Regulating Mechanism Of Secreted Protein Acidic And Rich In Cysteine In Human Trabecular Meshwork

Subject To study the regulating mechanism of secreted protein acidic and rich in cysteine(SPARC),one matricellular protein,in human trabecular meshwork cells(HTMC).Methods In vitro,HTMCs were cultured under 0,30 or 60 mm Hg culturing pressures,for 0,24 or 48 hours.Quantitative polymerase chain reaction(QPCR)was performed to evaluate mRNA levels of SPARC,matrix metalloproteinase-15(MMP-15)and zonula occludens-1(ZO-1).FITC-Phalloidin fluorescent staining was used to observe F-actin distributing in HTMCs.A slow virus containing SPARC-inhibited gene series named as pL_shRNA_mKate2-SPARC-543 was synthetizecd to invade HTMCs.After 48 hours,QPCR was performed to evaluate mRNA levels of SPARC,F-actin and ZO-1,western-bolt was performed to evaluate protein levels,FITC-Phalloidin fluorescent staining was used to observe F-actin distributing,and cell macrophage swallowing was also observed by fluorescence microscope.Results The expressions of SPARC-mRNA in HTMCs under different culturing pressures(0,30 or 60 mm Hg)for different culturing durations(0,24 or 48 hours)showed no obvious changes.MMP-15-mRNA decreased under 30 and 60 mm Hg culturing pressures at 24 hours time point,but recovered at 48 hours time point.ZO-1-mRNA only decreased under 60 mm Hg at 48 hours time point.F-actin fluorescent staining showed fluorescence dispersion,fuzzy structure and depolarizing distribution.After virus of pL_shRNA_mKate2-SPARC-543 invading HTMCs,the mRNA and protein levels of SPARC,F-actin and ZO-1 in HTMCs were depressed.F-actin fluorescent staining showed fluorescence dispersion,fuzzy structure,pseudopodium formation and obvious depolarizing distribution.The ability of Cell macrophage swallowing of HTMC was enhanced,correspondingly.Conclusions SPARC was not sensitive to culturing pressure.Downstream regulation of SPARC in HTMCs decreased the expression of cytoskeleton proteins ZO-1 and F-actin,led to the cytoskeleton rearrangement in cytoplasm,enhanced cell macrophage swallowing,and might play a role in altering outflow facility of trabecular mesh work.