叶酸受体介导188Re-siRNA干扰UbcH10治疗肿瘤的基础研究

The UbcH10 gene, which belongs to the ubiquitin-conjugation enzyme (E2) gene family, is significantly overexpressed in many different types of cancers including the breast cancer. Diminution of UbcH10 expression could markedly inhibit the tumor cell proliferation. UbcH10 may be used as a potential target in the breast cancer gene therapy. In this study, a 21nt UbcH10-targeted siRNA will be synthesized and radiolabeled with 188Re using the NHS-MAG3 method. Receptor mediated delivery of siRNA enables silencing of target genes in specific tissues. Folate receptor is an attractive target for tumor-selective gene delivery. Folate receptor-specific intracellular delivery of the 188Re-siUbcH10 into breast cancer cells overexpressing folate receptors will be explored by employing the folate-poly(ethylene glycol)-polyethylenimine (FOL-PEG-PEI) conjugate as a nonviral polymeric carrier. The FOL-PEG-PEI/188Re-siUbcH10 complexes prepared at different nitrogen/phosphate (N/P) ratios will be characterized with dynamic light scattering and gel electrophoresis and their cell specific gene silencing effect will be comparatively and quantitatively examined to optimize the delivery system. Then the 188Re-siUbcH10 will be formulated with FOL-PEG-PEI conjugate under the optimized condition. The therapeutic effect of the optimized FOL-PEG-PEI/188Re-siUbcH10 complexes on breast cancer cells will be systematically examined by folate receptor-mediated endocytosis. The dynamic biodistribution and therapeutic effect of the complexes will also be studied in breast tumor-bearing nude. In addition, the pharmacokinetics of the complexes in rabbit will be carried out. The focus of this study is to explore the co-therapeutic effect of 188Re and RNA interference by folate receptor-mediated endocytosis pathway and establish an effective and safe method for the gene therapy of malignant tumors.

以乳腺癌组织中高表达的泛素结合酶UbcH10为靶点，合成针对UbcH10基因编码区的21nt长度的siUbcH10，将其标记放射性核素188Re，优化188Re标记siUbcH10的反应流程。应用叶酸-聚乙二醇-聚乙烯亚胺阳离子多聚结合物（FOL-PEG-PEI）对188Re-siUbcH10装配包裹，得到不同N/P比值（即PEI所含铵根和siUbcH10中磷酸根的比值）的FOL-PEG-PEI/188Re-siUbcH10复合物，从中筛选出具有理想生物学性能的最佳N/P比值复合物。对最佳N/P比值复合物的理化特性、代谢和药动学进行研究，并研究其通过叶酸受体介导途径对乳腺癌细胞和荷乳腺癌动物的靶向治疗作用，阐明受体介导的188Re和RNA干扰（RNAi）对肿瘤的联合治疗作用机制，旨在建立起一种高效安全的受体介导的放射性核素RNAi靶向治疗肿瘤的方法，为肿瘤的基因治疗提供新的思路和方法。

为弥补RNAi技术的不足，增强RNAi对肿瘤的治疗效果，本研究提出受体介导放射性核素标记siRNA联合治疗肿瘤的概念。以乳腺癌组织中高表达的泛素结合酶UbcH10为靶点，合成针对UbcH10基因编码区的21nt长度的siUbcH10，将其标记放射性核素188Re，优化188Re标记siUbcH10的反应流程。应用叶酸-聚乙二醇-聚乙烯亚胺阳离子多聚结合物（FOL-PEG-PEI）对188Re-siUbcH10装配包裹，得到不同N/P比值的FOL-PEG-PEI/188Re-siUbcH10复合物，从中筛选出具有理想生物学性能的最佳N/P比值复合物。对最佳N/P比值复合物的理化特性、代谢和药动学进行研究，并研究其通过叶酸受体介导途径对乳腺癌细胞和荷乳腺癌动物的靶向治疗作用。研究显示，应用双功能螯合剂NHS-MAG3对siUbcH10进行标记，标记率可达70%以上，放化纯达95%以上；筛选出的最佳N/P比值FOL-PEG-PEI/188Re-siUbcH10复合物，通过叶酸受体介导的内吞作用，可大大提高乳腺癌细胞对188Re-siUbcH10摄取率，与未经叶酸受体介导的188Re-siUbcH10摄取率相比差异显著（p<0.05），188Re-siUbcH10进入细胞后能够有效抑制细胞生长及UbcH10 mRNA和蛋白的表达FOL-PEG-PEI/188Re-siUbcH10复合物在荷乳腺癌小鼠的体内分布研究显示，肿瘤组织摄取放射性随时间逐渐增加，而其它组织放射性分布逐渐减少；在各时间点，肿瘤组织对188Re-siUbcH10的摄取均明显低于FOL-PEG-PEI/188Re-siUbcH10（P<0.05）。对荷瘤裸鼠的治疗研究显示，FOL-PEG-PEI/188Re-siUbcH10组肿瘤组织的增长较188Re-siUbcH10组明显减缓，瘤组织中UbcH10 mRNA及蛋白的表达明显低于188Re-siUbcH10组。本研究显示，通过叶酸受体介导188Re-siUbcH10干扰UbcH10对乳腺癌细胞及荷瘤模型均显示了良好的治疗潜力，通过本研究可建立一种高效的受体介导的放射性核素RNAi靶向治疗肿瘤的方法，为肿瘤的基因治疗提供新的思路和方法。