Full text
| |
| Author(s): |
João Pedro Hübbe Pfeifer
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | Botucatu. 2021-07-08. |
| Institution: | Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária e Zootecnia. Botucatu |
| Defense date: | 2021-06-24 |
| Advisor: | Ana Liz Garcia Alves |
| Abstract | |
The paracrine function of stem cells (MSCs) is mainly performed by extracellular vesicles (EVs). Thus, the EV have functional impact on target cells and mechanisms of disease control through delivery microRNAs. For example, in osteoarthritis, in which joint homeostasis is regulated by microRNA-140. The objective of this work was to characterize extracellular vesicles (EVs) released by synovial membrane derived-mesenchymal stem cells (eqSMMSC) and to evaluate the expression of microRNA-140 in EVs and eqSMMSC cultivated in two cell culture models, in addition to comparing the secretion of EVs and the expression of microRNA-140 in an in vitro inflammatory environment. smMSCs were cultured and divided into four experimental groups. Control groups, one cultivation of 2D eqSMMSCs and one cultivation of 3D eqSMMSCs. Treated groups, also divided into 2D-OA and 3D-OA but were exposed to the culture medium simulating the “OA-Like” osteoarthritis inflammatory environment (IL-1β and TNF-α). Culture media samples from all groups were collected after 24, 72 and 120 hours of incubation for isolation and characterization of EVs. Isolation of EVs was performed with a commercial kit using the precipitation method. Characterization was performed by NTA, TEM and mRNA expression of tetraspanins CD9, CD63 and CD81. Analysis of microRNA-140 expression in EVs and cells by RT-qPCR and ADAMTS5 mRNA in EVs also by RT-qPCR. EVs could be isolated and characterized in all groups and time points. The expression of microRNA-140 in eqSMMSCs, the 3D-OA group had greater expression in relation to the other groups at time points 24 h; and in 72 h for both controls and 2D-OA; at 120 h, there was greater expression in the 3D group compared to those treated 2D-OA and 3D-OA. The expression of microRNA-140 in EVs stands out at 72 h, where the 3D group had significant expression in relation to the others, and at 120 h, the expression was also significant for 2D-OA in relation to the others. In ADAMTS5 analysis of mRNA expression, the 120 h time point can be 9 highlighted, where the 3D-OA group showed higher expression compared to control 2D and 3D. The data obtained showed that the model and the techniques used were effective for the isolation and characterization of EVs and for the identification of microRNA expression in different groups and time points. The expression of microRNA-140 in response to the inflammatory environment is performed by eqSMMSCs, and it was shown to be early in 3D cells and late in EVs from cells grown in 2D. (AU) | |
| FAPESP's process: | 19/05558-6 - Expression of microRNA-140 inside extracellular vesicles secreted by equine synovial membrane-derived mesenchymal stem cells |
| Grantee: | João Pedro Hübbe Pfeifer |
| Support Opportunities: | Scholarships in Brazil - Doctorate |