• Ditte Helene Lundsted
4. semester, Medicin med Industriel Specialisering, Kandidat (Kandidatuddannelse)
Background: Increasing evidence indicates that adipose-derived stem cells (ASCs) possess wound healing properties. The pro-angiogenic effect is mainly paracrine, exerted through cytokines, such as the vascular endothelial growth factor (VEGF). Hence, there has been great interest in the attempt to increase VEGF expression in order to optimize the effect of transplanted stem cells. One way to do this is to shortly expose the ASCs to trypsin, which induces the VEGF expression through activation of the protease-activated receptor 2. However, trypsin-induced response elements on the human VEGF promoter are yet to be elucidated.
Objective: The current work will focus on determining the most optimal experimental setup and conditions for analyzing trypsin-induced VEGF-promoter activity.
Methods/Results: Five deletion mutants of the human VEGF promoter cloned into the pGL3-Basic Vector were verified by restriction screening, polymerase chain reaction, and sequencing.
The ASCs were transfected with a green fluorescent protein reporter plasmid by the three different methods; lipofection, polyfection, and electroporation. For each method, the cell recovery and transfection efficiency were evaluated microscopically. Optimized electroporation conditions for the ASCs were found to be 300V, 1,000 µF, and 10 µg DNA per sample. With these conditions electroporation was found superior to both lipofection and polyfection.
In the optimization of the luciferase activity assay, the ASCs were electroporated with the vector containing the full-length VEGF promoter. When the transfected ASCs were shortly exposed to trypsin 18 h post electroporation, measurable level of luminescence was detected 36 h post electroporation.
Conclusion: The VEGF promoter deletion mutants cloned into pGL3 vectors were verified. Furthermore, optimized electroporation conditions were found superior to both lipofection and polyfection. Finally, the luciferase assay was designed and found suitable for measure the trypsin-induced VEGF promoter activity in ASCs. In order to clarify promoter regions involved in the trypsin dependent VEGF expression, further investigations are necessary.
SprogEngelsk
Udgivelsesdato2012
Antal sider55
ID: 66499405