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S.-Y. Chen, Y. Zhu, A. Kheirkah, S. C. G. Tseng; Differential Regulation of SDF-1, IL-8 and MCP-1 by Resting and Activated Normal Conjunctival Fibroblasts With or Without Mitomycin C Treatment. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2941.
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SDF-1 (stromal cell-derived factor-1), IL-8 and MCP-1 (chemoattractant protein-1), known expressed by cultured normal human conjunctival fibroblasts (HCF), are potent chemokines regulating the recruitment of pro-inflammatory and pro-immune cells from the bone marrow during wound healing, during which time fibroblasts turn into myofibroblasts. Mitomycin C (MMC) is an alkylating agent commonly used to inhibit fibroblast proliferation and myofibroblast differentiation in a number of ocular surface diseases. It remains unclear whether expression of these chemokines is affected when HCF are activated into myofibroblasts or whether both resting and activated HCF are treated with MMC.
After the surface epithelium was removed by dispase digestion at 4ºC for 16h, HCF were liberated from the remaining conjunctival stroma by digestion with 2 mg/ml collagenase A at 37ºC for 16h, and cultivated in either the supplemented hormonal epithelial medium (SHEM) or DMEM with 10%FBS (DMEM/FBS). At 70% confluence, Passage 2 HCF in either SHEM or DMEM/FBS were treated with or without 0.04 mg/ml MMC for 5 min, and then subjected quantitative real time PCR for comparing mRNA levels of SDF-1, IL-8 and MCP-1. Myofibroblast differentiation was determined by immunostaining to α-smooth muscle actin (α-SMA).
Expression of α-SMA was not noted in HCF cultured in SHEM, but vividly expressed when HCF were cultured in DMEM/FBS, suggesting that HCF were activated to myofibroblast differentiation in DMEM/FBS. Without MMC treatment, expression of SDF-1 in SHEM was 10-fold less than that in DMEM/FBS, while expression of IL-8 or MCP-1 in SHEM was 10-fold and 2-fold higher than in DMEM/FBS, respectively. Interestingly, after MMC treatment, expression SDF-1 was reduced to 0.5 fold in SHEM and to 4 fold in DMEM/FBS, while expression of MCP-1 was reduced to 5 fold in SHEM and 3 fold in DMEM/FBS. In contrast, expression of IL-8 was increased to 3 fold in SHEM but 17 fold in DMEM/FBS.
HCF expressed more IL-8 and MCP-1 at the resting state, but more SDF-1 when activated into myofibroblasts. Expression of SDF-1 was more dramatically reduced, while that of IL-8 was markedly promoted in activated HCF after MMC treatment. Expression of MCP-1 by resting HCF was more reduced than that by activated HCF. This information suggests that MMC treatment may alter wound healing by recruiting different subpopulations of pro-inflammatory/immune cells and progenitor cells from the bone marrow.
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