Non-Viral Co-Delivery Of Multi-Gene For Fibroblasts Differentiation Into Neural Cells

Direct reprogramming, a novel technology for directly switching terminally differentiated cells into another, provides a new source for cell transplantation in cinical application. However, this technology is restricted in neuron differentiation due to the gene vectors involoved, most of which are virus with potential risks, nanopaticles has been paid more and more attention because of its advantages of safety, high efficiency and so on. Herein, this study focused on the direct reprogramming of converting fibroblasts into nerve cells based on the non-viral vectors. In this study, ethylenediamine modified porphyra yezoensis polysaccharide was prepared as gene vector, and then combined with the nerve-related transcription factors and thereby directly reprogramed fibroblasts into nerve cells, mainly divided into four parts:Chapter One: ReviewThis section was dividied into two parts. The first part reviewed the development and applicaiton of gene vectors in gene therapy, including the comparision of different gene vectors. In addition, the cell sources for nervous system diseases therapy were introduced in the second part, such as their respective merits and demerits as well as problems unresolved.Chapter Two: Preparation and characterization of cationized Porphyra yezoensis polysaccharideIn this section, the Porphyra yezoensis polysaccharide was oxidated through potassium periodate followed by cationization with ethylenediamine(Ed) and spermine(Sm) respectively to obtain Ed-PYP and Sm-PYP. Moreover, cationized Porphyra yezoensis polysaccharides were characterized by FTIR(KBr) and Zeta potential particle size analyzer for further analysis. Chapter Three: Preliminary study on ethylenediamine modified Porphyra Yezoensis polysaccharide as a gene vectorThe cationized polysaccharide was used as the vector and the fibroblasts were used as seed cells. The Ed-PYP/pDNA complexes were characterized by agarose gel electrophoresis, and cytoxicity was tested by MTT, both of which indicated that Ed-PYP could potentially be used as a gene vector. Afterwards, EGFP delivery and ELISA were carried out to evaluate the expression of the interrelated factors in both visulization and protein level. The results showed that Ed-PYP/pDNA complexes achieved the optimum transfection efficiency in vitro at a weight ratio of 40:1, which was better than Polyethylenimine(PEI) and LipofectamineTM2000(Lip2000).Furthermore, YOYO-1 is a plasmid DNA fluorescence marker, as well as a variety of specific inhibitors were employed to investigate the mechanisms of cell uptake and intracellular trafficking of Ed-PYP/p DNA complexes in respective. And the results demonstrated that clathrin-mediated endocytosis and caveolin-mediated endocytosis which were the main pathway of cellular uptake of the Ed-PYP/pDNA complexes. In addition, the intracellular trafficking of Ed-PYP/pDNA complexes was participated with endosome-lysosome, cytoskeleton, intermediate filament and cytoplasmic dynein system.Chapter Four: The study of fibroblasts induced and differentiation into nerve cellsAccording to our previous work, Ed-PYP with nerve related transcription factors(Ed-PYP/pABF, Ed-PYP/pABT) were prepared at a weight ratio of 40:1 for fibroblasts neuronal differentiation, and characterized by agaroe gel electrophoresis, TEM analysis and Zeta potential particle size analyzer. The results are in accordance with tentative test.After cell morphological obsercation and ELISA analysis, the optimum efficiency was obtained at 14 days after transfection. Furthermore, immunofluorescence and western blotting were carried out to identify the induced fibroblasts. The results proved that the fibroblasts could be directly reprogrammed into nerve cells based on the induction of cationized porphyra yezoensis polysaccharide with neuron-related gene, which provide a rich supply of cells for clinical therapy of nervous system disease.