Hipóxia em fibroblastos humanos cultivados de derme normal e de quelóide
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Date
2003
Authors
Keira, Sidney Mamoru [UNIFESP]
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Ferreira, Lydia Masako [UNIFESP]
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Tese de doutorado
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Abstract
O presente estudo teve como objetivos comparar a origem dos fibroblastos humanos, derme normal ou queloide, em situacoes de normoxia e hipoxia, e comparar o efeito da hipoxia em fibroblastos humanos cultivados originados de derme normal e de queloide, atraves da mensuracao da biossintese de colageno. Os fibroblastos foram provenientes de derme normal (fragmentos cirurgicos de pele normal) e de queloide (fragmentos da regiao central de lesoes queloidianas) e foram mantidos em normoxia ou submetidos a uma sessao de hipoxia com uma mistura gasosa composta de nitrogenio e gas carbonico. Por outro lado, para a mensuracao do colageno produzido pelos fibroblastos, determinou-se uma sequencia de procedimentos fisico-quimicos de isolamento dos colagenos tipos I, II e III, no leito celular e no meio de cultura, atraves de reacao de precipitacao com sulfato de amonio, seguido de reacao de precipitacao cromogenica com o corante Sirius Red e finalizado com a mensuracao es pectrofotometrica da biossintese desta proteina. Os resultados submetidos a analise estatistica evidenciaram que a hipoxia, de forma isolada, nao alterou a biossintese de colageno. Contudo, observou-se que os fibroblastos derivados de queloide apresentaram uma biossintese de colageno maior do que os derivados de derme normal, quando mantidos em normoxia ou quando submetidos a hipoxia
The objective of this study was to compare the effect of hypoxia on cultivated fibroblasts from normal dermis and keloid by measuring the biosynthesis of collagen. Culture of fibroblasts derived from normal dermis (surgical fragments of normal skin) and keloids (biopsies of the central zone of keloids) were established. These fibroblasts were maintained in normoxia or submitted to a hypoxia session with a gaseous mixture of nitrogen and carbonic dioxide. A sequence of physicochemical procedures was determined to isolate the collagen type I, II and III from the cellular layer and the culture medium, consisting in a precipitation reaction with ammonium sulfate. In a subsequent step, a chromogenic precipitation reaction with the dye Sirius Red and an elution procedure was used to measure the biosynthesis of collagen with the aid of spectrophotometry. The results have shown that hypoxia, as an isolated stress factor, is not able to modify the fibroblast collagen synthesis. However, it was noted that the fibroblasts from keloids presented a greater collagen biosynthesis than those from normal dermis when they were maintained in normoxia as well as when submitted to hypoxia.
The objective of this study was to compare the effect of hypoxia on cultivated fibroblasts from normal dermis and keloid by measuring the biosynthesis of collagen. Culture of fibroblasts derived from normal dermis (surgical fragments of normal skin) and keloids (biopsies of the central zone of keloids) were established. These fibroblasts were maintained in normoxia or submitted to a hypoxia session with a gaseous mixture of nitrogen and carbonic dioxide. A sequence of physicochemical procedures was determined to isolate the collagen type I, II and III from the cellular layer and the culture medium, consisting in a precipitation reaction with ammonium sulfate. In a subsequent step, a chromogenic precipitation reaction with the dye Sirius Red and an elution procedure was used to measure the biosynthesis of collagen with the aid of spectrophotometry. The results have shown that hypoxia, as an isolated stress factor, is not able to modify the fibroblast collagen synthesis. However, it was noted that the fibroblasts from keloids presented a greater collagen biosynthesis than those from normal dermis when they were maintained in normoxia as well as when submitted to hypoxia.
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Citation
KEIRA, Sidney Mamoru. Hipóxia em fibroblastos cultivados de derme normal e de quelóide. 2003. 140 f. Tese (Doutorado) - Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2003.