Self-assembly Of Embryonic Mouse Salivary Gland Epithelial Cell

Objectives:In this study, we used an three-dimensional culture system to study the branching process and the self-organizing machannism of embryonic salivary cells in order to assess their potential for use in salivary gland regeneration.Methods:(1) The embryos from timed pregnant female mice (with the day of finding a vaginal plug defined as EO) were harvested at E15. Embryonic mouse submandibular gland tissues were cultured. Submandibular gland epithelial cells were dissociated,purified and subcultured. Passage 2 cells were identified with CK19,Vimentin and Laminin by immunocytochemistry. Cells ultrastructure were observed using transmission electron microscopy. (2) Passage 2 cells were inoculated in matrigel for three-dimensional culture,either with anti-E-cadherin and anti-β-catenin (group A) or without (group B). The constructs of these cells aggregation were observed under inverted microscope at 1,3,6,9 day. The cells/matrigel constructs were harvested at 6,12 day, respectively. The constrcuts were fixed for PAS staining and amylase immunohistochemistry staining. The structure of the constructs were observed with CK19 immunofluorescence. The constructs were indentified with E-cadherin,B-catenin immunostaining and confocal laser microscopy. MOD values were measured,then analysed with statistical software.Results:(1) We harvested submandibular gland cells expressing CK19 and Laminin. Passage 2 cells were observed under transmission electron microscope can be seen in the large nucleus, the cytoplasm contains a large number of zymogen granules and abundant mitochondria, rough endoplasmic reticulum and Golgi complex.(2) The submandibular gland epithelial cells inoculated in matrigel with anti-E-cadherin and anti-B-catenin (Group A) showed small number of branches, and acinar-like structure were not observed. On the other hand, the cultured cells without anti-E-cadherin and anti-B-catenin (Group B) showed elongated morphology and aggregated branch tissue, acinar-like structures at 12 day. (3) Specimens were selected randomly from group A and B at 6,12 day. Group A was weakly positive for PAS and amylase,and group B was positive for them. (4) At 12 day with CK19 immunofluorescence assay, Group A cells were disordered spatial structure and had no obvious acinar lumens. Group B cells grew as orderly arranged network and formed acinar lumen in three-dimensional space. (5) Under confocal microscopy,Group A expressed weak green fluorescence of E-cadherin,β-catenin mainly in cell membranes and cytoplasm,but group B is strong in the cytoplasm. At 6,12 day, E-cadherin,β-catenin immunocytochemistry staining were analysed. MOD, Group A< Group B,P<0.01.Conclusion:Embryonic mouse submandibular gland epithelial cells we harvested had the characteristics of stem/progenitor cell. They can aggragate to form a branching-acinar-like structure in three-dimensional culture conditions. E-cadherin-β-catenin complex-mediated the Wnt signaling pathway involved in cell-cell signal transduction,thereby contributing to the occurrence of branching patterns.