| Gene therapy is a method that introduces healthy genes into cells to replace the disease-causing genes by utilization of the vectors. There are two common gene vectors be used for Gene therapy:viral gene vectors and non-viral gene vectors. And Nonviral gene delivery is currently a subject of increasing attention because of its relative safety and simplicity. The researchers find that non-viral gene vectors into cells mainly depend on pinocytosis, and the size, shape, surface charge and other physicochemical properties of the vectors has an effect on its way into cells; Furthermore, there are different ways for the same vectors to entry into different cells. There will be helpful for improving transport efficiency by studying the interaction between the carrier and the celler uptake. As a class of non-viral vectors, Metal complexes are concerned in recent years. In this work, we mainly studied the endocytic pathways into COS-7cells for the aggegates of [Co(NTB)Cl]+-DNA.Before the study of cell membrane pathways, we have first detected the plasmid DNA which we have extracted by using the US-visible spectroscopy and the Agarose gel electrophoresis. The results show that the purity and concentration of the plasmid DNA is higher, and the most structural forms are supercoiled DNA.In the cell experiment, We sutdy the cytotoxity of inhibitors which used in experiments by MTT method firstly. And we find that either alone inhibitor or inhibitors and complexes or aggregates, cytotoxicity is not large. Secondly, We study the cellular uptake pathways of aggregates by the luciferase gene expression method. The results show that the [Co(NTB)Cl]+/DNA aggregates into COS-7cells are mainly dependent on Caveolae-mediated endocytosis and Macropinocytosis. There have no effect on the uptake pathways when NLS are added, aggregates into COS-7cells still rely on Caveolae-mediated endocytosis and Macropinocytosis. Finally, we study the transfection of aggregates on HepG2cells, and the transfection of aggregates on COS-7cells are compared. The results show that aggregates on HepG2cells can transfect and have the highest transfection when the molar ratio of complexe and DNA on1:1. But in general, compared with COS-7cells, the aggregates have a lower transfection on HepG2cells. |
