The Construction And Evaluation Of A Novel Non-viral Gene Transfection System And Its Application In Mesenchymal Stem Cells Gene Recombination

Objective:To enhance the level and prolong the duration of gene expression for gene-engineered rat mesenchymal stem cells (MSCs) using non-viral vector, and to provide a better environment for the proliferation of MSCs for the clinical therapy of cartilage defect.Methods:1. Pullulan-spermine was synthesized and incubated with pDNA to make gene complexes. The particle size and Zeta potential of the gene complexes were investigated.2. The transfection effect of gene complexes were evaluated in various types of cells. A reverse transfection system based on anionic gelatin was developed. The reverse gene transfection system was evaluated for their transfection efficiency as compared with the conventional transfection methods. 3. Collagen sponge and polyethylene terephthalate (PET) non-woven fabric were introduced as scaffolds to perform three-dimensional (3D) cell culture with reverse transfection. pDNA coding for TGFβ-1 was delivered to MSCs to assess its ability in inducing chondrogenesis.4. The biodegradable chitosan thermo-sensitive hydro gel and gelatin sponge were used as 3D scaffolds to implant the pullulan-spermine/pTGFβ-1 transfected MSCs to induce the cartilage regeneration.Results:Pullulan-spermine/pTGFβ-1 complexes are nanoparticles with positive charge which can successfully transfect MSCs. The reverse transfection method induced higher transgene levels than the conventional transfection method in the presence of serum. The electric charge of the anionic gelatin plays an important role in reverse gene transfection system by affecting the release pattern of the gene complexes and the adsorption of serum protein to the substrate. Different transfection methods might change the cellular uptake pathway of gene complexes in a cell type and vector dependent manner. During a long-time in vitro culture, MSCs cultured on 3D scaffolds exhibited a higher transgene expression level and more sustained transgene expression than those cultured and transfected on the two-dimensional (2D) substrate. When chitosan thermo-sensitive hydrogel was used as the scaffold to implant pTGFβ-1 transfected MSCs to treat the cartilage defect, the treatment group had no evident difference with the control group. pTGFβ-1 incorporated gelatin sponge or pTGFβ-1 transfected MSC seeded on gelatin sponge can induce better articular cartilage regenerative effect than the control group.Conclusion:The reverse transfection system permits the presence of serum in the transfection process and the combination of this method with 3D cell culture scaffold benefit the cell proliferation and long-time gene transfection of MSCs in vitro. pTGFβ-1 transfected MSC can promote the articular cartilage regeneration of the rat when using gelatin sponge as a scaffold.