荧光碳点介导的纳米基因传递及其诱导脑胶质瘤干细胞分化研究

This project focuses on the establishment of a highly efficient, low toxicity and self-tracking non-viral gene delivery system and the investigation of its gene transfer mechanisms as well as its effect on the induced differentiation of glioma stem cells. The main contents of this study include the following aspects: firstly, the development of positively charged fluorescent carbon dots (PCFCDs) from arginine and glucose via “one-step” microwave-assisted pyrolysis, which will then complex with genes encoding neuronal transcription factor and WNT protein to form a self-assembly DNA-PCFCDs nanoparticle; secondly, using CD133 positive U251 glioblastoma stem cells as the model cell, the transfection efficiency and gene transfer mechanisms of the DNA-PCFCDs nanoparticle as well as its impact on neural differentiation of U251 stem cells will be comprehensively addressed; thirdly, a thermo-sensitive gel which incorporates the DNA-PCFCDs nanoparticle and U251 stem cells will be prepared, followed by the evaluation of gene transfection efficiency and duration with ELISA tests, then assessment of the cell growth and neural differentiation in this PCFCDs-labeling three-dimensional system, thus conjecturing the correlations among the transfection efficiency, duration and neural differentiation of U251 stem cells; fourthly, an orthotopic implantation model of the DNA-PCFCDs nanoparticle and U251 stem cells -incorporating thermo-sensitive gel will be established in nude mice, thus the U251 stem cells’ migration and proliferation in vivo could be tracked by the PCFCDs. This in vivo model allows investigation of neural differentiation of U251 stem cells in situ and its tumorigenicity as well as the related molecular mechanisms. Altogether, this project can provide a new strategy and vehicle for the induced differentiation of tumor cells.

本项研究重在构建高效、低毒、可示踪的非病毒纳米基因传递系统，研究其基因转运机理及胶质瘤细胞的诱导分化。主要研究内容包括：以精氨酸和葡萄糖为原料，一步法制备荷正电的荧光碳量子点，以神经转录因子和WNT通路蛋白为模型基因，自组装制备DNA-碳点纳米粒；选取CD133+分选的人脑胶质瘤干细胞为模型细胞，研究纳米粒介导的基因转染效率和传递机理，评价U251干细胞的神经分化；制备温敏型凝胶，ELISA法测定三维基因转染效率和持续时间，碳量子点标记示踪三维体系中U251干细胞的生长特性和神经分化，推测基因转染效率、持续时间与U251干细胞神经分化之间的相关关系；建立裸鼠人胶质瘤原位移植模型，碳量子点示踪U251干细胞在体内的迁移、增殖，考察胶质瘤干细胞的原位神经分化和致瘤性，并进行相关的分子机理研究。本项研究可为肿瘤细胞的诱导分化提供新方法、新载体。

胶质瘤的治疗与控制一直是人们研究的重大难题，本项研究以精氨酸和葡萄糖为原料，采用一步法水热合成荷正电荷荧光碳量子点。以优化后的碳点为载体，筛选最佳神经转录因子组合，以人脑胶质瘤干细胞为模型细胞，成功实现体外二维条件下U251干细胞向正常神经细胞的转化，并研究碳点介导的基因传递机理。在二维培养的基础上，制备温敏型壳聚糖凝胶，实现了三维高效的U251干细胞细胞诱导分化。与二维培养相比，三维传递系统具有更好的高效、缓释效果。最后建立裸鼠人胶质瘤干细胞模型，研究了碳量子点体内诱导U251干细胞神经分化效果，体内分化结果表明三维纳米传递系统能成功介导GBSCs原位分化。利用全基因谱分析/WB/PCR尝试探索碳点介导的纳米基因传递系统诱导U251向神经分化的机理，解决肿瘤干细胞治疗难题。该项研究的顺利进展，可为胶质瘤干细胞治疗开辟新途径，提供新手段，更加安全有效，为肿瘤的临床治疗及新药研发提供新的视角。