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目的 旨在观察球结膜成纤维细胞中Piezo1的表达是否参与结膜松弛症(CCH)的发生发展。方法 采用组织块贴壁法获得CCH患者和正常对照组(NC)的原代球结膜成纤维细胞，纯化后行结膜成纤维细胞鉴定。免疫组化检测NC组和CCH组球结膜组织中Piezo1的表达情况。免疫荧光检测CCH组和NC组球结膜成纤维细胞中Piezo1的表达情况，以及Piezo1激动剂Yoda1干预后基质金属蛋白酶-9(MMP-9)的表达情况。qRT-PCR检测CCH组和NC组中Piezo1与MMP-9的mRNA表达情况，以及Yoda1干预后MMP-9的mRNA表达情况。此外，Yoda1干预后采用共聚焦显微镜检测NC组球结膜成纤维细胞中Ca2+内流情况；Yoda1干预后膜片钳检测NC球结膜成纤维细胞的电流幅度，验证Piezo1功能性表达。结果 成功培养原代球结膜成纤维细胞。免疫组化发现CCH组中Piezo1蛋白表达升高(P<0.01)。qRT-PCR检测发现：相较于NC组，CCH组中Piezo1的mRNA表达升高(P<0.01),MMP-9的mRNA表达升高(P<0.01)；相较于CCH组，CCH+Yoda1组MMP-9的mRNA表达升高(P<0.01)。免疫荧光检测Piezo1发现CCH组相对于NC组细胞膜上的荧光增强(P<0.05)，加入Yoda1刺激后MMP-9的荧光较CCH组增强(P<0.05)。NC组球结膜成纤维细胞加入Yoda1后，共聚焦显微镜显示Piezo1的荧光增强(P<0.05)；膜片钳检测NC组球结膜成纤维细胞加入Yoda1后激活的电流幅度增强(P<0.000 1)。结论 CCH中结膜组织产生的机械损伤上调了Piezo1的表达，而Piezo1的激活可能放大了CCH造成的机械性损伤。
Abstract：Objective To investigate whether the expression of Piezo1 in bulbar conjunctival fibroblasts contributes to the development of conjunctivochalasis(CCH).Methods Primary bulbar conjunctival fibroblasts were obtained from CCH patients and normal controls(NC) 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 metalloproteinase-9(MMP-9) after the intervention of the Piezo1 agonist, Yoda1. qRT-PCR was used to detect the expression 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 fibroblasts 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 elevated Piezo1 protein expression in the CCH group(P<0. 01). qRT-PCR analysis showed that Piezo1 mRNA expression 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 conjunctival 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 conjunctival fibroblasts(P<0. 000 1).Conclusion Mechanical damage produced by conjunctival tissues in conjunctivochalasis upregulates Piezo1 expression, and Piezo1 activation may amplify the mechanical damage caused by CCH.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2026.07.006
中图分类号:R777.3
引用信息:
[1]夷成龙,陶霓霞,杨灿,等.机械敏感离子通道Piezo1在结膜松弛症中的表达[J].安徽医科大学学报,2026,61(07):1200-1207.DOI:10.19405/j.cnki.issn1000-1492.2026.07.006.
基金信息:
国家自然科学基金项目(编号：82074495)~~
2026-05-11
2026-05-11
2026-05-11