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Endothelial microparticles detection: in vitro studies using huvec
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Orientador(es)
Resumo(s)
Endothelial microparticles (EMP) are microparticles originated from activated or apoptotic endothelial cells, measuring 0,1 to 1 µm, and containing cell material, such as proteins, mRNA and lipoproteins.
The causal role of EMP in the development and progression of cardiovascular diseases is now beginning to be well documented. In fact, circulating EMP levels are elevated in several types of cardiovascular diseases and therefore a methodology allowing their accurate quantification is of clinical importance. However, the analytical and preanalytical steps and variables affecting EMP detection have only been examined by few studies and there is a need to define specific endothelial markers that allow their detection in vitro and in vivo.
With the aim of establishing a protocol to detect EMP by flow cytometry, we compared different analytical and preanalytical steps to understand which conditions affect the accuracy of this analysis in vitro. To investigate this, we used Human Umbilical Vein Endothelial Cells (HUVEC) as a study model. We studied different centrifugation protocols used to isolate EMP from HUVEC culture medium. We concluded that a high-speed centrifugation is necessary to successfully isolate EMP. We additionally investigated whether freezing the cell culture medium would affect EMP posterior identification and quantification. We concluded that one-week freezing of samples did not affect EMP detection when compared to fresh ones. Finally, we studied whether our protocol would affect the expression of one endothelial cell marker (CD31) in EMP. Surprisingly, we were unable to detect CD31 in our EMP, suggesting the occurrence of problems in the cytometer calibration or in the staining procedure, demanding future investigation.
In conclusion, quantification of circulating EMP may be used as important tools to study the physiopathology and progression of cardiovascular diseases. Further studies are necessary to optimize methodologies allowing their accurate detection in the clinical setting.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2016
Palavras-chave
Cardiovascular diseases Endothelial dysfunction Endothelial microparticles Flow cytometry Human umbilical vein endothelial cells Mestrado Integrado - 2016