Study On The Biological Properties And Therapy Of Hepatic Injury Of Broiler Adipose-derived Stem Cells And Sheep Amniotic Mesenchymal Stem Cells

The genetic resources of livestock and poultry are closely related to human beings,which is the basis of agricultural production and food security,it affects the development of society and economy.The self-renewal of stem cells and the ability to differentiate into multiple germ layers has arised a new idea for the protection of genetic resources of livestock and poultry.Adipose-derived mesenchymal stem cells(ADSCs)have been widely recognized in the field of conservation and utilization of genetic resources,tissue engineering and so on.In this study,the MSCs from broiler chickens and sheep amnion were used as experimental objects to study the isolation,culture,biological characteristics,induction and differentiation and treat liver injury in mice by cell transplantation.The results are descripted as follows:1.The ADSCs were obtained from broiler adipose using type I collagenase,they were long fusiform shape,were cultured for 37 generations in vitro.The surface markers of ADSCs,CD29,CD31,CD44,CD71,and CD73,were detected by RT-PCR and immunofluorescence.The result showed that CD29 CD44,CD71 and CD73 were positive in different passages cells.Flow cytometry showed that ADSCs were high expression for CD29,CD44,CD71 and CD73.The growth of different passages cells typically appeared in S shape by and large,which in accordance with the general characteristics and regularity of cell growth in vitro.The population doubling time and the ability of clone-forming showed that the cell proliferation ability was decreased with the increase of generation time.The analysis of cell cycle denoted that the cell ratio of G0/G1 phase increased gradually,but the cell ratio of S phase decreased gradually with the increase of cell generation times.Broiler ADSCs were induced into osteoblasts,adipocytes and hepatocytes under suitable conditions in vitro,which proved by specific staining,RT-PCR and immunohistochemistry.The results showed that the multiple differentiation potential of ADSCs was verified.2.Sheep AMSCs were isolated from amnion using type II collagenase and trypsin,mainly showing long fusiform shape,and were cultured for 25 generations in vitro.The gene of Oct-4,Rex-1,CD29,CD44,CD71,and CD73 were identified by RT-PCR and immunofluorescence technique.The result showed that they were positive in AMSCs.Flow cytometry showed that AMSCs were high expression for Oct-4,CD44,CD73 and CD90.The growth of different passages cells typically appeared in S curve,which in accordance with the general characteristics and regularity of cell growth in vitro.The population doubling time and the ability of clone-forming showed that the cell proliferation ability was decreased with the increase of generation time.Through the analysis of cell cycle,it showed that with the increase of cell generation times,the cell ratio of G0/G1 phase increased gradually,but the cell ratio of S phase decreased gradually.in vitro Sheep AMSCs were induced into osteoblasts,adipocytes and hepatocytes under suitable conditions in vitro,which proved by specific staining,RT-PCR and immunofluorescence.The results showed that the multiple differentiation potential of AMSCs was verified.3 Mouse livers were injured with cisplatin,morphological changes.scattered bleeding points and hard texture was observed.Liver tissue sections showed that the normal structure has been destroyed and fibrous tissue hyperplasia has occurred;and that serological liver index was significantly increased.ADSCs labeled with fluorescent were injected into mice by tail intravenous.The transplanted ADSCs were found in the hepatic lobule around the central vein and hepatic lobules in the connective tissue.Histological observation and serological analysis showed that ADSCs exert a therapeutic influence on liver damage at some extent.In summary,broiler ADSCs and sheep AMSCs were successfully isolated and studied in this experiment,the biological characteristics and the capacity of differentiation were also verified.These results indicated that the ability of self-renewal and differentiation of stem cells,which is of great significance for the conservation and utilization of genetic resources of livestock and poultry,and it provide an experimental basis for the application of MSCs in tissue engineering and regenerative medicine.