基于乳腺癌核内标志物的信使RNA响应性分子探针的构建及影像实验研究

Molecular characteristic is very important for the precise therapy of breast cancer. Imaging-guided detection of biomarkers is one of the hotspots of molecular imaging, due to the unique advantages like noninvasive, dynamic, and comprehensive. However, major biomarkers, such as estrogen, progesterone receptors and cell proliferation related antigen, are mainly in the nucleus, resulting in great difficulties for the living imaging. So looking for suitable method for the imaging of markers within the nucleus is the key issue. Herein, this project designs novel estrogen, progesterone receptors and cell proliferation related antigen mRNA-recognize molecular beacons based on the reports that mRNA located in the cytoplasm, and the expression levels of mRNA are highly consistent with the corresponding protein levels, which can indirectly indicate the characteristics of proteins through imaging of mRNA expression. The molecular beacon consists of a stem-and-loop structure doubly labeled with a fluorophore (Cy5) and a quencher (gold nanoparticle). In the absence of targets, molecular beacon acts like switches that are normally closed by the stem part, and in the ‘‘off’’ position, little fluorescence background is observed because of quenching. However, upon binding with their targets, conformational changes open the hairpin, and fluorescence is turned ‘‘on’’. This kind of molecular beacon with specific detection of mRNA and near infrared light property is expected to achieve non-invasive detection of biomarkers, guiding precision treatment of breast cancer.

乳腺癌具有高度异质性，分子标志物的表达水平检测是乳腺癌精准诊疗的关键技术之一。利用影像方法在体实时检测其表达水平能为精准治疗提供重要依据。然而多数乳腺癌分子标志物，如雌、孕激素受体和细胞增殖相关抗原等，主要在细胞核内表达，不易在体实时检测。因此寻找对核内标志物进行成像的新方法是乳腺癌精准治疗的关键科学问题。本项目基于核内标志物相应的信使RNA在细胞质内表达，且与核内标志物蛋白表达水平高度一致的特点，提出可通过对信使RNA成像间接反映核内标志物蛋白表达水平假设拟构建能特异性识别雌、孕激素受体和细胞增殖相关抗原等信使RNA的新型分子信标，一端标记荧光基团Cy5，另一端是淬灭剂金纳米颗粒。通过Cy5荧光的淬灭与复燃进行实时监测分子信标在生物体中的靶向定位能力及生物分布，实现对核内标志物的间接成像。预期该具有特异性响应信使RNA分子信标可为在体检测乳腺癌分子标志物水平指导乳腺癌精准治疗提供新策略。

乳腺癌是临床重大疾病，严重威胁我国人民生命健康。乳腺癌具有高度异质性，分子标志物的表达水平检测是乳腺癌精准诊疗的关键技术之一。利用影像方法在体实时检测其表达水平能为精准治疗提供重要依据。然而多数乳腺癌分子标志物，如雌、孕激素受体和细胞增殖相关抗原等，主要在细胞核内表达，不易在体实时检测。因此寻找核内标志物进行成像等新方法是乳腺癌精准治疗的关键科学问题。本课题基于核内标志物相应的信使RNA在细胞质内表达，且与核内标志物蛋白表达水平高度一致的特点，通过对信使RNA成像间接反映核内标志物蛋白表达水平。实验结果显示所构建的分子信标在体外具有较高的稳定性，在细胞水平和动物水平具有很好的生物相容性，未见明显的毒性作用。流式细胞检测和共聚焦显微镜结果显示在细胞水平所构建的分子信标对靶向mRNA具有很好的结合能力。裸鼠皮下肿瘤模型显示靶向雌激素受体的分子信标在活体水平对雌激素受体mRNA具有很好的成像效果。本项目可为间接检测乳腺癌分子标志物水平提供新策略。