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S. Zhang, H. He, S. C. G. Tseng; Expression and Regulation of Heavy Chains of II, TSG-6 and PTX3 in Epithelial and Stromal Cells of Human Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4860.
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HC•HA complex formed between heavy chains (HCs) of inter-α-trypsin inhibitor (IαI) and hyaluronic acid (HA) has been purified from the human amniotic membrane (AM) and shown to suppress inflammation and scarring. But it remains unclear whether HC can be locally produced by AM epithelial or stromal cells, and whether cellular expression of HCs as well as PTX3 and TSG-6, of which the latter is involved in HC•HA formation, is upregulated by TNFα.
Expression of HC of IαI, TSG-6 and PTX3 in AM tissues were detected by immunostaining. Primary AM epithelial and stromal cells were isolated and cultured in SHEM, and treated with 20 ng/ml TNFα for 4 or 24 h before subjecting to RT-PCR and Western blotting analysis for the expression of HCs, TSG-6 and PTX3 transcripts and proteins, respectively.
Positive immunoreactivity to HCs of IαI and TSG-6 was present in AM epithelial and stromal cells with the former also present in the stromal extracellular matrix, while that of PTX3 was found only in AM epithelium. HC1, HC2, HC3, TSG-6 and PTX3 mRNAs were expressed by both cultured epithelial and stromal cells. The levels of HC1 and HC2 mRNAs were higher in epithelial cell and that of HC3 mRNA was higher in stromal cells; the level of TSG-6 mRNA expressed in stromal cells was higher than that in epithelial cells. TNFα treatment increased HC1 and HC2 mRNA in both epithelial and stromal cells, and HC3 mRNA in epithelial cells, but decreased HC3 mRNA in stromal cells. TNFα also induced stronger expression of TSG-6 mRNA and PTX3 in stromal cells. Western blot analysis revealed expression of a dominant 50 kD TSG-6-like protein and a weak 30 kD TSG-6 protein as well as PTX3 by both epithelial and stromal cells, with the latter upregulated by TNFα. Furthermore, HC1, but not HC3 and HC4, protein was found to be present in HC•HA.
HCs, TSG-6 and PTX3 are expressed and differentially regulated by TNFα in both AM epithelial cells and stromal cells. These baseline characterization will help us explore the likelihood that AM epithelial and/or stromal cells may be a new source other than the liver to provide HCs and TSG-6-like protein may be a AM-specific protein responsible for HC•HA formation.
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