Introduction
Extracellular membrane vesicles (EVs) are released into the extracellular environment by various cell types. They are identified into three classes (exosomes, microvesicles and apoptotic bodies) depending on their biogenesis. Evidently, EVs contain various components, including microRNAs [1]. It is thought that they have important roles in many biological processes. However, the role of EVs , especially vesicle-related miRNAs, in wound healing is poorly understood. This project aims to understand how wound healing processes are influenced by EVs and what species of vesicle-associated miRNAs participate in this healing.
Materials and Methods
The method of cell culture is used to harvest EVs and investigate the functional effects of vesicles on dermal healing. To isolate EVs, ultracentrifuge and differential centrifugation will be utilized. The EVs will be characterised by Western blot and microscopy. Candidate miRNAs associated with EVs will be isolated, detected and analysed by using miRCURYTM RNA, qRT-PCR and then next generation sequencing using Ion TorrentTM. Identified miRNAs will be investigated for their functional effect on epidermal cell proliferation, migration and protein expression.
Result and Discussion
Our results revealed that both sucrose gradient ultracentrifugation and the differential centrifugation successfully isolate EVs from cell culture media. Data revealed the presence of Hsp70, TSG 101 and CD81 is different in fractions and vesicles. Microscope analysis identified small vesicles with the size ranging from 40-100nm (exosomes), 100-1000nm (microvesicles) and 1000nm (apoptotic bodies). qRT-PCR identified the expression of miRNA 29a, miRNA 21, miRNA 203 and miRNA 141 from keratinocyte-derived extracellular membrane vesicles. These miRNAs express differently in different fractions and vesicles.
Conclusions
Extracellular membrane vesicles from keratinocytes have been characterized and expressed the vesicle markers Hsp70, TSG 101 and CD81. Additionally, microscopy has identified small vesicles as exosomes, microvesicles and apoptotic bodies. miRNAs also have been identified in vesicles.