An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-1 in Salivary Gland Epithelial and Mesenchymal Differentiation
Figure 5
The expression of TGF-1 ligand and receptors in Col1a1-GFP derived submandibular salivary gland tissues and cultured cells. (a)–(h) Immunofluorescence of Col1a1-GFP derived submandibular salivary gland tissues taken by Zeiss fluorescence microscope. Salivary gland epithelium, in particular ductal epithelium, strongly expressed TGF-1 (in cytoplasm) (a and b), TGF- receptor 1 (TGF-R1) (in nuclei and cytoplasm) (c and d), and TGF- receptor 2 (TGF-R2) (in nuclei and cytoplasm) (in red) (e and f). GFP-positive salivary gland mesenchyme (in green) did not seem to highly express either TGF-1 or TGF-R1, in normal salivary gland tissues. (e) and (f) Rabbit IgG control was used as negative control to confirm the specificity of TGF-1, TGF-R1, and TGF-R2 antibodies. (i)–(n) Cultured salivary gland cells in DMEM plus serum medium from early (passage 1) and late (passage 9) cultures showed the different expression of TGF-1, TGF-R1, and TGF-R2. In the early culture, TGF-1 and TGF-R2 were strongly expressed in the cytoplasm of epithelial cells (i and m, resp.) whereas the strong TGF-R1 expression was found in cultured salivary gland epithelial cells in cytoplasmic and nuclear areas (k), but not in mesenchymal cells. In the late culture, the expression of TGF-1, TGF-R1, and TGF-R2 was seen in both salivary gland epithelial and mesenchymal cells (j, l, and n). The salivary gland mesenchymal cells increased the expression of TGF-1 (j), TGF-R1 (l, inset), and TGF-R2 (n) after late culture. The staining pattern of TGF-1 and TGF-R1 in late cell passage was similar to early cell passage (j and l) whereas that of TGF-R2 was shown in both membrane and nuclei (n). (o) and (p) Mixed salivary gland cells cultured in N2 media which contained a majority of salivary gland epithelium showed higher levels of both Tgf-b1 and Tgf-br1 expression, compared to that in DMEM media plus serum. Relative expression was normalized to the expression of Gapdh which was used as the reference gene. Values were represented as mean ± SEM from three independent experiments (). Student’s -test was analyzed to compare between cells cultured in N2 and DMEM media plus serum, *. Scale bars = 100 μm.
