Abstract
Purpose To evaluate the role of SPARC in the antiproliferation effect of MMC on human Tenon's fibroblasts (HTF). Method Sixteen PACG patients aged 59 ± 10 years (31–72 years), including 6 males and 10 females, were recruited. Tenon tissue was harvested during filtering surgery. Cell density was evaluated after MMC application with different concentrations and application times, by which the optimized MMC application modality was determined. MMC, si-SPARC, or SPARC protein was used when needed to evaluate the cell densities under different conditions, by which the role of SPARC in MMC-mediated antifibrotic process was identified. Results Considering that the cell densities, as well as SPARC expression on mRNA and protein levels, are relatively stable when the MMC concentration is higher than 0.02% and exposure time longer than 90 s, we chose the MMC application pattern with 0.02% and 90 s as an optimized pattern for the downstream work. Compared to control, the si-SPARC and MMC downregulated the SPARC protein by 91% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (P < 0.01) and 65% (Conclusion This study demonstrates that in HTF, (1) MMC downregulates the expression of SPARC in protein and mRNA levels; (2) SPARC depletion has synergistic effect on the antifibrotic effect of MMC; and (3) reactive oxygen species are the possible mediator in the antifibrotic effect of MMC and si-SPARC.
Highlights
Trabeculectomy was first introduced by Cairns in 1968 [1] and has been a widely used therapy strategy for glaucoma
After incubation for 1 day, cell attachment was observed, and the cells were treated with 0.01%, 0.02%, or 0.04% MMC solution in phosphate-buffered saline (PBS) for 30, 60, 90, or 120 s and/or transfected with a previously designed siRNA targeted to Secreted protein acidic and rich in cysteine (SPARC) (100 nM; si-SPARC, 5′-AACAAGACCUUCGACUCUUCC-3′) [8], or a scrambled control siRNA (100 nM; si-Scram, 5′-GCUCACAGCUCAAUCCUAAUC-3′; synthesized and purified by Bioneer, General Biosystems, China), using Lipofectamine 2000
SPARC known as osteonectin and BM-40 is participated in the modulation of cell interactions [22]
Summary
SPARC Levels Modulate the Capacity of Mitomycin to Inhibit the Proliferation of Human Tenon’s Capsule Fibroblasts. MMC, si-SPARC, or SPARC protein was used when needed to evaluate the cell densities under different conditions, by which the role of SPARC in MMC-mediated antifibrotic process was identified. Considering that the cell densities, as well as SPARC expression on mRNA and protein levels, are relatively stable when the MMC concentration is higher than 0.02% and exposure time longer than 90 s, we chose the MMC application pattern with 0.02% and 90 s as an optimized pattern for the downstream work. Is study demonstrates that in HTF, (1) MMC downregulates the expression of SPARC in protein and mRNA levels; (2) SPARC depletion has synergistic effect on the antifibrotic effect of MMC; and (3) reactive oxygen species are the possible mediator in the antifibrotic effect of MMC and si-SPARC Conclusion. is study demonstrates that in HTF, (1) MMC downregulates the expression of SPARC in protein and mRNA levels; (2) SPARC depletion has synergistic effect on the antifibrotic effect of MMC; and (3) reactive oxygen species are the possible mediator in the antifibrotic effect of MMC and si-SPARC
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