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Master's Dissertation
DOI
https://doi.org/10.11606/D.10.2020.tde-28012020-105305
Document
Author
Full name
Rafael Garcia Karam
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Ambrosio, Carlos Eduardo (President)
Freitas, Silvio Henrique de
Vidane, Atanásio Serafim
Title in Portuguese
Avaliação do padrão de secreção e morfologia de vesículas extracelulares derivadas de células-tronco da membrana amniótica canina
Keywords in Portuguese
Exossomos
Mesenquimais
Microvesículas
Abstract in Portuguese
O estudo das vesículas extracelulares (VEs) secretadas pelas células-tronco mesenquimais (CTM) em cultura pode ajudar a entender de maneira mais concisa o seu funcionamento. Este trabalho tem como objetivo comparar a quantidade e tamanho das vesículas extracelulares pequenas (até 150nm) derivadas de célulastronco da membrana amniótica canina. Foram isoladas células da membrana amniótica de cadelas, cultivadas e caracterizadas. Para responder ao nosso objetivo, o número de células foi normalizado em cada passagem. As CTM isoladas apresentaram uma curva de crescimento crescente até atingir a segunda passagem, seguindo-se uma diminuição até a quinta passagem. As células foram diferenciadas em tecido adipogênico, condrogênico e osteogênico. Análises por citometria de fluxo mostraram que as CTM foram positivas para marcadores mesenquimais CD90 (62,6%) e CD105 (89,5%) e negativas para marcadores hematopoiéticos CD34 (5,2%) e CD45 (3,1%). Para a análise de VEs, as células em diferentes passagens (P0-P2) foram cultivadas até atingir 80% de confluência, em seguida, o meio foi substituído por meio livre de VEs (previamente ultracentrifugado). Após 48h, o meio de cultura foi removido e centrifugado para isolar as VEs, seguido por análise de nanosight para medir a concentração e o tamanho das VEs. A caracterização por western blot também foi realizada. As VEs foram positivas para Alix e negativos para Citocromo C. Os resultados demonstraram que a concentração nessas diferentes passagens analisadas foi aumentada em P0 comparado a P1 e P2 (P0=1,2x109±71220315, P1=3,8x108±54173333, e P2=3,3x108±170128967). No entanto, não foram encontradas diferenças no tamanho das VEs (P0 = 132nm, P1 = 130nm e P2 = 120nm). Estes resultados demonstram que a passagem inicial é enriquecida de VEs quando comparada com as passagens posteriores, indicando um efeito de deficiência in vitro no padrão de secreção das VEs.
Title in English
Evaluation of secretory pattern and morphology of extracellular vesicles derived from canine amniotic membrane stem cells
Keywords in English
Exosome
Mesenchymal
Microvesicles
Abstract in English
The study of the extracellular vesicles (EVs) secreted by stem cells in culture may help to understand in a more concise way their functioning. This work aims to compare the amount and size of the small extracellular vesicles (up to 150nm) derived from stem cells derived from the canine amniotic membrane. Bitches cells from the amniotic membranes were isolated, cultured and characterized. In order to answer our aim the number of cells was normalized at each passage. The isolated stem cells presented an increasing growth curve until reach the second passage, following by a decrease until the fifth passage. The cells were differentiated into adipogenic, chondrogenic and osteogenic tissue. Flow cytometry analysis showed that the MSC were positive for CD90 (62.6%) and CD105 (89.5%) and negative for CD34 (5.2%) and CD45 (3.1%), mesenchymal and hematopoietic marks, respectively. For EV analysis, cells in different passages (P0-P2) were culture until reach 80% of confluence, then the medium was replaced by EVs depleted medium (previously ultracentrifugated). After 48h, culture medium was removed and centrifuged to isolate EVs, followed by nanosight analysis to measure the EVs concentration and size. The EVs were also characterized by western blot. They were positive for Alix and negative for Cytochrome C. Our results demonstrated that the concentration in those different passages analyzed was increased in P0 compared to P1 and P2. However, no differences were found in EVs size (P0=132nm, P1=130nm and P2=120nm). These results demonstrate that earlier passage is enriched of EVs when compare to later passage, indicating an in vitro impairment effect on EVs secretion pattern.
 
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Publishing Date
2020-04-14
 
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