microRNA132反义核酸-纳米金复合物促进胚胎干细胞向多巴胺神经元分化的研究

Embryonic stem cells have good application prospect in cell replacement therapy in Parkinson's disease. However, how to improve the efficiency of dopamine neurons differentiation from embryonic stem cells has important scientific significance and application value. Previous study of our research center showed that miR-132 negatively regulated the differentiation of embryonic stem cells into dopamine neurons. Therefore, using miR-132 antisense nucleic acid silencing of endogenous miR-132 could promote the differentiation. Conventional transfection methods are not efficient enough and have certain toxicity. My pre-work confirmed that gold nanoparticles could highly efficient delivery of nucleic acids into cells at low dose, and significantly increase the biological activity of nucleic acid(Angew. Chem. Int. Ed., IF=13.734). In this project, we intend to develop gold nanoparticle- based polyvalent miR-132 antisense nucleic acid nanoconjugates by using self-assembled method, and study its ability to promote embryonic stem cells differentiation into dopamine neurons. After treatment with miR-132 antisense nucleic acid-gold nanoparticle complexes, we will elucidate the biological effect of embryonic stem cells differentiate into dopamine neurons and the underlying mechanism to target gene, and in-depth evaluate its application value. The results of the study will provide theoretical basis and experimental support for using functional nano-complexes to efficiently improve the dopamine neuronal differentiation efficiency.

胚胎干细胞在帕金森病的细胞替代治疗中具有很好的应用前景。如何提高胚胎干细胞向多巴胺神经元的分化效率具有重要的科学意义和应用价值。我中心的前期研究显示，miR-132反向调控胚胎干细胞向多巴胺神经元的分化。因此，利用miR-132反义核酸沉默内源性miR-132将达到促进分化的作用。但常规方法转染效率不高且有一定毒性。我的前期工作已证明，纳米金能够低剂量、高效率的递送功能性核酸进入细胞且显著提高其生物活性(Angew.Chem.Int.Ed., IF=13.734)。据此，本项目拟采用天然多聚腺嘌呤嵌段核酸与纳米金自组装的方法，合成不同表面核酸密度的miR-132反义核酸-纳米金复合物，评估其促进胚胎干细胞向多巴胺神经元分化能力，阐明该复合体系的生物学效应和对靶基因的作用机制并深入论证其应用价值。该研究结果将为利用功能性纳米复合体系高效提高多巴胺神经元分化效率提供理论依据和实验支持。

胚胎干细胞具有多种分化潜能，为神经退行性疾病的治疗和神经系统的损伤修复带来了新的希望，细胞替代治疗帕金森病被认为是比较有前景的治疗手段。如何提高胚胎干细胞向多巴胺神经元分化的效率具有十分重要的意义。我们课题组前期研究发现，microRNA132反向调控胚胎干细胞向多巴胺神经元的分化，沉默内源性microRNA132将达到促进分化的作用。本项目中，我们针对microRNA132设计microRNA132反义核酸-纳米金复合物，研究结果发现，该纳米复合物能够提高小鼠胚胎干细胞向多巴胺神经元的分化效率。同时我们也发现，纳米金本身对小鼠胚胎干细胞向多巴胺神经元的分化也具有较好的效果。我们扩展研究内容，发现不同尺寸的纳米金（5-60nm）均能够进入小鼠胚胎干细胞细胞并具有较好的生物相容性， 30nm左右的纳米金能够显著促进小鼠胚胎干细胞向多巴胺神经元的分化。进一步研究发现，纳米金是通过mTOR/p70S6K信号通路调节小鼠胚胎干细胞向多巴胺神经元分化的。研究结果将为发展高效、低毒、低成本的胚胎干细胞分化方法及今后细胞移植治疗的临床应用奠定实验基础。相关研究结果发表在Nanomedicine和Journal of Nanobiotechnology等杂志上。