，通过可逆腙键将模板多肽修饰在磁性纳米颗粒表面，制备了能选择性识别细胞色素C的表面分子印迹纳米材料（MIPs）。所制备的MIPs具有快速的结合动力学，显示出对目标肽段以及细胞色素C特异性识别能力，结合质谱检测与石英晶体微天平传感分析，实现了细胞裂解液中细胞色素C的选择性富集检测。进一步选择通过活化PI3K-AKT信号通路在抵抗凋亡中发挥重要作用的LAPTM4B蛋白为靶蛋白，发展了高效、高选择性筛选新方法，筛选出特异性多肽探针，实现了活体靶向成像分析与肿瘤多药耐药研究。本项目在Angew. Chem. Int. Ed.、Anal. Chem.、Chem. Commun.等杂志发表论文12篇，授权中国发明专利2项。