부산대학교 도서관

Objective·To establish a culture system of oviductal epithelial organoids from wild type (WT) mice and miR-34b/c-/- and miR-449-/- double knockout (dKO) mice, and verify the phenotypes.Methods·The oviduct epithelial cells of WT mice and dKO mice were isolated and purified by enzyme digestion and differential adhesion method, and the purity of the isolated oviduct epithelial cells was identified by immunofluorescence staining. The numbers, growth rates and sizes of oviductal epithelial organoids between WT mice and dKO mice were compared by counting and diameter measurement. Hematoxylin-eosin (H-E) staining and transmission electron microscope (TEM) were used to observe the morphology and structure of the oviductal epithelial organoids. The proportions of ciliated cells and secretory cells in the oviductal epithelial organoids from WT mice and dKO mice were observed and counted by immunofluorescence staining. Immunohistochemistry (IHC), real-time quantitative PCR (RT-qPCR) and Western blotting were used to observe the expression levels of marker genes of ciliated cells and secretory cells in the oviductal epithelial organoids.Results·The purity of the isolated and purified oviduct epithelial cells was high. Compared with the organoids from WT mice, the oviductal epithelial organoids from dKO mice grew faster and larger, and were more in number. But they developed more slowly than those from WT mice, as the invaginations of the dKO mice organoids appeared on the 28th day of culture, while the WT mice organoids exhibited the same structures on the 16th day. The oviductal epithelial organoids showed similar structures as those of the oviduct in vivo under hematoxylin-eosin (H-E) staining and TEM. Immunofluorescence staining showed that the ciliated cells of oviductal epithelial organoids from dKO mice were significantly reduced and the secretory cells were significantly increased (both P