机械敏感离子通道Piezo1在结膜松弛症中的表达OA

Objective To investigate whether the expression of Piezo1 in bulbar conjunctival fibroblasts contrib-utes to the development of conjunctivochalasis(CCH).Methods Primary bulbar conjunctival fibroblasts were ob-tained from CCH patients and normal control(NC)group by tissue block applanation.The cells were then purified and subjected to fibroblast identification.Immunohistochemical analysis was performed to assess Piezo1 expression in the bulbar conjunctival tissue of the NC and CCH groups.Immunofluorescence was used to detect the expression of Piezo1 in bulbar conjunctival fibroblasts in the CCH and NC groups,as well as the expression of matrix metallo-proteinase-9(MMP-9)after the intervention of the Piezo1 agonist,Yoda1.qRT-PCR was used to detect the expres-sion of mRNA of Piezo1 and MMP-9 in the CCH and NC groups,as well as the expression of mRNA of MMP-9 after the intervention of Yoda1.In addition,confocal microscopy was used to detect Ca2+influx in bulbar conjunctival fi-broblasts in the NC group after Yoda1 intervention.Membrane clamp was used to detect the current amplitude of NC bulbar conjunctival fibroblasts after Yoda1 intervention to verify the functional expression of Piezo1.Results Primary bulbar conjunctival fibroblasts were successfully cultured.Immunohistochemical analysis revealed el-evated Piezo1 protein expression in the CCH group(P＜0.01).qRT-PCR analysis showed that Piezo1 mRNA ex-pression was higher in the CCH group than in the NC group(P＜0.01),as was MMP-9 mRNA expression(P＜0.01).Furthermore,MMP-9 mRNA expression was higher in the CCH+Yoda1 group than in the CCH group(P＜0.01).Immunofluorescence staining for Piezo1 revealed enhanced fluorescence on the cell membranes of the CCH group compared with the NC group(P＜0.05),and the fluorescence of MMP-9 was enhanced by the addition of Yoda1 stimulation compared with that of the CCH group(P＜0.05).Confocal microscopy of NC group bulbar con-junctival fibroblasts after the addition of Yoda1 showed enhanced fluorescence of Piezo1(P＜0.05).The addition of Yoda1 activated enhanced current amplitude,showed by membrane clamp detection of NC group bulbar conjunc-tival fibroblasts(P＜0.000 1).Conclusion Mechanical damage produced by conjunctival tissues in conjunctivo-chalasis upregulates Piezo1 expression,and Piezo1 activation may amplify the mechanical damage caused by CCH.