Résumé
The purpose of this work was to investigate the isolation, culture process of chicken gonadal primordial germ cells (PGCs) and study their biological characterization. PGCs were harvested from 5.5-day-old chicken embryonic genital ridges and explanted onto chicken embryonic fibroblasts (CEFs). The results showed that the primary cultivation of chicken PGCs on their own gonadal stroma cells were better than CEFs at first two days for reproduction. The conditioned media supported the growth and colony formation of PGCs for a prolonged time in vitro and maintained a normal diploid karyotype, which were positively stained by alkaline phosphatase (AKP), periodic acid Schiff (PAS) and reacted with anti-SSEA-1, SSEA-3, Oct4, Blimp1 and Sox2. Real-time PCR showed that they expressed the stage specific genes CVH, Blimp1 and Dazl, the stem cell specific genes Sox2, Pouv and Nanog. They also formed the embryoid bodies (EBs). These results suggested that the chicken PGCs cultured in vitro not only had strong self-renewal ability, but also had the potential capability of multi-lineage differentiation.
Résumé
BACKGROUND: Heavy metals can cause great harm to Siberian tigers in the natural environment. Cadmium (Cd2+) is an environmental contaminant that affects multiple cellular processes, including cell proliferation, differentiation, and survival. It has been shown to induce apoptosis in a variety of cell types and tissues. RESULTS: We investigated the apoptotic effects of Cd2+ on Siberian tiger fibroblasts in vitro. Our research revealed the typical signs of apoptosis after Cd²+ exposure. Apoptosis was dose- (0-4.8 µM) and duration-dependent (12-48 h), and proliferation was strongly inhibited. Cd²+ increased the activity of caspase-3, -8, and -9 and disrupted calcium homeostasis by causing oxidative stress and mitochondrial dysfunction. It also increased K+ efflux and altered the mRNA levels of Bax, Bcl-2, caspase-3, caspase-8, Fas, and p53. CONCLUSIONS: Our results suggest that Cd2+ triggers the apoptosis of Siberian tiger fibroblasts by disturbing intracellular homeostasis. These results will aid in our understanding of the effects of Cd2+ on Siberian tigers and in developing interventions to treat and prevent cadmium poisoning.
Sujets)
Animaux , Cadmium/toxicité , Apoptose/effets des médicaments et des substances chimiques , Espace intracellulaire/effets des médicaments et des substances chimiques , Tigres , Fibroblastes/effets des médicaments et des substances chimiques , Homéostasie/effets des médicaments et des substances chimiques , Sibérie , Altération de l'ADN , Cycle cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Réaction de polymérisation en chaîne , Espèces réactives de l'oxygène/analyse , Apoptose/génétique , Caspases/analyse , Caspases/effets des médicaments et des substances chimiques , Test des comètes/médecine vétérinaire , Microscopie électronique à transmission , Transcription inverse , Potentiel de membrane mitochondriale/effets des médicaments et des substances chimiques , Fibroblastes/physiologie , Homéostasie/physiologieRésumé
Flow cytometry (FCM) is a technology for fast qualitation, quantitative analysis and separation of single cells or other biological particles. The method is rapid, sensitive and accurate, and its objective and direct result can be analyzed with multi-parameters simultaneously. The morphological, biochemical and molecular changes during apoptosis include cellular shrinkage, permeability transition of plasma membrane, caspases activation, dissipation of mitochondrial trans-membrane potential, phosphatidylserine redistribution, calcium flux and DNA fragmentation and content. The review outlined the methods to characterize, identify and quantify apoptoUc cells by flow cytometry to further determine cell apoptosis.