细胞外基质仿生的脂质体/凝胶复合型基因活化支架的构建与评价

Incorporating transfectable plasmid DNA encoding growth factors into the bioscaffolds to build gene-activated matrix (GAM) has been considered as a potential strategy for tissue regeneration. After implantation into the tissue defect, DNA can be released to transfect surrounding cells in situ and then the cells continuously secret the encoded growth factors for a period of time. This novel technique will overcome the shortages of traditional growth factors, such as the short half-life, the frequent drug administration and the high cost. However, there are some problems of GAM, such as the lack of molecular signals for cell adhesion, hard to control the gene release and the low transfection efficiency. In this project, therefore, we propose a novel GAM mimicking the structure and function of extracellular matrix (ECM), abbreviated as ECM-m-GAM. The cell penetrating peptide TAT modified liposomes, employed as efficient gene transfer vectors, will be incorporated into RGD-hyaluronic acid hydrogels cross-linked by matrix metalloproteinase (MMPs) sensitive peptides. The obtained liposome/hydrogel composite scaffolds are ECM-m-GAM. Firstly, cells can adhere to ECM-m-GAM induced by RGD peptides. Secondly, the hydrogels will be degraded by cell-secreted MMPs, creating space for cell proliferation and releasing TAT modified gene vectors. Finally, the cells will be transfected effectively by TAT modified gene vectors. In this project, the relationship between the bionic performance of GAM and the transfection efficiency will be explored. The rat calvarial defect model will be employed to study the application of ECM-m-GAM for bone repair. This project will provide a new strategy for the design of gene delivery system for tissue regeneration.

将编码有生长因子并具有转染能力的DNA融合于生物支架，即得基因活化支架（GAM），植入组织缺损区域后可对周围细胞进行原位转染，一段时间内持续分泌DNA编码的生长因子，克服传统生长因子半衰期短、用药频繁、价格昂贵等缺点，是极具潜力的组织修复手段。但目前GAM存在细胞粘附不足、基因难以控释、转染效率低等问题。本项目拟用细胞穿膜肽TAT修饰脂质体作为高效的基因转染载体，并将其包载到以基质金属蛋白酶（MMPs）敏感肽为交联剂制备的RGD-透明质酸凝胶中，构建一种模拟细胞外基质结构与功能的GAM（ECM-m-GAM）。它利用RGD肽促进细胞在支架中的粘附，然后依靠细胞分泌的MMPs降解凝胶，为细胞的增殖创造空间，并同步释放TAT修饰的基因载体，介导高效的基因转染。本项目将探索GAM仿生性能与转染效率的关系，并建立大鼠颅骨缺损模型，评价其在骨修复中的应用，为组织再生修复中基因给药系统的设计提供新策略。

本研究构建了一种细胞外基质仿生的基因活化支架（ECM-m-GAM），并表征了它的机械性能和释放行为，评价其体外转染能力以及体内引导骨再生修复的能力。结果显示，ECM-m-GAM具有典型的凝胶结构，机械强度随着透明质酸用量的增加而增强，透明质酸用量为4%时其弹性模量约为1600 Pa，适合支架的植入；DNA（报道基因pEGFP-N1）从ECM-m-GAM中的释放呈现明显的MMPs敏感性，并且释放的DNA仍以纳米粒的形式存在；ECM-m-GAM能够体外转染大鼠骨髓间充质干细胞（BMSCs）并使其表达DNA编码的绿色荧光蛋白，证明ECM-m-GAM具备作为转染支架的可行性。. 研究过程中我们发现jetPEI具有更好的转染效率，因此，我们以jetPEI/DNA代替TAT-LPD构建ECM-m-GAM并开展后续研究。细胞毒性实验表明空白凝胶支架的细胞毒性很低，ECM-m-GAM的细胞毒性主要来源于内部包载的转染载体（如jetPEI）；最后，以表达骨形成蛋白BMP-2的质粒pDNA-BMP-2为治疗基因，制备了包载jetPEI/pDNA-BMP-2纳米复合物的ECM-m-GAM，它能够在体外转染MC3T3成骨细胞并使其表达BMP-2蛋白。建立大鼠颅骨缺损模型，将ECM-m-GAM植入到缺损部位，结果显示ECM-m-GAM组的新生骨体积明显大于空白对照组，表明ECM-m-GAM具有引导骨再生的能力。. 研究结果为该新型支架在组织工程中的应用提供了理论基础和实验依据，也为基因活化支架的仿生设计与构建提供了新的思路。此外，负载pDNA-BMP-2的ECM-m-GAM可以在植入部位实现对细胞的原位转染，持续分泌BMP-2，引导骨组织的再生修复，有望克服临床使用的重组人骨形成蛋白rhBMP-2蛋白半衰期短、需大剂量反复用药、价格昂贵等缺点，为骨缺损提供高效、价廉、切实可行的治疗方法。