Research Of Biological Characteristics And Transplantation Of Mesenchymal Stem Cells From Duck Embryo In Treatment Of Liver Injury

The diversity of animal genetic resources is very important to human survival and economic development, so their conversation becomes especially significant. Because stem cells have the characteristics of the "infinite" proliferation and self-renewal, which make it the seeding cell to preserve the animal genetic resources has been widely used. In this study, we established the culture system of duck embryo derived mesenchymal stem cells. Through the research of the biological characteristics of mesenchymal stem cells(MSCs) and the cell therapy for the liver injury in mice, this work provided theoretical and experimental basis for animal genetic resources preservation and laid a solid foundation for the use of stem cells for cell therapy. The results are shown below:1. The umbilical cord mesenchymal stem cells(UCMSCs) were isolated from duck embryo with collagenase type IV digestion method. Primary UCMSCs were sub-cultured to passage 39 in vitro. The morphology of the cells focused on elongated fusiform and irregular shape.MSCs-related gene CD29 and CD44 were detected by immunofluorescence test, and the result of RT-PCR showed CD44, CD71 and CD73 positive in different passages. The above results suggested the isolated and cultured cells were UCMSCs. The induced osteoblasts showed red calcium nodules by alizarin red staining, and RT-PCR test showed Collagen typeⅠand Osteopontin positive expression; The induced adipocytes showed red lipid droplets by oil red O staining, and RT-PCR test showed LPL and PPAR-γ positive expression. These results indicated UCMSCs possess the multiple differentiation potential.2. The liver mesenchymal stem cells(LMSCs) were isolated from duck embryo with collagenase type IV digestion method. Primary LMSCs were sub-cultured to passage 56 in vitro. The morphology of the cells focused on elongated fusiform and irregular shape, with a small number of rounded cells. The nuclei were small. Growth curves of LMSCs indicated that LMSCs possess the strong proliferation ability. MSCs-related gene CD44, CD105,CD166,IL-7 were detected by immunofluorescence test, and the result of RT-PCR showed CD71, CD73,SOX2,KLF4 positive in different passages. The above results suggested the isolated and cultured cells were LMSCs.The induced osteoblasts showed red calcium nodules by alizarin red staining, and RT-PCR test showed Collagen typeⅠand Osteopontin positive expression; The induced adipocytes showed red lipid droplets by oil red O staining, and RT-PCR test showed LPL and PPAR-γ positive expression; The induced hepatocyte-like cells showed deep red by Periodic acid-schiff staining and RT-PCR test showed AFP and ALB positive expression. These results indicated LMSCs can be differentiated into different blastoderm cells and possess the multiple differentiation potential.3.The livers of male Kunming mice were injured with carbon tetrachloride(CCl4). H&E-stained sections revealed massive necrosis and hepatic lobule damage, and MT staining showed that liver fibrosis had increased significantly. The levels of ALT and AST in serum were significantly higher than the normal. After cell-transplantation, pathological examination and serological test showed liver function improved. The results suggested the induced hepatocyte-like cells have therapeutic effect on liver injury in mice at some extent.In conclusion, this article introduced the research progress of mesenchymal stem cells, including isolation and culture in vitro, the biological characteristics, and the cell therapy for the liver injury in mice. This work verified the self-renewal and multipotent capacity of stem cells, which was of great significance to the preservation of animal genetic resources, and provided experimental basis for the clinical application of stem cells preliminarily.