自主神经通过小梁细胞骨架调控小梁网房水外流的实验研究

Numerous studies indicated that the trabecular meshwork (TM) could actively participate in the regulation of aqueous humor outflow resistance. Meanwhile cytoskeleton of the TM cell could influence the systolic and diastolic state of the TM and was the regulation target of aqueous humor outflow resistance. In addition, this regulation function was associated with the autonomic nervous and exploring the effect of autonomic nerve on the cytoskeleton of the TM cell could be the key point to reveal the reason for the alteration of aqueous humor outflow resistance. According to our previous studies, we found that the excitement of autonomic nervous could induce expansion of the TM and decrease in the intraocular pressure (IOP); autonomic neurotransmitters could be found distributed in the TM; the autonomic neurotransmitters, cytoskeleton of the TM cell and opening state of the TM were abnormal in the glaucomatous rat model and cytoskeleton of the TM cell, opening state of the TM and IOP could change after local autonomic nervous medicine application. Based on these, we hypothesize that autonomic nervous could influence cytoskeleton of the TM cell, then altering the systolic and diastolic state of the TM，maintaining the normal configuration of aqueous outflow channel and finally regulating the aqueous humor outflow resistance. In this project, we would like to observe the morphology of the TM cell cytoskeleton in vitro under the intervention of different pressure stress and autonomic nervous medicine by atomic mechanics microscope and immunofluorescence technique, verifying that autonomic nervous could change the abnormal cytoskeleton of the TM cell induced by high pressure and to study the association between the excitability of the autonomic nervous ( local eye dropping of the autonomic nervous medicine ) and the cytoskeleton of the TM cell, opening state of the TM, ultrastructure of the TM and IOP by scanning electron microscope and immunofluorescence technique, and finally exploring new thought for clinical treatment of glaucoma and physiological mechanism of autonomic nervous regulate aqueous humor outflow by influencing cytoskeleton of the TM cell.

大量研究表明小梁网能主动调节房水外流，且与自主神经相关。同时小梁细胞骨架可影响小梁网舒张和收缩，是房水外流调节的靶点。探索自主神经重构小梁细胞骨架是揭示房水外流阻力变化的关键问题。我们研究发现：自主神经兴奋可使小梁网扩张、眼压下降；小梁网存在自主神经递质分布；青光眼模型鼠自主神经递质、小梁细胞骨架、小梁网开放状态异常；眼局部给予自主神经工具药后，小梁细胞骨架、小梁网开放状态及眼压改变。由此假设：自主神经重构小梁细胞骨架，改变小梁网舒张或收缩状态，维持房水流出通道正常构型，调节房水外流。本项目拟用免疫荧光、原子力学显微镜观察压力刺激、工具药干预下的体外小梁细胞骨架，验证自主神经可改变压力所致的异常小梁细胞骨架状态；青光眼模型鼠眼局部给予工具药，用免疫荧光、扫描电镜观察小梁细胞骨架与小梁网开放状态、表面结构及眼压变化关系。为自主神经通过小梁细胞骨架调控房水外流的生理机制及青光眼治疗开拓新思路。

大量研究表明，小梁网能主动调节房水外流，且与自主神经相关。小梁细胞骨架在眼压调控、青光眼发病和药物调节房水外流阻力中扮演重要角色。研究证实小梁细胞骨架重构是影响小梁网舒张和收缩状态、维持房水外流通道正常构型的关键。我们前期的临床研究运动实验发现自主神经兴奋性发生变化时，可观察到小梁网扩张及眼压下降。因此，我们想探索自主神经调节小梁细胞骨架状态，进而调控小梁网舒张或收缩状态，影响房水外流。当自主神经调节紊乱，小梁细胞骨架异常，小梁网舒张减弱或收缩增强，致眼压升高。通过改变局部自主神经递质含量，重构小梁细胞骨架，维持小梁网房水流出通道的正常构型。目前研究结果：1、青光眼模型鼠小梁网房水流出通道血管活性肠肽VIP受体VPAC2含量增加、小梁网形态压缩、F-actin 排列紊乱、块状 F-actin 取代连续线状 F-actin。2、VIP通过与VPAC2受体结合，调节小梁网房水流出通道F-actin骨架构象，促使F-actin向G-actin转变，调节F-/G-actin比例，使细胞骨架疏松、间隙增大，扩张小梁网房水外流通道，达到降低眼压的作用。3、使用工具药VIP后，小梁网房水流出通道中Sp1发生核转移及LRRK2表达增加；F-/G-actin比值降低，F-actin向G-actin的转化，使二者保持动态平衡，从而维持细胞骨架的稳定性；使用Sp1抑制剂后，VIP作用下的LRRK2表达增加被抑制，进而抑制VIP对F-/G-actin比例的调节。提示VIP通过Sp1核转移上调LRRK2的表达，进而改变 F-actin和G-actin间的比例，并调节F-actin的稳态结构。推测VIP 通过Sp1-LRRK2信号通路调控小梁网房水流出通路细胞骨架从而达到调控眼压的目的。4、扫描电镜发现青光眼模型鼠小梁网房水流出通道细胞间连接显著致密，提示高眼压下小梁网房水流出通道细胞连接致密化，导致房水流出阻力增加。5、给予青光眼鼠VIP点眼，眼压下降的同时伴其异常增多的小梁网房水流出通道细胞连接蛋白ZO-1可以重新降至正常水平。提示VIP可调控细胞连接，使得细胞间的连接松弛。以上结果为自主神经递质VIP通过小梁网房水流出通道细胞骨架维持房水流出通道的正常构型来调控房水外流的环路机制及临床干预提供新思路。