MiR-155对角膜损伤修复过程中角膜上皮细胞通透性的影响及其分子机制

The physical barrier formed by corneal epithelial cell layer can resist external invasion of pathogenic factors. Corneal wound can cause deletion of epithelial cells, disappearance of tight junction structure, loss of barrier function, increase of permeability. In our previous study, the epithelial cells covered corneal epithelial defect via cell proliferation and migration, but its permeability was higher than that of the control group, immunofluorescence analysis showed that the intercellular tight junction was arranged in disorder and not formed the normal structure. Studies have found that the MLCK inhibitors can significantly reduce the corneal epithelial cell permeability increased by benzalkonium, the miR-155 can inhibit the expression of MLCK, while changes of MLCK dependent ZO-1 is necessary in regulation of epithelial barrier function. To sum up, we put forward a hypothesis, which is during the process of the wound healing in corneal epithelial cell, miR-155 inhibits the expression of MLCK with decreasing of MLC phosphorylation level. At the same time, in order to remodel, the tight junction proteins expressed and transferred to cell membrane, which promote the formation of corneal epithelial barrier and decrease its permeability. This project aims to explore and reveal the role of miR-155 on the permeability during corneal wound healing in rats and the relationship with MLCK expression, tight junction remodeling.

角膜上皮细胞形成的物理屏障可抵御外界致病因子的侵袭。角膜损伤后，缺损处上皮细胞缺失，紧密连接结构消失，屏障功能丧失，通透性增加。我们先前的研究表明当上皮细胞经增殖、迁移修复损伤使得上皮细胞完全覆盖缺损处，但仍可观察到通透性明显高于正常，免疫荧光分析表明此时细胞间紧密连接蛋白排列紊乱未形成正常结构。有研究发现MLCK抑制剂可明显降低苯甲烃铵促进角膜上皮细胞通透性增加的作用；miR-155可抑制细胞MLCK的表达；而MLCK依赖的ZO-1变化对上皮细胞屏障功能的调节是必需的。为此，我们提出假说：角膜上皮细胞损伤修复过程中，miR-155抑制MLCK表达，MLC磷酸化水平下降，表达的紧密连接蛋白转移至细胞膜而重塑，促进角膜上皮细胞屏障的形成而降低通透性。本项目拟探讨并揭示miR-155对实验性大鼠角膜损伤修复过程中角膜上皮细胞通透性的影响是否与调控MLCK表达、紧密连接重塑有关。

背景：角膜上皮细胞在角膜功能中起着至关重要的作用，可通过形成物理屏障来保护眼睛免受外部病原体的侵袭。最近的一项研究表明 miR-155可促进皮肤伤口愈合，然而，其在角膜上皮伤口愈合中的作用尚不清楚。本研究探讨在角膜上皮损伤修复过程中miR-155-5p是否能通过重塑角膜上皮细胞的紧密连接来降低角膜上皮通透性。..材料与方法：在解剖显微镜下，用钝刀刀片切除角膜中央上皮，形成大鼠角膜创面。在角膜上皮损伤前后3天，每只老鼠的一只眼睛接受了miR-155-5p滴眼液治疗，另一只眼睛用局部agomir滴眼液阴性对照治疗。用大分子渗透法法评价角膜上皮通透性。用Westernblot检测ZO-1、occludin和肌球蛋白轻链激酶（MLCK）的表达和MLC的磷酸化。人角膜上皮（HCE）细胞在Transwell过滤器上室培养，用电压表测量上皮电阻（TER）。ZO-1和occludin的分布用免疫荧光法测定miR-155-5p处理的HCE细胞。..结果：miR-155-5p对角膜上皮损伤的修复有显著的促进作用，在角膜损伤修复过程中它可降低角膜上皮的通透性，显著降低MLCK的表达和MLC的磷酸化，增加伤口愈合过程中的角膜上皮细胞的紧密连接蛋白ZO-1和occludin的表达。miR-155-5p可显著增加TER，降低MLCK表达和MLC磷酸化，增加ZO-1和occludin表达，促进紧密连接蛋白在细胞膜中的锚定和HEC细胞的重塑。..结论：我们的结果表明miR-155-5p通过降低MLCK的表达和MLC的磷酸化来降低角膜通透性，加速角膜上皮伤口的恢复 通过重塑紧密的连接。