FIGURE 9.

siRNA against orbital fibroblast HAS2 prevents mast cell-derived PGD₂ induction of HA. Orbital fibroblasts were transfected with HAS1, HAS2, or SC siRNA using Lipofectamine 2000 (Invitrogen). Following this, the fibroblasts were co-cultured for 24 h with HMC-1 cells in a transwell system at a ratio of 20:1 (mast cells/orbital fibroblasts), and the media were collected for HA detection. Untransfected orbital fibroblast co-cultured with HMC-1 cells significantly increased HA production (***, p < 0.0001; compared with untransfected without co-culture). Co-culture of fibroblasts with HMC-1 cells also induced a significant increase in HA in SC siRNA-transfected (**, p < 0.001) and HAS1 siRNA-transfected (*, p < 0.05) orbital fibroblasts. HAS2 siRNA-transfected orbital fibroblasts failed to increase HA synthesis when co-cultured with HMC-1 cells (###, p < 0.0001 compared with untransfected, control siRNA, and HAS1 siRNA). Results are expressed as mean ± S.D. (n = 4–6).