论文部分内容阅读
本文拟构建一种模拟细胞外基质结构与功能的基因活化支架(ECM-m-GAM),并对其结构、机械强度和释放性能进行表征。首先制备细胞穿膜肽TAT修饰的脂质体基因载体TAT-LPD,然后将TAT-LPD与RGD修饰的透明质酸混合,加入交联剂MMPs敏感肽(HS-MMP-SH),使透明质酸凝胶化,在凝胶化过程中实现对TAT-LPD的负载,从而将细胞黏附因子RGD、MMPs敏感底物和高效的基因转染载体TAT-LPD有机地整合到一个结构中,完成ECM-m-GAM的构建。利用PicoGreen试剂盒考察ECM-m-GAM在不同释放介质中DNA的释放行为。研究结果表明:TAT-LPD在透射电镜下呈球形,平均粒径为(263.0±4.30)nm,可被成功地包埋在ECM-m-GAM中;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1 600 Pa,适合支架植入和组织修复;DNA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,能够转染大鼠骨髓间充质干细胞(BMSCs)并表达绿色荧光,为后续的细胞转染和组织修复研究奠定了基础。
In this paper, we will construct a gene-activated scaffold (ECM-m-GAM) that simulates the structure and function of extracellular matrix and characterize its structure, mechanical strength and release properties. Firstly, TAT-LPD, a cell-penetrating peptide TAT modified liposome, was prepared and mixed with RGD-modified hyaluronic acid (TAT-LPD). Then the crosslinker MMPs sensitive peptide (HS-MMP-SH) Acid gelation and the loading of TAT-LPD in the process of gelation, so as to organically integrate the cell adhesion factor RGD and MMPs sensitive substrates and the efficient gene transfection vector TAT-LPD into a structure to complete the ECM -m-GAM build. The release of DNA from ECM-m-GAM in different release media was investigated by PicoGreen kit. The results showed that TAT-LPD was spherical under transmission electron microscope with an average diameter of (263.0 ± 4.30) nm and could be successfully embedded in ECM-m-GAM. ECM-m-GAM had typical gel structure , The mechanical strength increased with the increase of the dosage of hyaluronic acid. When the dosage of hyaluronic acid was 4%, the elastic modulus was about 1 600 Pa, which was suitable for stent implantation and tissue repair. The release of DNA from ECM-m-GAM Showing obvious MMPs sensitivity, and the released DNA still exists in the form of nanoparticles. It can transfect rat bone marrow mesenchymal stem cells (BMSCs) and express green fluorescence, which lays the foundation for subsequent cell transfection and tissue repair research .