Trypsin-induced VEGF expression in adipose-derived stem cells; optimization of assays

Trypsin induceret VEGF ekspression i adipøse stamceller; optimering af analysemetoder

Lundsted, Ditte Helene

4. term

Medicine with Industrial Specialisation, Master

2012

55

Baggrund: Nyere forskning tyder på, at fedtvævsafledte stamceller (ASCs) kan fremme sårheling ved at stimulere dannelsen af nye blodkar (angiogenese), primært via udskilte signalstoffer som VEGF (vascular endothelial growth factor). For at styrke effekten af transplanterede stamceller ønsker man at øge VEGF-produktionen. En kort eksponering for trypsin kan øge VEGF via aktivering af proteaseaktiveret receptor 2 (PAR2), men de præcise områder på den humane VEGF-promotor (et DNA-kontrolelement) er endnu ikke kortlagt. Formål: at fastlægge en optimal forsøgsopsætning til at analysere trypsin-induceret aktivitet fra VEGF-promotoren. Metoder og resultater: Fem deletionsvarianter af den humane VEGF-promotor blev klonet i pGL3-Basic-vektoren og verificeret med restriktionsanalyse, PCR og sekventering. For at finde den bedste måde at introducere DNA i ASCs (transfektion) blev et grøn fluorescerende reporterplasmid (GFP) indført ved tre metoder: lipofektion, polyfektion og elektroporation (korte elektriske pulser, der midlertidigt gør cellemembranen gennemtrængelig). Celleoverlevelse og transfektionseffektivitet blev vurderet mikroskopisk. Elektroporation var bedst, med optimale betingelser på 300 V, 1.000 µF og 10 µg DNA pr. prøve. Til optimering af luciferase-assayet (en lysbaseret reporter til at måle promotoraktivitet) blev ASCs elektroporeret med vektoren, der indeholder VEGF-promotoren i fuld længde. Når cellerne kort blev udsat for trypsin 18 timer efter elektroporation, kunne der måles luminescens 36 timer efter elektroporation. Konklusion: De klonede VEGF-promotor-deletionskonstruktioner blev bekræftet. Elektroporation under de angivne betingelser var bedre end lipofektion og polyfektion. Den udviklede luciferase-opsætning er velegnet til at måle trypsin-induceret aktivitet fra VEGF-promotoren i ASCs. Yderligere undersøgelser er nødvendige for at identificere de promotorregioner, der ligger bag den trypsinafhængige VEGF-ekspression.

Background: Growing evidence shows that adipose-derived stem cells (ASCs) support wound healing by promoting new blood vessel formation (angiogenesis), mainly through signaling proteins they secrete, such as VEGF (vascular endothelial growth factor). To enhance the effect of transplanted stem cells, researchers aim to boost VEGF expression. Brief exposure to trypsin can increase VEGF by activating protease-activated receptor 2 (PAR2), but the exact response elements on the human VEGF promoter (a DNA control switch) remain unknown. Objective: to determine the optimal experimental setup to analyze trypsin-induced VEGF promoter activity. Methods and results: Five deletion variants of the human VEGF promoter were cloned into the pGL3-Basic vector and verified by restriction analysis, PCR, and sequencing. To identify the best way to introduce DNA into ASCs (transfection), a green fluorescent protein (GFP) reporter plasmid was delivered using three methods: lipofection, polyfection, and electroporation (short electrical pulses that temporarily open the cell membrane). Cell recovery and transfection efficiency were evaluated microscopically. Electroporation performed best, with optimal settings of 300 V, 1,000 µF, and 10 µg DNA per sample. For the luciferase assay (a light-based reporter to read promoter activity), ASCs were electroporated with the vector containing the full-length VEGF promoter. A brief trypsin exposure 18 hours after electroporation led to measurable luminescence 36 hours after electroporation. Conclusion: The VEGF promoter deletion constructs were validated. Electroporation under the stated conditions outperformed lipofection and polyfection. The luciferase setup is suitable for measuring trypsin-induced VEGF promoter activity in ASCs. Further studies are needed to identify the promoter regions involved in the trypsin-dependent VEGF expression.

[This abstract was generated with the help of AI]

Adipøse stamceller ; Sårheling ; Angiogenese ; Vascular endothelial growth factor (VEGF) ; Trypsin ; Transkriptionel regulation ; Promoter struktur ; Celledyrkning ; Tranfeltion ; Rekombinante DNA teknikker ; PCR

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Lundsted, Ditte Helene:

• Trypsin-induced VEGF expression in adipose-derived stem cells; optimization of assays (2012)