| Fibroblasts have been widely used in research and clinical practice,because they are easy to obtain and proliferate well. Recently, with the study of cloning and transgenic animal, more and more attentions have been paied to the quality of donor cells and the impact on the efficiency of nuclear transfer. Fetal fibroblasts, which present stronger capacity of divisions in vitro, have been widely applied in selection of transgenic donor cells and induction of induced pluripotent stem cells (IPSCs). But adult fibroblasts are easier to cellect compared with the fetal fibroblasts and the genetic characters are identified, which is very important for the reproduction of excellent domestic animal individuals. At the same time, adult fibroblasts have the advantages of preserving animal species which are close to disappear.Thus, this study aimed to find differences between adult cells and fetal cells. In detail, the following items were examined, including telomere length, genes about apoptosis, anti-apoptosis and pluripotency. According to differences found between adult and fetal fibroblasts and related references, we choosed Vc to treat adult cells and achived results of this study were as follows:1. At molecular levels, the telomere length and expression of genes associated with apoptosis, anti-apoptosis and pluripotency were analyzed. The results showed that adult fibroblasts grew very slower than fetal fibroblasts (P<0.05). As for the telomere length, fetal fibroblasts were longer than adult fibroblasts (P<0.05). Apoptosis related genes, including P53and Caspase3, were expressed higher in adult cells than fetal cells (P<0.05), while the former had lower expression of Bax-inhibitor (P<0.05), an anti-apoptosis gene. At the same time, expression of pluripotent gene Sox2was higher in fetal cells than adult cells (P<0.05). The above data might reveal the main reason that contributed to the poor ability of adult fibroblasts to proliferate in vitro.2. Vc was selected to treat adult fibroblasts and its effects on cell growing state, gene expression, epigenetic modification were examined. As revealed by the results, the ratio of G1phase was decreased by Vc treatment (42.16±0.40%vs65.89±0.39%, P<0.05), along with increased expression of cell cycle related gene Survivin, which was1.91-fold higher than control (P<0.05). Vc treatment increased the expression of Sox2and Oct4(P<0.05). The level of5hmC in Vc treated fibroblasts was1.43-fold higher than control (P<0.05). The level of DNA methylation was decreased.3. The SCNT efficiency was improved significantly by using Vc treated donor cells and the quality of cloned embryos was much better than control. Results showed that Vc treatment increased cloned embryos’cleavage and blastocyst rates (83.13±3.34%vs69.67±2.79%;39.09±1.61%vs28.13±1.14%, respectively, P<0.05), elevated the percentage of inner cell mass (ICM) in blastocysts (42.40±1.21%vs33.25±1.03%, P<.05), leaded lower apoptotic rate (P<0.05) and higher expression of pluripotent genes Sox2and Oct4(P<0.05). Therefore, with the introduction of Vc during culture of adult fibroblasts, cell division could be accelerated obviously and SCNT efficiency was improved. |
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