Decorin/fusin双基因修饰骨髓间充质干细胞趋化动力学及抗纤维化的研究

目的 观察双基因(Decorin/fusin)修饰骨髓间充质干细胞(MSCs)体外趋化动力学及对纤维合成的影响.方法 pLVX-puro为载体,pLTR-CMV-DCN、pLTR-CMV-FIB-DCN、pLTR-CMV-FIB-Fusin为质粒,构建慢病毒嵌合体融合基因载体;HEK293细胞包装,滴度1×107cfu/ml病毒液转染MSCs,puromycin抗性筛选、纯化.检测转染效率、基因表达情况、修饰MSCs体外趋化动力学及Transwell纤维合成.结果 基因转染效率为11%,筛选纯化后达30%;外源基因可稳定表达;修饰MSCs具有微环境依赖性体外趋化、穿膜及抗纤维合成能力.结论 双基因修饰MSCs可影响其基因表达和趋化动力学并因微环境变化而降低体外纤维合成.

In vitro Study of chemotaxis dynamics and anti-fibrillar synthesis of Decorin/fusin dual-gene-modified mesenchymal stem cells

Objective To investigate in vitro chemotaxis dynamics and anti-fibrillar synthesis of dual-genes-modified mesenchymal stem cells ( MSCs). Methods A recombinant lentiviral-mediated chimera fusion genes was constructed using pLTR-CMV-DCN, pLTR-CMV-FIB-DCN, pLTR-CMV-FIB-Fusin plasmids and a pLVX-puro vector. Viral particles titer 1×107 cfu/ml obtained through HEK 293 cells packages were transfected into MSCs followed by puromycin selection. Transfection efficiency, gene expression were analyzed;chemotaxis,Transwell in vitro assay of fibrillar synthesis were assessed. Results Transfection efficiency 11%, after selection and purification 30%. Migrational ability and anti-fibrillar synthesis were shown under various conditions. Conclusion Dual-gene-modified mesenchymal stem cells could undergo a conformational structural change under specific microenvironment, affecting its genes expression, chemotaxis dynamics,and in vitro fibrillar synthesis.