Isolation And Identification Of Mongolian Sheep Chorionic Trophoblastic Cells In Vitro

Aimed to study Mongolian sheep trophoblast cells in vitro and identification, and to explore its biological characteristics, to create a stable, easy access to high-purity sheep chorionic trophoblast cells for subsequent cell transfectionand and RNA interference experiments, provided a cytological basis.Develop different stages of gestation (early, middle and late) Mongolian sheep trophoblast cells and found that the placenta collection time influenced the rate of adherent cells and cell activity, the placenta collection period for the second trimester is the best time. By adding different concentrations of serum screening in DMEM, found containing15%(Fetal Bovine Serum FBS) fetal bovine serum culture system is better,.The early of cells culture, we used different trypsin digestion time to remove the fibrous cells mixed among the epithelioid cells and purification within5generations. Application Yihong-hematoxylin staining (HE) can be observed in the purified cells, which have diverse polygonal, oval, clear cell boundaries. The cells have transparent cytoplasm, round nuclear or a small bubble. Using immunohistochemical method was used to detect the different pregnancy and normal sheep placental villi, and trophoblast cells in vitro. The result is cell cytokeratin positive expression, vimentin negative expression, proved the purity of the cells more than90%, and can satisfied with the follow-up experiment requirements. By transmission electron microscope observing sheep trophoblast cells, the result indicates that the cultured cells with typical trophoblast cells structure of, many microvillis on the cell surface, mitochondria and the high electron density of lipid droplets are more visible in the cytoplasm, nuclei is larger, gap junction structure between the cells,suggesting that the cultured cells is the sheep trophoblast cell. By RT-PCR detected of trophoblast protein-1, found the purified cells were able to express trophoblast-protein-1.The results showed that this test used tissue culture method to culture primary cells, which is simple, effective to obtain high purity and biological activity of sheep trophoblast cells, can provide experimental basis in follow-up vitro research experiments in our laboratory.