Trypsin-induced VEGF expression in adipose-derived stem cells; optimization of assays
Translated title
Trypsin induceret VEGF ekspression i adipoøse stamceller; optimering af analysemetoder
Author
Lundsted, Ditte Helene
Term
4. term
Publication year
2012
Pages
55
Abstract
Background: Adipose-derived stem cells (ASCs) have demonstrated wound-healing potential, with pro-angiogenic effects largely mediated paracrinely through vascular endothelial growth factor (VEGF). Brief exposure to trypsin can increase VEGF expression via activation of protease-activated receptor 2, but the trypsin-responsive elements on the human VEGF promoter remain undefined. Objective: To determine the most suitable experimental setup and conditions for analyzing trypsin-induced VEGF promoter activity in ASCs, supporting efforts to enhance the therapeutic performance of stem cells for chronic wound treatment. Methods and results: Five deletion mutants of the human VEGF promoter were cloned into the pGL3-Basic vector and verified by restriction screening, polymerase chain reaction, and sequencing. ASCs were transfected with a green fluorescent protein reporter by lipofection, polyfection, and electroporation, and cell recovery and transfection efficiency were assessed microscopically. Electroporation at 300 V, 1,000 µF, and 10 µg DNA per sample was identified as optimal and outperformed lipofection and polyfection. In an optimized luciferase activity assay using the full-length VEGF promoter, brief trypsin exposure 18 hours after transfection produced a measurable luminescence signal at 36 hours. Conclusion: VEGF promoter constructs were verified, electroporation conditions were optimized and superior to alternative methods, and the luciferase assay was suitable for detecting trypsin-induced VEGF promoter activity in ASCs. Further studies are needed to map the promoter regions responsible for trypsin-dependent regulation.
Baggrund: Adiposederiverede stamceller (ASC) har dokumenterede sårhelende egenskaber, hvor den pro-angiogene effekt primært medieres parakrint via vaskulær endotelial vækstfaktor (VEGF). En kort eksponering for trypsin kan øge VEGF-udtryk via aktivering af proteaseaktiveret receptor 2, men de trypsin-responsive elementer i den humane VEGF-promotor er endnu ikke klarlagt. Formål: At fastlægge det mest optimale forsøgssetup og betingelser til analyse af trypsin-induceret VEGF-promotoraktivitet i ASC med henblik på at understøtte udviklingen af mere effektive stamcellebehandlinger af kroniske sår. Metoder og resultater: Fem deletionsmutanter af den humane VEGF-promotor blev klonet i pGL3-Basic-vektoren og verificeret ved restriktionsscreening, polymerasekædereaktion og sekventering. ASC blev transficeret med en grøn fluorescerende protein-rapportør ved tre metoder (lipofektion, polyfektion og elektroporation), og cellegenvinding samt transfektionseffektivitet blev vurderet mikroskopisk. Elektroporation med 300 V, 1.000 µF og 10 µg DNA pr. prøve blev identificeret som optimale betingelser og var bedre end både lipofektion og polyfektion. I en optimeret luciferase-aktivitetstest med fuldlængde VEGF-promotor gav kort trypsin-eksponering 18 timer efter transfektion et målbart luminescenssignal 36 timer efter. Konklusion: VEGF-promotor-konstruktionerne blev verificeret, elektroporationsbetingelserne blev optimeret og var overlegen de øvrige metoder, og den udviklede luciferaseassay var egnet til at måle trypsin-induceret VEGF-promotoraktivitet i ASC. Yderligere undersøgelser er nødvendige for at identificere de promotorregioner, der medierer den trypsinafhængige regulering.
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Keywords
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