Expression and Characterization of Antimicrobial Peptide LL-37 in Dog Peripheral Blood Endothelial Progenitor Cells In Vitro

Abstract

This study aimed to construct antimicrobial peptide LL-37 expressed lentiviral vector and explore the expression of LL-37 in endothelial progenitor cells (EPCs) from dog periphery mononuclear cells (PBMCs). The methodology on obtaining EPCs from dog PBMCs was explored and infection approach about the expression of LL-37 in EPCs was optimized. LL-37 was amplified, digested by Age I and constructed into pGC-FU vector. Meanwhile, EPCs were generated from PBMCs using endothelial growth medium. Markers including acetylated low density lipoproteins (Dilac-LDL), Ulex lectin (UEA-1), von Willebrand factor (vWF), VE-cadherin, CD34 and CD133 were used to identify EPCs. Then, LL-37 was introduced into EPCs using lentivirus infection with green fluorescent protein (GFP) as the biomarker. Finally, we used western-blot to examine the expression of LL-37 in EPCs. The pGC-FU-LL-37-GFP was successfully constructed. Viral particles existed in 10−4 μL group with titer of 2 × 108 TU/mL. Both early (round-shaped morphology) and late (pebble-shaped appearance) EPCs were observed. And these EPCs were confirmed by Dil-acLDL uptake and FITC-UEA-1 binding. An approximate 50 % of infection efficacy was confirmed by immunofluorescence. Furthermore, we successfully observed LL-37 expression in EPCs via western blot analysis. In conclusions, LL-37 expressed in the EPCs was successfully achieved. However, the anti-infection and angiogenesis of LL-37 expressed EPCs need further research in future.