聚ADP核糖聚合酶的高通量检测及其生物医学应用研究

PARP-1 is the prototypical and founding member of the Poly (ADP-ribose) polymerase (PARP) family. It is an important mediator in physiological activities, occurrence and development of disease. The levels in normal and cancer cells and their change upon employed with drugs and/or PARP-inhibitor is important to diagnosis, treatment and prognosis of tumor. Firstly, sensitive, specific and label-free PARP-1 activity detection methods based on the specific interaction between PARP-1 and DNA and its PARylation will be developed. Secondly, high throughput SPR and fluorescence detection chips will be constructed by using microfluidics. Detection sensitivity, specificity and signal resolution will be improved by optimizing the captured efficiency from complex matrix, polymerization conditions, and the interactions between nano probes and the PAR. Thirdly, based on the above constructed high throughput detection strategy, PARP-1 activity in normal cells/tissues versus tumor cells/tissues will be detected. The correlation between their levels in nucleus, cytoplasm and the occurrences of tumors will be investigated extensively. When PARP-1 inhibitor are used or in conjunction with anti-cancer drugs, their level changes in nuclear versus cytoplasm will be monitored and be used to evaluate the development, treatment and prognosis of tumors. Based on these researches, a high throughput PARP-1 inhibitor screening strategy and the new drug research platform will be constructed.

PARP-1是一种聚ADP核糖聚合酶，其在正常细胞和肿瘤细胞中表达水平的差异、以及该差异在临床药物或PARP-1抑制剂共存时的变化对研究肿瘤的发生发展和疗效评价有重要意义。本项目拟利用PARP-1与DNA或其抗体之间的特异性相互作用以及催化合成聚腺苷二磷酸核糖（PAR）带有大量负电荷的特性，发展无标记、高灵敏和高特异性的PARP-1活性检测新方法；利用微流控芯片技术构建PARP-1高通量表面等离子体共振（SPR）和荧光检测的阵列芯片，研究复杂生物样品中PARP-1捕捉效率及其在芯片上的催化聚合性能以及PAR与金纳米棒、荧光分子间的相互作用，提高检测的时空分辨率和灵敏度；将该高通量方法用于细胞与药物或药物-抑制剂共存时PARP-1在肿瘤细胞核、细胞浆中的活性检测，研究其在正常组织与癌旁组织中的表达差异，阐述其表达水平在特定肿瘤发生中的关键作用，为肿瘤药物和抑制剂提供高通量筛查方法与研发平台。

聚ADP核糖聚合酶（PARPs）是一类存在于多数真核细胞中的蛋白质翻译后修饰酶。这个家族包含18种酶，其中PARP-1含量最高，是一种在正常生理活动和疾病的发生发展中都起关键作用的细胞功能调节剂。本项目主要研究PARP-1活性检测新方法以及其在生物医学应用研究中的应用。我们利用PARP-1催化合成聚ADP核糖链的特性，结合聚合链上带有大量负电荷的特性以及其含有大量磷酸根、大量ADP单体等特点，利用多种纳米材料和荧光探针等建立了多种PARP-1定量检测的电化学、荧光、化学发光以及细胞内荧光成像或基于LSPR效应的单颗粒成像等多种检测方法。以乳腺癌或卵巢癌正常细胞和肿瘤细胞为模型细胞，通过在细胞培养过程中添加如炎症治疗药物或抗肿瘤药物、PARP-1活性抑制剂等进行共培养，通过胞浆和核内PARP-1的活性和表达水平的检测，阐述了加药剂量、暴露时间等对胞浆、细胞核内PARP-1表达水平和活性的影响与作用规律；通过比较PARP-1在肿瘤细胞核、细胞浆及核浆共表达与相应的正常细胞或癌旁组织中的表达差异性，揭示了PARP-1表达水平和活性在特定肿瘤发生中的关键作用，建立了PARP-1抑制剂筛查的灵敏快速检测方法。