紧密连接在家兔自体颌下腺移植远期腺体分泌增多中的作用及机制研究

Microvascular autologous transplantation of submandibular gland is an effective strategy for treating severe dry eye syndrome, which provides a continuous, endogenous source of ocular lubrication. However, more than 40% of patients experience epiphora 4 months after surgery, and the mechanism has not yet been fully elucidated. Paracellular pathway which mediated by tight junction is one of the most important way in fluid transport of submandibular gland. But whether it is involved in hypersecretion of long-term transplanted gland and its underlying mechanism is still unclear. In this study, we used rabbit autologous transplantation model, isolated perfused submandibular gl to clarify the effect of tight junction in hypersecretion of long-term transplanted gland, and cytological experiments to verify that activated extracellular signal-regulated kinase might promoted phosphorylation of myosin light chain 2, and then inducing fibros actin reorganization and change of tight junction structure, resulting in open of paracellular pathway with an increased secretion of gland. This study will provide better understanding in the mechanism of secretion in transplanted submandibular glands, and enrich the knowledge of mechanism of secretion in normal submandibular gland, and improved the effect of submandibular gland transplantation for treating severe dry eye syndrome.

血管化自体颌下腺移植以颌下腺分泌液替代泪液治疗重症干眼症，但移植远期出现泪溢现象，严重影响手术疗效。而目前有关移植远期分泌调控机制尚不完全清楚。紧密连接介导的旁细胞途径是颌下腺水转运的重要方式之一，但其是否在移植远期腺体高分泌中发挥作用及其调控机制目前尚未有研究。本课题拟采用家兔自体颌下腺移植远期模型、离体颌下腺灌流相结合，明确紧密连接介导的旁细胞途径在移植远期腺体分泌增多中的作用；再通过细胞学实验验证移植远期腺体中ERK1/2激活，磷酸化下游底物MLC2，进而引起与之相连的细胞骨架蛋白F-actin的重排，导致紧密连接结构的变化，增加旁细胞途径的开放而引起分泌的增多的可能机制。该研究有助于全面认识移植颌下腺分泌的机制，丰富了正常颌下腺的分泌机制，对提高颌下腺移植治疗重症角结膜干燥症的成功率和临床疗效具有明显的应用价值。

血管化自体颌下腺移植以颌下腺分泌液替代泪液治疗重症干眼症。但移植6个月后，40%患者出现泪溢现象。紧密连接介导的旁细胞途径是颌下腺水转运的重要方式之一，但其是否在移植远期腺体高分泌中发挥作用及其调控机制目前尚未有研究。本项目通过建立家兔自体颌下腺移植远期模型模拟临床泪溢现象，证实家兔移植远期腺体分泌明显增多。进一步研究显示移植远期腺体中紧密连接宽度明显增加，旁细胞途径开放，同时与紧密连接复合物相连的细胞骨架蛋白F-actin发生重排，导致紧密连接结构的变化。家兔颌下腺离体灌流实验证实M受体激动剂卡巴胆碱模拟的高分泌状态下腺体紧密连接开放和F-actin在顶侧膜分布发生改变。揭示F-actin 重排，紧密连接开放引起旁细胞途径的通透性增加参与移植腺体的高分泌状态。此外，本项目还了证实家兔自体颌下腺移植远期高分泌腺体中M受体敏感性增加。局部涂抹M受体拮抗剂阿托品凝胶后，移植180天腺体的分泌量、紧密连接宽度以及F-actin在顶侧膜附近的聚集也显著减少。这表明激活M受体，引起 F-actin分布改变，介导紧密连接介导的旁细胞途径开放可以引起移植远期腺体分泌增加，局部涂抹阿托品可以抑制移植远期腺体的高分泌。本项目的研究成果揭示移植远期高分泌状态颌下腺中旁细胞途径的作用以及相应的调控机制，不仅有助于全面认识移植颌下腺分泌的机制，也对改善血管化自体颌下腺移植术后疗效，并提高术后患者的生活质量有重要意义。