RESUMO
Primary cell cultures were investigated as a tool for molecular diagnostics in a forensic setting. Fibroblast cultures had been established from human Achilles tendon resected at autopsies, from cases of sudden infant death syndrome and control infants who died in traumatic events (n=41). After isolation of primary cultures cells were stored at -135°C, and re-established up to 15 years later for experimental intervention. Growth characteristics in cultures were evaluated in relation to the age of the donor, the post mortem interval before sampling, and the storage interval of cells before entry into the study. High interpersonal variation in growth rates and cell doubling time was seen, but no statistically significant differences were found with increasing age of the donor (mean 19 weeks), length of post-mortem interval prior to sampling (6-100 h), or increase in years of storage. Fibroblast cultures established from post-mortem tissue are renewable sources of biological material; they can be the foundation for genetic, metabolic and other functional studies and thus constitute a valuable tool for molecular and pathophysiological investigations in biomedical and forensic sciences.
Assuntos
Tendão do Calcâneo/patologia , Fibroblastos/citologia , Mudanças Depois da Morte , Biópsia , Estudos de Casos e Controles , Contagem de Células , Proliferação de Células , Células Cultivadas , Patologia Legal , Humanos , Lactente , Manejo de Espécimes , Morte Súbita do Lactente , Fatores de TempoRESUMO
The aim of the present study was to investigate stress gene expression in cultured primary fibroblasts established from Achilles tendons collected during autopsies from sudden infant death syndrome (SIDS) cases, and age-matched controls (infants dying in a traumatic event). Expression of 4 stress responsive genes, HSPA1B, HSPD1, HMOX1, and SOD2, was studied by quantitative reverse transcriptase PCR analysis of RNA purified from cells cultured under standard or various thermal stress conditions. The expression of all 4 genes was highly influenced by thermal stress in both SIDS and control cells. High interpersonal variance found in the SIDS group indicated that they represented a more heterogeneous group than controls. The SIDS group responded to thermal stress with a higher expression of the HSPA1B and HSPD1 genes compared to the control group, whereas no significant difference was observed in the expression of SOD2 and HMOX1 between the two groups. The differences were related to the heat shock treatment as none of the genes were expressed significantly different in SIDS at base levels at 37 °C. SOD2 and HMOX1 were up regulated in both groups, for SOD2 though the expression was lower in SIDS at all time points measured, and may be less related to heat stress. Being found dead in the prone position (a known risk factor for SIDS) was related to a lower HSPA1B up-regulation in SIDS compared to SIDS found on their side or back. The study demonstrates the potential usefulness of gene expression studies using cultured fibroblasts established from deceased individuals as a tool for molecular and pathological investigations in forensic and biomedical sciences.
Assuntos
Chaperonina 60/genética , Proteínas de Choque Térmico HSP70/genética , Transtornos de Estresse por Calor/genética , Heme Oxigenase-1/genética , Proteínas Mitocondriais/genética , Morte Súbita do Lactente/genética , Superóxido Dismutase/genética , Tendão do Calcâneo/citologia , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Genética Forense , Patologia Legal , Temperatura Alta , Humanos , Lactente , Recém-Nascido , Decúbito Ventral , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para CimaRESUMO
The upregulation of stress proteins is an important step in the cellular defense against various endogenous and exogenous stressors, and the stress response systems are therefore essential to cell maintenance and survival. In order to evaluate the cellular capability to cope with exogenous stressors, we investigated stress response profiles in cultured human fibroblasts exposed to short-term heat shock or oxidative stress. Quantitative PCR analysis as well as Western blotting demonstrated a specific upregulation of Hsp70 mRNA and protein after heat shock, while heme oxygenase 1 (HO1 or Hsp32) mRNA and protein was specifically upregulated following H2O2-induced oxidative stress. Hsp70 and HO1 should therefore be considered as useful biomarkers for acute heat stress and oxidative stress, respectively. Moreover, the results indicate that the expression of Hsp70 and HO1 in response to exogenous stressors may be regulated through different mechanisms.
Assuntos
Fibroblastos/metabolismo , Resposta ao Choque Térmico/genética , Biologia Molecular/métodos , Estresse Oxidativo/genética , Western Blotting , Células Cultivadas , DNA Complementar/biossíntese , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/isolamento & purificação , Proteínas de Choque Térmico/metabolismo , Humanos , Reação em Cadeia da Polimerase , RNA/isolamento & purificaçãoRESUMO
Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding, and preliminary experiments suggested that the variant protein displayed prolonged association with chaperonins and delayed formation of active enzyme. Accordingly, the molecular pathogenesis of SCAD deficiency may rely on intramitochondrial protein quality control mechanisms, including degradation and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate with mitochondrial hsp60 chaperonins; however, the variant SCAD proteins remained associated with hsp60 for prolonged periods of time. Biogenesis experiments at two temperatures revealed that some of the variant proteins (R22W, G68C, W153R, and R359C) caused severe misfolding, whereas others (R147W, G185S, and Q341H) exhibited a less severe temperature-sensitive folding defect. Based on the magnitude of in vitro defects, these SCAD proteins are characterized as folding-defective variants and mild folding variants, respectively. Pulse-chase experiments demonstrated that the variant SCAD proteins either triggered proteolytic degradation by mitochondrial proteases or, especially at elevated temperature, aggregation of non-native conformers. The latter finding may indicate that accumulation of aggregated SCAD proteins may play a role in the pathogenesis of SCAD deficiency.