Effects On Ovine Embryonic Stem Cell Pluripotency By Adding Different Factors

Embryonic stem cells, isolated and cultured from the inner cell mass, can differentiate into the cells of all cell types under appropriate conditions. Screened the factors to maintain the Ovine embryonic stem cell pluripotency, our laboratory built oESC-like for material, according to the morphology, proliferation AKP and pluripotency candidate gene Oct4, Sox2, Klf4, c-Myc, Lin28mRNA expression changes to analyze the pluripotent embryonic stem cells of Ovine in order to determine the optimal culture system. The results are as follows:On the base of N2B27/CH, added PD, PD/hLIF, PD/BMP4or PD/hLIF/BMP4to the medium respectively. In these four different mediums and compared with the oESC-like cultured8d in the culture system(N2B27/CH/bFGF), results showed that, cells of the four groups all expressed AKP, Sox2, Oct4protein. Cells with PD, PD/hLIF added grew compacter, but slower. Cells adding PD/BPM4, PD/hLIF/BMP4bind loosely, but grew quicker. With PD, PD/hLIF, the pluripotent candidate genes Lin28and c-Myc expression levels were significantly high (P<0.01). Nestin expression was significantly reduced(P<0.01). with PD/hLIF added, Oct4expression was significantly up-regulated(P<0.01). To add PD/BPM4, PD/hLIF/BMP4, multi-candidate genes Oct4, Sox2, c-Myc, Klf4, the expression of which were significant difference (P<0.01), Nestin and Lin28expression was significantly decreased (P<0.01);The growth factors PD, PD/hLIF, PD/BPM4, PDhLIF/BMP4were added respectively to the constructed training system N2B27/CH/bFGF of oESC-like for8d. The results showed that, the AKP staining and Sox2and Oct4immune fluorescence staining showed positive, mRNA level of Oct4and Klf4was significantly higher with PD than that of N2B27/CH/bFGF(P<0.01); Nestin and Lin28decreased significantly down-regulated(P<0.01). Sox2and c-Myc was higher. Our results showed that mRNA level of Oct4was significantly higher with PD/BMP4than that of N2B27/CH/bFGF(P<0.01); c-Myc, Klf4and Lin28was higher. Nestin and Sox2was down-regulated, the cells growth better and pluripotency. Added PD/BPM4, PD/hLIF/BMP4, Oct4, Sox2, Nestin expression levels reduced significantly (P<0.01), Lin28, c-Myc, and Klf4expression didn’t decreased significantly.Of PD and PD/hLIF group, while added bFGF, the cells were more compact. Expression of alkaline phosphatase activity was positive, so were the expression of pluripotency-designate to select the gene Sox2and Oct4immuno fluorescence protein, pluripotency mark. Cells grew faster than that of the PD group. The PD group, being added the bFGF, expressed higher than that of other groups. Expression level of Oct4, Klf4, and Lin28differed significantly(P<0.01). Sox2and c-Myc also had regulatory effect. Nestin, marker gene of the nerve cells, expressed significantly lower than that of the group without bFGF.