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Previous issue date: 2015-03-13 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Condroitim sulfato (CS) ? um glicosaminoglicano natural presente na matriz
extracelular de tecidos conectivos, podendo ser extra?do e purificado desses tecidos.
O CS est? envolvido em diversas fun??es biol?gicas, o que pode estar relacionado ?
variabilidade estrutural que possui, apesar da simplicidade da cadeia linear dessa
mol?cula. Pesquisas na ?rea biotecnol?gica e farmacol?gica com rejeitos
provenientes da aquicultura vem sendo desenvolvidas no Brasil. Nas ?ltimas
d?cadas, a til?pia (Oreochromis niloticus), peixe nativo da ?frica, tem sido uma das
esp?cies mais cultivadas em v?rias regi?es do mundo, inclusive no Brasil. A
tilapicultura ? uma atividade economica cujo principal inconveniente ? a grande
quantidade de res?duos que s?o descartados pelos produtores. Entende-se que o
material descartado pode ser aproveitado em pesquisas como fonte de mol?culas
com importante aplica??o biotecnol?gica, o que tamb?m contribui na redu??o de
impactos ambientais e favorece o desenvolvimento dessa atividade de maneira
ecologicamente correta. Dessa forma, v?sceras de til?pias nil?tica foram submetidas
? prote?lise, em seguida os glicosaminoglicanos foram complexados com resina de
troca i?nica (Lewatit), fracionados com volumes crescentes de acetona e purificados
atrav?s da cromatografia de troca i?nica DEAE-Sephacel. Na sequ?ncia, a fra??o foi
analisada atrav?s de eletroforese em gel de agarose e resson?ncia magn?tica
nuclear (RMN). O perfil eletrofor?tico do composto, a an?lise dos espectros 1H de
RMN e a correla??o do HSQC permitem afirmar que o composto corresponde a uma
mol?cula de condroitim sulfato. O ensaio de MTT foi utilizado para avalia??o da
viabilidade celular na presen?a do CS isolado de til?pia e mostrou que o composto
n?o ? citot?xico a c?lulas normais provenientes do fibroblasto de embri?o de
camundongos (3T3). Ent?o, o composto foi testado quanto a capacidade de reduzir
o influxo de leuc?citos em modelo de peritonite aguda (in vivo) induzida por
tioglicolato de s?dio. Nesse contexto, foi realizada a contagem total e diferencial de
leuc?citos do sangue e l?quido peritoneal coletadas, respectivamente, da veia cava e
do lavado peritoneal de cada animal submetido ao experimento. O condroitim
sulfato, pela primeira vez isolado de til?pia (CST
), foi capaz de reduzir a migra??o de
leuc?citos ? cavidade peritoneal de camundongos inflamados em at? 80,4% na dose
de 10?g/kg. Os resultados mostram ainda que houve redu??o significativa (p<0,001)
da popula??o de polimorfonucleares do lavado peritoneal nas tr?s doses testadas
(0,1?g/kg; 1?g/kg e 10?g/kg) quando comparado ao controle positivo (apenas
tioglicolato). Portanto, uma vez que a estrutura e o mecanismo de a??o do CST
tenham sido totalmente elucidados, esse composto pode apresentar potencial para
uso terap?utico em doen?as inflamat?rias. / Chondroitin sulfate (CS) is a naturally glycosaminoglycan found in the extracellular
matrix of connective tissues and it may be extracted and purified those tissues. CS is
involved in various biological functions, which may be related to the having structural
variability, despite the simplicity of the linear chain structure from this molecule.
Researches in biotechnology and pharmaceutical field with wastes from aquaculture
has been developed in Brazil. In recent decades, tilapia (Oreochromis niloticus),
native fish from Africa, has been one of the most cultivated species in various regions
of the world, including Brazil. The tilapia farming is a cost-effective activity, however,
it generates large amount of wastes that are discarded by producers. It is understood
that waste from tilapia can be used in research as a source of molecules with
important biotechnological applications, which also helps in reducing environmental
impacts and promote the development of an ecofriendly activity. Thus, nile tilapia
viscera were subjected to proteolysis, then the glycosaminoglycans were complexed
with ion exchange resin (Lewatit), it was fractionated with increasing volumes of
acetone and purified by ion exchange chromatography DEAE-Sephacel. Further, the
fraction was analyzed by agarose gel electrophoresis and nuclear magnetic
resonance (NMR). The electrophoretic profile of the compound together the analysis
of 1H NMR spectra and the HSQC correlation allow to affirm that the compound
corresponds to a molecule like chondroitin sulfate. MTT assay was used to assess
cell viability in the presence of CS tilapia isolated and showed that the compound is
not cytotoxic to normal cells such as cells from the mouse embryo fibroblast (3T3).
Then, this compound was tested for the ability to reduce the influx of leukocytes in
model of acute peritonitis (in vivo) induced by sodium thioglycolate. In this context, it
was done total and differential leukocytes counting in the blood and peritoneal fluid
collected respectively from vena cava and the peritoneal cavity of the animals
subjected to the experiment. The chondroitin sulfate for the first time isolated from
tilapia (CST
) was able to reduce the migration of leukocytes to the peritoneal cavity of
inflamed mice until 80.4 per cent at a dose 10?g/kg. The results also show that there
was a significant reduction (p<0.001) of the population of polymorphonuclear
leukocytes from peritoneal cavity in the three tested doses (0.1?g/kg; 1?g/kg and
10?g/kg) when it was compared to the positive control (just thioglycolate). Therefore,
since the CST structure and mechanism of action has been completely elucidated,
this compound may have potential for therapeutic use in inflammatory diseases
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/19791 |
| Date | 13 March 2015 |
| Creators | Pinto, L?via de Lourdes de Sousa |
| Contributors | 01843965496, http://lattes.cnpq.br/9951316929526841, Dore, Celina Maria Pinto Guerra, 03383860418, http://lattes.cnpq.br/2279068301048550, Pedrosa, Matheus de Freitas Fernandes, 96728647449, http://lattes.cnpq.br/2929963416385218, Souza, Giulianna Paiva Viana de Andrade, Filgueira, Luciana Guimar?es Alves |
| Publisher | Universidade Federal do Rio Grande do Norte, PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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