RESUMO
Embryonic germ [EG] cells are the results of reprogramming primordial germ cells [PGC] in vitro. Studying these cells can be of benefit in determining the mechanism by which specialized cells acquire pluripotency. Therefore in the current study we have tried to derive rat EG cells with inhibition of transforming growth factor-beta [TGFbeta] and mitogen-activated protein kinase kinase [MEK] signaling pathways. In this experimental study, rat PGCs were cultured under feeder free condition with two small molecules that inhibited the above mentioned pathways. Under this condition only two-day presence of stem cell factor [SCF] as a survival factor was applied for PGC reprogramming. Pluripotency of the resultant EG cells were further confirmed by immunofluorescent staining, directed differentiation ability to neural and cardiac cells, and their contribution to teratoma formation as well. Moreover, chromosomal stability of two different EG cells were assessed through G-banding technique. Formerly, derivation of rat EG cells were observed solely in the presence of glycogen synthase kinase-3 [GSK3beta] and MEK pathway inhibitors. Due to some drawbacks of inhibiting GSK3beta molecules such as increases in chromosomal aberrations, in the present study we have attempted to assess a feeder-free protocol that derives EG cells by the simultaneous suppression of TGFbeta signaling and the MEK pathway. We have shown that rat EG cells could be generated in the presence of two inhibitors that suppressed the above mentioned pathways. Of note, inhibition of TGFbeta instead of GSK3beta significantly maintained chromosomal integrity. The resultant EG cells demonstrated the hallmarks of pluripotency in protein expression level and also showed in vivo and in vitro differentiation capacities. Rat EG cells with higher karyotype stability establish from PGCs by inhibiting TGFbeta and MEK signaling pathways
RESUMO
Pectin is composed of complex polysaccharides that can inhibit cancer metastasis and proliferation with no evidence of toxicity. In the present study, the apoptotic and necrotic effects of pectic acid [PA] on the rat pituitary GH3/B6 tumor cells has been investigated. GH3/B6 cells were cultured in the Ham's F12 medium enriched with 15% horse serum and 2.5% fetal bovine serum for 3 days. Then, they were treated by various amounts of PA in different periods [6, 24 and 48 hours]. Bromocriptine was used as positive control and the cell viability was detected by MTT test. The nuclear morphology of cells was explored by florescent stains including acridine orange [AO]/ethidium bromide [EB]. In addition, percentages of apoptotic and necrotic cells were studied with triphosphate nick-end labeling [TUNEL] assay, cell cycle analysis and propidium iodide [PI] staining. Long-term incubation with PA results in increased cell death and DNA damage as detected by MTT assay and AO/EB staining. TUNEL assay showed that PA [100 micro g/ml to 1 mg/ml] could induce apoptosis in a dose-dependent manner, while higher concentrations of PA [2.5 and 5 mg/ml] induced necrosis which was confirmed by PI staining. Furthermore, cell cycle analysis indicated that PA induced sub G1 events, and DNA fragmentation was also correlated with the number of the apoptotic cells. It can be concluded that PA is responsible for apoptosis in the rat pituitary tumor cells. Therefore, one may suggest that this group of polysaccharides can be used in treatment of pituitary tumors