新型miR21靶向肿瘤反义核素显像与治疗研究

miRNAs, non-coding RNA which close related with tumorigenisis, could be new tumor biomarkers. Targeted miRNAs for tumor diagnose and therapy may explored a new way for tumor theranostic. Howerver,existed miRNA imaging depends on the reported gene through genetherapy, but its broadly use will be limited because of the dangours of virus in gene therapy. Target miRNA for therapy always adopt single modified antisense(antagmir), its disadvantages of lacking of tumor targeting and difficult in passing through cell membrane resulted uneffective inhibiting the tumor growth. To solve those critical problems in tumor imaging and therapy by target miRNA, this study will introduce the tumor angiogenesis target peptide NGR and the cell penetrating peptide TAT to antagmir to prepare a completely new peptide nuclide acid PNA target miR21 which is broadly over expressed in many solid tumors. By labeling the new probe with 99mTc, it will be used for imaging miR21 expressed in tumor for tumor diagnose and evaluation the therapy. By labeling the new probe with 177Lu, the tumor growth will be significantly inhibited by combing the PNA inhibit expression of miR21 and β particles from 177Lu which will completely destroy the cells. The utmost aim of this study will established a new method in tumor imaging and therapy, which will be open a new window for studies of both the miRNAs and tumors.

miRNAs是调节基因表达的非编码RNA，与肿瘤发生及发展关系密切，为潜在的肿瘤标志物。开展肿瘤组织异常表达miRNAs的可视化及靶向治疗，有望为肿瘤的诊断和治疗开辟新途径。现有miRNAs显像采用报告基因，通过基因治疗进行显像，但病毒载体的不安全性使其难以推广；miRNAs靶向治疗采用与miRNAs互补的反义核酸，该法缺乏靶向性、难以进入肿瘤细胞，无法有效抑制肿瘤。本研究拟以反义核酸互补结合为理论依据，利用NGR靶向肿瘤新生血管及穿膜肽TAT介导细胞转入的作用，以肿瘤过度表达的miR21为靶点，合成肿瘤靶向性抗miR21反义肽核酸PNA。以99mTc标记新型反义探针对肿瘤进行显像，开展肿瘤诊断及疗效评价的研究；以177Lu标记，通过联合PNA对miRNA的抑制作用及核素的电离辐射效应，开展肿瘤治疗研究。最终建立miRNA靶向的核素反义显像与治疗新方法，为提高肿瘤诊断与治疗效果开辟新途径。

本研究主要是以肿瘤异常表达的miRNAs为标志物，建立针对异常表达miRNAs的核素显像及靶向治疗新方法。其中miRNA21在多种实体肿瘤中异常表达，本研究设计合成与miRNA21互补的肽核酸（PNA），并将其与肿瘤靶向性短肽—低pH插入肽（pH-Low Insertion Peptide，pHLIP）连接，通过与DOTA偶联实现核素标记。该复合物进入细胞后能与miRNA21特异性结合，并滞留在肿瘤细胞，利用其所携带的显像核素（68Ga）进行PET显像；对该复合物进行131I标记，利用131I β射线的电离辐射生物效应对肿瘤细胞进行杀伤，实现对肿瘤的核素靶向治疗。本研究初步检验了miRNA21在肿瘤患者的表达，通过检测多种不同实体瘤患者（46例）血液中miRNA21的含量，并与18例健康志愿者血液miRNA21进行比较，结果显示肿瘤患者血液miRNA21的量是健康志愿者的2.78倍，表明miRNA21在肿瘤患者异常表达，可以作为肿瘤标志物。在此基础上成功的合成了与miRNA21特异结合的PNA，在细胞水平利用荧光检测结果显示随靶向PNA加入的增多荧光信号不断增强，而对照组PNA并不与miRNA21结合，增加对照PNA的用量，荧光信号并未增强。该研究显示合成的PNA能与miRNA21互补结合，具有较好的miRNA21靶向性，但miRNA21位于肿瘤细胞内，PNA只有到达肿瘤组织，进入肿瘤细胞才能与miRNA21有效结合。为此，我们根据肿瘤细胞代谢旺盛导致肿瘤微环境pH值较正常组织低的特点，选用pHLIP短肽，该短肽不仅能在低pH高度聚集，同时在低pH时能插入细胞的特性，利用该短肽与PNA结合，从而有效的将PNA携带至miRNA21异常表达的肿瘤细胞内。最终我们成功的制备了68Ga-DOTA-pHLIP-Anti-miR21-PNA正电子核素标记探针，利用该探针成功的对肿瘤进行了显像，与不含pHLIP短肽的68Ga-DOTA-Anti-miR21-PNA相比，肿瘤靶向的68Ga-DOTA-pHLIP-Anti-miR21-PNA在肿瘤有明显的聚集（SUV为76.05Kbq/ml），而68Ga-DOTA-Anti-miR21-PNA几乎未见肿瘤显像（SUV仅为20.84Kbq/ml）。本方法成功的对异常表达miRNA21的肿瘤进行了显像，显示了以miRNA为靶点对肿瘤进