microRNA非病毒纳米传递系统构建及其促进体细胞重编程研究

The purpose of this research is to develop a novel 3 dimensional nanoparticle mediated gene delivery systems based on natural cationized polysaccharide-calcium phosphate hybrid nanoparticles(NPCPHN), which can be used to deliver microRNAs at high efficiency and produce safely induced pluripotent stem cells(iPS cells). The main contents include incorporating miR-302 and miR-302-367 cluster into miR-NPCPHN using cationized polysaccharide with good adhesive ability to human somatic cells as a source of carrier material; The mechanism of NPCPHN transferring miRNA to cells; preparation and evaluation of 3 dimensional nano-scaffolds; The RNA expression level and transgene duration time based on miRNA delivery in 2D and 3D cultured cells would be determined with RT-PCR technique, and the correlation between influence factors such as miRNA delivery efficiency and action time on iPS formation rate and reprogramming efficiency would be analyzed by calculating colony with alkaline phosphatase positive staining. The iPS cells would also be identified and characterized via detecting ES cell specific surface markers, gene expression related to pluripotency and three-layer differentiation in vitro, generation teratomas with mesoderm, endoderm and ectoderm-like structures. The safety of the iPS cells would further be evaluated by human genome gene chip to compare oncogene expression profiling of iPS induced with Yamanaka four factors or c-Myc instead of microRNAs. The mechanism of miRNA promoting iPS cell reprogramming miRNA inhibits NR2F2 expression and targets the transforming growth factor, beta receptor II (TGFBR2) is to be studied as well. The research may provide a novel strategy and carrier to benefit the generation of safer iPS cells and non viral efficient gene delivery.

本项研究重在构建基于阳离子化多糖-磷酸钙混杂纳米粒的三维纳米基因传递系统，并藉此实现微核糖核酸（microRNAs）的高效传递和多能干细胞的安全诱导。主要内容包括： 以miR-302及miR-302-367簇为对象、黏附性能好的阳离子多糖为材料，制备miR-混杂纳米粒，研究其高效传递机理；构建三维纳米支架；RT-PCR测定RNA在三维体系中的表达水平、持续时间；探寻二维、三维体系中重编程速度、效率与RNA表达水平、作用时间等影响因素之间的相关关系；分别检测ES细胞标志分子、全能基因和三胚层分化基因表达、体内三胚层分化能力评价iPS细胞的多能性；分析基因谱中癌基因表达变化以考察iPS细胞的安全性；研究microRNAs对NR2F2基因、TGFβ2通路的抑制，探讨其促进体细胞重编程的机理。本项研究可为microRNAs高效传递、iPS细胞安全诱导提供新载体、新方法。

以提高iPS细胞的安全性和体细胞重编程效率为目标，以非致癌基因miRNA替代c-Myc，利用阳离子化多糖-磷酸钙混杂纳米粒，构建安全、高效miRNA 非病毒纳米传递系统。主要内容：1. 以miR-302及miR-302-367簇为对象、黏附性能好的阳离子化杏鲍菇多糖为材料，制备了粒径在30~50 nm的miRNA-阳离子化天然多糖-磷酸钙混杂纳米粒；2. 对miRNA转导性能与混杂纳米粒体细胞转运机制进行研究，考察了针对不同细胞摄入途径的抑制剂对纳米粒入胞行为的影响，结果表明：miRNA混杂纳米粒主要通过由网格蛋白和陷穴小泡介导的内吞途径进入体细胞，并通过内涵体-溶酶体系统对进入细胞内的纳米粒进行基因转运和释放；3. 制备了直径为100nm～2μm、疏松多孔的三维纳米支架，纳米粒均匀分布在支架上，三维纳米支架为粘附于支架上的细胞转染提供了良好的环境；4. 研究了miRNA在二维和三维体系中的表达水平、持续时间及影响因素，结果表明：不同纳米粒及其用量、细胞数量、培养基的配方、转染次数、支架黏附性能均影响miRNA的表达水平和持续时间，可以实现对miRNA转染效率和持续时间的定量调控；5. 考察了二维和三维体系中miRNA介导的体细胞重编程速度、效率及影响因素，结果表明：在二维和三维体系中，影响转染效率的因素都影响重编程效率，与二维体系相比，三维纳米支架的重编程效率显著增加；6. 对iPS细胞的多能性与安全性进行了评价，将miRNA-多糖-磷酸钙混杂纳米粒转染人源脐带间充质干细胞HUMSCs，对形成的iPS克隆进行多能性鉴定，结果表明：所形成的iPS具有多能干性及分化为三胚层细胞的潜能；通过对miRNA介导形成的iPS进行基因芯片分析，与Yamanaka因子形成的iPS细胞相比，miRNA介导形成的iPS细胞中，ROS1、CRLF1、BCL2L2等癌基因表达下调，KLF10、SOX30、PDGFRA等抑癌基因表达上调，说明miRNA介导形成的iPS细胞具有安全性；7. 研究了miRNA对NR2F2基因、TGFβ2受体的作用，探讨了miRNA介导的体细胞重编程机理，结果表明：miRNA通过抑制NR2F2基因表达，从而抑制TGF-β2受体的活化，促进体细胞重编程；miRNA也可以直接抑制TGFβ2受体的活性，下调smad2/3和psmad2/3的表达，促进体细胞重编程。