一种仿病毒结构的无机纳米基因转运系统在干细胞三维组织工程中的应用研究

Induced pluripotent stem cells (iPSCs) has provided an excellent cell resource for stem cell based therapies. Gene delivery systems are required for the generation of iPSCs with gene reprograming, yet the currently used virus based gene delivery systems have some biological risks, such as tumorigenesis..Mesoporous silica nanoparticles (MSNs) have been proved as promising gene carriers recently due to good biocompatibility, tunable pore structure, adjustable particle size and abundant surface chemistry. Compared to virus based systems, they are much safer and can be degraded inside cells after several cell cycles, which can be potentially applied to deliver plasmids in both reprogramming and differentiation steps. However, the delivery efficacy of most current MSNs is not satisfactory due to limited gene payload and moderate cellular uptake ability. In addition, it is desirable that MSNs can be released from scaffolds and protect loaded genes until delivery into targeted cells, which is key for tissue engineering..The general aim of this project is to provide a safe and efficient non-viral nano-delivery system for generating iPSCs from somatic cells and differentiating them into designed functional cells after proliferating. This proposal will use bioengineering method with precisely arranged the reprogramming of oral mucosa cells into iPSCs and then differentiate to the neuron cells. The scaffolds containing multiple plasmids in the nano-delivery systems with be fabricated through a three dimension (3D) bioprinting method and nerve tissue regeneration will be assessed by both in vitro and in vivo models..The proposed system is a revolutional technology to improve the efficiency of gene delivery in a safe way, which could potentially solve the challenge in the stem cell resources.

诱导性多能干细胞（iPSCs）技术为组织工程提供了较为理想的种子细胞来源。体细胞经重编程获得iPSCs和iPSCs定向分化所需的目的基因的导入需借助质粒实现，但目前用来转运质粒（含有目的基因）的基因转运系统存在缺陷。因此，开发高效的基因转运系统是十分必要的。本项目将首次制备一种安全高效的仿病毒结构的新型纳米基因转运系统，能高效侵入细胞、大量携载基因且安全可靠。新型基因转运系统首先携载质粒转染HEK 293T细胞检测该基因转运系统的基因转运效率；接着，利用该基因转运系统转运目的基因使得口腔黏膜上皮细胞重编程获得iPSCs和实现iPSCs的成神经分化；最后用3D生物打印技术将获得的iPSCs和含有纳米基因递送系统（携载iPSCs成神经分化所需的基因）的支架材料装配用于周围神经损伤的修复和功能重建。新型纳米基因转运系统的开发将提高基因转运效率、解决干细胞来源等问题造福社会。

诱导性多能干细胞(iPSCs)技术为组织工程提供了较为理想的种子细胞来源。体细胞经重编程获得iPSCs和iPSCs定向分化所需的目的基因的导入需借助质粒实现，但目前用来转运质粒(含有目的基因)的基因转运系统存在缺陷。因此，开发高效的基因转运系统是十分必要的。本项目将首次制备多种安全高效的仿病毒结构的新型纳米基因转运系统，能高效侵入细胞、大量携载基因且安全可靠。新型基因转运系统携载荧光质粒高效稳定体外转染。通过新型基因转运法对DPSCs进行重编程，从而得到iPSCs。在牙髓干细胞的应用方面我们发现使用碱性成纤维细胞生长因子（bFGF）能够明显促进冷冻复苏DPSCs的快速增殖，这对于临床实际应用DPSCs具有重要意义。本项目尝试了利用DPSCs为种子细胞修复长距离外周神经缺损。含有DPSCs的实验组有明显的新生神经纤维以及血管形成，且形成的新生神经纤维排列比较规则，与自体神经移植组类似；而单纯材料组新生神经纤维的量较少，同时排列较为松散。这个研究成果指出DPSCs在修复神经损伤的方面具有巨大的潜力，依靠这个实验成果，本项目进一步在大动物食蟹猴模型中进行了大段的坐骨神经修复的预实验，实验成果提示DPSC组能明显促进神经组织的修复形成。利用腺相关病毒研究表达bFGF的缺氧反应元件(5HRE)调控下的DPSCs在脊髓损伤修复中的作用，研究结果发现DPSCs能够分化为CD13+血管周细胞，上调n-钙粘蛋白表达，促进CD13+周细胞重返血管内皮细胞；CD13+周细胞重新附着在血管内皮细胞上，促进脊髓损伤修复。另外，该项目构建了一种天然高分子材料来源的HSPS复合神经导管，并携载过表达VEGF-A的雪旺细胞，携载有VEGF-A组HSPS神经导管在再生神经组织中发现CD31+内皮细胞明显增加，导致VEGFR2/ERK信号通路的激活，从而明显促进周围神经缺损的有效修复。该项目成功构建了具有高效基因转运能力的"纳米卡车"，完成了DPSCs诱导成iPSCs，并进一步证明了DPSCs作为种子细胞修复神经损伤的优秀能力，为DPSCs修复外周神经缺损和脊髓损伤的临床实验提供了充足证据。

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