| Summary: | 碩士 === 國立中興大學 === 動物科學系所 === 101 === Cell cycle of donor cells would affect the efficiency of somatic cell nuclear transfer (SCNT), as well as the nuclear reprogramming efficiency. Recent researches have demonstrated that the Xenopus laevis egg extracts (XEE) treated cells, called extract treated cells (ETCs), could undergo reprogramming and regain the expressions of pluripotent genes, such as oct4, sox2 and nanog. Therefore, the aims of this study were to investigate the effects of cell cycle on the efficiency of reprogramming into the pluripotent stage by treating mouse somatic cells with XEE and assess the possibility to define the embryonic grem like (EG-like) cells from the reprogrammed cells. Experiment 1, a mouse NIH/3T3 cell line and mouse embryonic fibroblasts (MEFs) were incubated with 0.2% or 0.5% bovine serum (BS) for 3 or 5 days would significantly stay at G0/G1 phase, while NIH/3T3s and MEFs treated with various concentrations of colchicine (0.5, 1.0 or 1.5 μg/ml) for 1 or 2 days, would significantly stay at G2/M phase. Experiment 2, NIH/3T3s were treated 0.5% BS for 3 days or 0.5 μg/ml colchicine for 1 day before treatment with XEE. On Day 7 and 8 after XEE treatment, all groups showed the expressions of tumor suppressors (p53, p21 and p16), pluripotent markers (oct4, sox2 and nanog) and primordial germ cells (PGCs) markers (blimp1 and stella), although the expressions were low and unstable. However, there showed more round morphology cells, and the expressions of tumor suppressors, pluripotenct and PGC markers were increased in colchicine-treated NIH/3T3s. The expressions of Oct4 and Nanog protein were also shown by analysis of immunocytochemical (ICC) staining. MEFs treated with 0.2% BS for 5 days or 0.5 μg/ml colchicine for 2 days before XEE treatment showed pluripotent markers on Day 7 to Day 9, but the expressions of tumor suppressors, pluripotent and PGC markers still remained at low level. Furthermore, the expressions of PGC markers were low. Experiment 3, cell cycle-regulated NIH/3T3s were treated with XEE for 7 or 8 days, then incubated in PGCM for 24 h. For groups changed to PGCM on Day 7, colchicine-treated NIH/3T3s had significantly higher expressions of Blimp1, Stella and Sox2, while for groups changed to PGCM on Day 8, serum-starved NIH/3T3s had significantly higher expression of Blimp1. After cultured in PGCM for 24 h, the cells were passaged and seeded on feeder layer and then cultured in 2i-LIF culture system. It was found that groups changed to PGCM on Day 7, the colchcine group formed more EG-like colonies, although the expressions of pluripotent and PGC markers in the EG-like colonies showed no differences with ES cells in the goups changed to PGCM on Day 7 and Day 8. By analysis of immunocytochemistry, all groups of EG-like colonies expressed pluripotent markers, including Oct4, Nanog, SSEA1 and a PGC-specific marker Stella. These studies showed that after serum starvation and colchicine treatment, NIH/3T3s and MEFs could significantly stay at G0/G1 and G2/M, respectively. Further, we treated cell cycle-regulated cells by XEE, we found colchicine had more round morphology cells and Oct4/Nanog expressions in NIH/3T3s; but there were less round morphology cells and Oct4/Nanog expressions in MEFs. Then we changed to 2i-LIF culture system, the d7 colchicine formed more EG-like colonies, otherwise, all groups of colonies expressed PGC marker Stella and pluripotent markers Oct4, Nanog and SSEA1 in NIH/3T3s.
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