基于SELEX技术筛选靶向Dockkopf-1核酸适配体及抗多发性骨髓瘤的研究

Multiple myeloma is a kind of common incurable hematological tumor and its morbidity increases year by year. And the progressive sclerotin-damage is the extrusive characteristic of MM, which influence the quality of life and prognosis of MM patients seriously. The Wnt/β-catenin pathway is germane to the pathogenesis of MM; as the signal transduction inhibitor of Wnt pathway, the expression of Dockkopf-1(DKK1) increases in many MM patients and then it inhibits the Wnt/β-catenin pathway through which the differentiation from bone marrow mesenchymal stem cells(BMSCs) to osteoblasts is inhibited; in a final, the secretion of IL-6 is advanced and the tumor develops increasingly; so DKK1 has been the potential target in the therapy of MM. The aptamer which is screened based on SELEX can interact with the target specially and then inhibits the target’s function, which had been applied in the treatment of many diseases. On the base of prokaryotic expression of DKK1 in this study, we will screen aptamer which interacts with DKK1 specially and inhibits the function of DKK1 through SELEX and then test that if the aptamer has the role of anti-multiple myeloma through bone marrow micro-environment or not, and we will elucidate the role the aptamer plays on the impact of differentiation potential of BMSCs differentiating into osteoblasts and osteoclasts and the related mechanism. This study will promote the development of new anti-multiple myeloma nucleic acid drugs which have self-contained intellectual property.

多发性骨髓瘤（multiple myeloma，MM）是常见且难治的血液系统恶性肿瘤，发病率逐年升高；进行性骨质破坏是突出临床特点，严重影响MM 患者生存质量及预后。Dockkopf-1（DKK1）作为Wnt信号通路抑制剂，在多发性骨髓瘤发生发展中发挥重要作用，能抑制Wnt/β-catenin通路和骨髓间充质干细胞（mesenchymal stem cells，MSCs）向成骨细胞分化，促进IL-6分泌和肿瘤细胞生长。DKK1是MM重要的潜在治疗靶点。 SELEX技术筛选出的与多种功能蛋白结合的核酸适配体能阻断其功能，被用于多种疾病的靶向治疗。本课题在原核表达DKK1基础上，采用SELEX技术筛选能与DKK1特异结合的核酸适配体，研究其能否通过影响骨髓微环境发挥抗MM作用及其机制；明确其对MSCs向成骨细胞和破骨细胞分化潜能的影响及机制。本研究将促进具有自主知识产权的MM核酸类新药开发。

多发性骨髓瘤（multiple myeloma，MM）是常见且难治的血液系统恶性肿瘤，发病率逐年升高；进行性骨质破坏是突出临床特点，严重影响MM 患者生存质量及预后。Dockkopf-1（DKK1）作为Wnt信号通路抑制剂，在多发性骨髓瘤发生发展中发挥重要作用， SELEX技术筛选出的与多种功能蛋白结合的核酸适配体能阻断其功能，被用于多种疾病的靶向治疗。本课题构建了DKK1的原核表达载体，并进行了诱导表达，通过Protein-SELEX筛选条件的优化，筛选并验证了与DKK1蛋白特异结合的核酸适配体DA3，DA3核酸适配体体外半衰期约为8h，能与表达dockkopf-1的MM细胞特异性结合，通过体外pull-down实验发现DA3适配体能与Dockkopf-1蛋白特异结合，DA3核酸适配体能抑制HS-5共培养MM细胞增殖活性，并能抑制D与HS-5共培养MM细胞分泌IL-6 ，抑制MM细胞Wnt/β-catenin 信号通路。该研究能为以DKK1为靶点的核酸药物研发具有重要意义。