光学分子影像技术在活体小动物示踪细胞周期的实验研究

It is well recognized the aberration in cell cycle regulation will leads to development and progression of cancer. perturbing the proliferative cycle of tumor cells at diverse stages of the cell cycle is an important strategy for cancer treatment. Most of the current drugs used to treat cancer is the so-called anti-DNA synthesis，which arrest cell cycle progression at G1 phase or S phase. However, how to detect and identify cell cycle? To date, cell cycle has mostly been observed either after nuclear bromodeoxyuridine (BrdU) staining, or by flow cytometry. It is difficult to observe in live samples in vivo..Bioluminescence imaging (BLI) is an emerging approach that is based on detection of light emission from cells or tissues. In our previous study, we have established methods through bioluminescence to observe biological responses, such as signal transduction, gene expression. We also generated a p27-luciferase fusion protein used as an optical reporter to visualize CDK2 change in vivo, which published in Nature Medicine. Since currently lack of noninvasive and real-time methods for detecting cell cycle at G1 phase of S phase in vivo, we are going to generate a construct dual-color ?uorescent indicator serving as a reporter of G1 phase or S phase to monitor cell cycle change. In this study, we will establishe pGL3-E1Pro-E1-DR that can stand for G1 phase, pGL3-A2Pro-NA2-DR for S phase. Both of them are consisted of the N-terminus of cyclin E1 or cyclin A2 fused to both luciferase and EGFP (enhanced green fluorescent protein) to develop dual-color ?uorescent fusion protein, controlled by the cyclin E1 promoter or cyclin A2 promoter. In order to demonstrate that this two reporters are regulated in a cell cycle dependent manner and are accumulated in G1 or S pahse in cellular assay, we will use in vitro and in vivo imaging to detect whether G1 of S1 inhibitor can induces cell cycle arrest in G1 or S1 phase, whether up-regulating cyclin E or cyclin A reporter can be observed in breast cancer cell lines in both hollow fiber and subcutaneous models with non-invasive longitudinal measurement. .In short, the aim of this study is to develop a non-invasive and real-time reporter of cell cycle of G1 phase or S phase in living animals. It would be also helpful to validate/identify any agents potentially to inhibit DNA replication and arrest cell cycle in G1 or S phase.

光学分子影像技术是应用光学成像在分子或细胞水平上研究活体状态下的生理或病理过程，为疾病机制的研究、新药研发等提供了新的契机。我们曾构建了p27-Luc融合蛋白，应用光学分子影像技术在活体小动物监测细胞周期依赖性激酶CDK2的活性。本项目针对癌细胞无限增殖的特性，以在活体小动物监测细胞周期分布的理论方法为主线，基于细胞周期蛋白泛素化依赖性降解机制，构建受转录和翻译后修饰调控的细胞周期蛋白荧光素酶融合蛋白，体外研究这些融合蛋白报告细胞周期时相的特异性和调控机制，进一步在活体小动物验证它们监测细胞周期的准确性及在抗肿瘤药物疗效中的指示作用，期望在小动物活体成像监测细胞周期分布的理论方法上取得突破。旨在创建在活体小动物研究肿瘤的发生进展机制、加速抗肿瘤靶向药物研发的技术平台，实现在同一生物体监测细胞周期的不同时相，并为在体监测肿瘤的相关事件提供理论依据。

建立分子影像平台使之能够用于高通量筛选特异性抑制细胞周期的抗肿瘤药物，并能在体评估抗肿瘤药的疗效，是目前肿瘤治疗领域的研究热点之一。目前缺乏能够在活体条件下筛选针对细胞周期DNA合成期（S期）期的抗肿瘤药物筛选模型。本研究以泛素依赖性降解途径为理论基础，利用荧光素酶基因能够应用于活体动物成像这一特点，通过基因工程技术将G1期和S期特异性蛋白cyclin E和Cyclin A2克隆于荧光素酶基因上游，形成可表达融合蛋白Cyclin E-Luciferase （CYCE-Luc）和CyclinA2-Luciferase （CYCA-Luc）真核重组表达载体。通过上述研究工作，将融合蛋白“CYCE-Luc”和“CYCA-Luc”作为监测G1期和S期的生物发光报告蛋白。在培养的稳定表达该融合蛋白的肿瘤细胞株中验证CYCE-Luc和CYCA-Luc报告基因是否受泛素化依赖性的蛋白体系来调控，比较G1和S期阻滞前后报告基因的变化，并进一步通过生物发光分子影像技术在活体小动物验证目前临床使用的特异性抑制G1期和S期的抗肿瘤药物是否可以上调报告基因。建立了一个在体内体外都能实时、反复、非侵袭性地监测G1和S期的光学分子影像报告系统，对于今后更加深入地研究细胞周期调控机制及筛选抗肿瘤药物具有重要意义。