气道平滑肌被动刚度和收缩力维持及相关调控机制的研究

发布时间：2019-05-15 12:18

【摘要】：哮喘病（Asthma）是目前世界上最常见的慢性呼吸道疾病之一，并已经被公认为是人类公共卫生健康的重大威胁，但令人遗憾的是，我们至今还不能很好地解释哮喘病的发病机理和发展过程。哮喘病最主要的病理学特征是气道受到刺激之后的过度收缩反应，又被称为气道的高反应性(Airway Hyperresponsiveness,AHR)。气道平滑肌作为主导气道收缩的因素，它的力学行为及其收缩性能，包括其刚度，收缩力，力-速率关系等力学特性，对于气道的力学反应和哮喘病研究具有重要的生理病理学意义，然而目前我们对于平滑肌力学性质的了解目前还存在很大的空白，近期研究结果显示，哮喘病人的气道平滑肌在受到拉伸之后，维持收缩力和恢复收缩力的能力显著高于健康人；另一方面，气道平滑肌的刚度，作为调节气道可扩张性的重要因素，一直以来被作为哮喘研究的重要目标。因此，本研究在众多的气道平滑肌力学性质之中选择了气道平滑肌刚度和维持收缩力的能力作为研究方向，采用了气道平滑肌组织，探索了刚度和维持收缩力作用以及其背后的信号通路和超微结构，主要的研究内容和结果如下： 1）气道平滑肌刚度可以独立于主动收缩力被调控，而Rho激酶（ROCK）是调控这一机制的信号通路。为了观察细胞骨架刚度的变化，我们建立了细胞膜透化的气道平滑肌组织，分离了肌球蛋白磷酸化和主动收缩对刚度产生的影响，实验中搭配使用钙离子和乙酰胆碱作为调控因子，观察记录并分析被动刚度变化。结果表明细胞骨架刚度并非完全被动,在pCa=10-9的情况下10-4M乙酰胆碱仍然可以显著提高通透化平滑肌的刚度，更重要的是，在平滑肌刚度增加的同时，并没有检测到主动收缩力的产生，肌肉继续维持静息张力，与此同时未侦测到任何肌球蛋白轻链（MLC20）磷酸化。进一步研究显示， ROCK抑制剂H1152(1μM)完全沉默了这一被动刚度变化，而调控主动收缩过程的肌球蛋白轻链激酶（MLCK）抑制则对这一刚度反应没有影响。 2）气道平滑肌维持收缩力的能力，特别是在收缩中对抗机械拉伸的机制是由ROCK通路调节的。为了研究平滑肌维持收缩力的能力及其信号通路，本试验中使用了四种不同的抑制剂，分别是：蛋白激酶C（PKC）抑制剂GF109023x(15μM),肌球蛋白轻链激酶（MLCK）抑制剂ML-7(5μM)，Rho激酶（ROCK）抑制剂Y27632(3μM)以及H1152(3μM)，这些抑制剂分别抑制了平滑肌收缩中最重要的三个通路。为了排除不同抑制剂对主动收缩力的影响，试验中的每一组肌肉收缩力都通过不同浓度的抑制剂（实验组）或乙酰胆碱（对照组）匹配到50%的最大等长收缩力（Fmax）。实验记录气道平滑肌产生的等长收缩力达到峰值之后100s内维持等长收缩力的能力，数据结果显示GF109023x以及ML-7没有影响平滑肌维持等长收缩力的能力，然而两个不同的ROCK抑制剂都显著的降低了平滑肌维持收缩力的能力。为了模拟深呼吸（DI）对气道的舒张（Bronchodilation effect）作用，进一步试验对正在收缩的平滑肌加入了25%Lref的机械拉伸，100s连续的周期拉伸数据显示了和之前试验相似的数据：对照组数据显示平滑肌在第一个次牵拉之后收缩力对抗力学拉伸的反应停留在一个稳定的水平，，GF109023以及ML-7的反应与对照组相同，而Y27632和H1152显著地抑制了气道平滑肌在收缩中对抗力学拉伸的能力。进一步研究集中于力学拉伸之后收缩力的恢复过程，数据表明只有ML-7没有影响收缩力的恢复，而ROCK抑制剂和PKC抑制剂都显著减缓了气道平滑肌受到拉伸后恢复收缩力的能力。 3）气道平滑肌维持收缩力的结构基础是肌球蛋白的聚合，而ROCK是调控这一过程的信号通路。为了研究气道平滑肌维持收缩力和在收缩过程中对抗机械拉伸能力的超微结构基础，并研究调节该结构变化的机制，试验同时使用了ROCK抑制剂Y27632和PKC抑制剂GF109023x，我们在肌肉达到收缩力峰值时固定组织并做肌肉横截面的电镜超薄切片，取得图像后分析不同实验组的肌球蛋白丝密度。结果显示在等长收缩力峰值相同的前提下，GF109023x抑制组和对照组的肌球蛋白重链密度没有显著差异，而ROCK通路的抑制显著地降低了气道平滑肌横截面中肌球蛋白重链的密度。本研究发现了平滑肌被动刚度可独立于主动收缩被调节；肌球蛋白丝的聚合组装能帮助平滑肌维持其收缩力，而这些机制均由ROCK通路调节。上述结果完善了平滑肌的收缩单元模型，发现了平滑肌细胞骨架刚度的变化机理，进一步解释了气道平滑肌收缩原理。Rho激酶信号通路在气道平滑肌生理学功能和哮喘病病理学机制中扮演重要角色，相关调节机制的发现对未来开发哮喘新药具有重要意义。
[Abstract]:Asthma (Astma) is one of the most common chronic respiratory diseases in the world, and has been recognized as a major threat to the health of human health, but it is regrettable that we have not yet done well to explain the pathogenesis and development of asthma. The most important pathological feature of asthma is the over-systolic reaction after the airway is stimulated, and is also known as the high reactivity (AHR) of the airway. airway smooth muscle as a factor leading to the contraction of the airway, its mechanical behavior and its contracted performance, including its mechanical properties such as its stiffness, the contractive force, the force-rate relationship and so on, has important physiological and pathological significance for the mechanical response of the airway and the study of asthma, However, at present, we have a great gap in the understanding of the mechanical properties of the smooth muscle, and the recent study shows that the ability of the airway smooth muscle of the asthma person to maintain the contractive force and the recovery contraction force after being stretched is significantly higher than that of the healthy person, and on the other hand, The stiffness of the airway smooth muscle, as an important factor for regulating the expansionary of the airway, has been an important target for the study of asthma. Therefore, this study chose the ability of airway smooth muscle stiffness and the ability to maintain the contractility as the research direction among the many mechanical properties of the airway smooth muscle, and adopted the airway smooth muscle tissue, and explored the effect of the rigidity and the maintenance and contraction force as well as the signal path and the ultrastructure behind it. The main contents and results are as follows: 1) The stiffness of the airway smooth muscle can be regulated independently of the active contracting force, and the Rho kinase (ROCK) is the signal that regulates this mechanism. In order to observe the changes of the cell skeleton stiffness, we established a cell membrane permeabilized airway smooth muscle tissue, isolated the effect of myosin phosphorylation and active contraction on the stiffness, and in the experiment, calcium ion and acetylcholine were used as the control factor. son, observe the record and analyze the passive stiffness change The results show that the stiffness of the cytoskeleton is not completely passive, and the 10-4 M acetylcholine in the case of pCa = 10-9 can obviously improve the rigidity of the permeabilized smooth muscle, and more importantly, the rigidity of the smooth muscle is increased, and the generation of the active contracting force is not detected, and the muscles continue to maintain the rest. Force, while no myosin light chain (MLC20) phosphoric acid was detected Further studies showed that the ROCK inhibitor-152 (1. mu.M) completely silent the change in the passive stiffness, while the inhibition of myosin light-chain kinase (MLCK), which regulates the active contraction, has no shadow on this stiffness reaction. in response to that ability of the airway smooth muscle to maintain a contractive force, especially in the contraction, the mechanism for resisting mechanical tension is by the ROCK pathway In order to study the ability of smooth muscle to maintain a contractile force and its signal path, four different inhibitors were used in this test: protein kinase C (PKC) inhibitor GF109023x (15. mu.M), myosin light chain kinase (MLCK) inhibitor ML-7 (5 . mu.M), Rho kinase (ROCK) inhibitor Y27632 (3. mu.M), and REGN152 (3. mu.M), which respectively inhibit the most important three of smooth muscle contraction In order to exclude the effect of different inhibitors on the active contractile force, each set of muscle contractility in the test was matched to 50% of the maximum isometric contractility (Fm) by a different concentration of inhibitor (experimental group) or B-choline (control group) The results showed that GF109023x and ML-7 did not affect the isometric contractility of smooth muscle. The ability of the two different ROCK inhibitors, however, to significantly reduce the contraction of the smooth muscle In order to simulate the effect of deep breathing (DI) on the relaxation of the airway, a further test was performed on the mechanical stretching of 25% Lref to the smooth muscle being contracted, and the 100 s continuous cycle tensile data showed similar to the previous test The data of the control group showed that the response of the contraction force to the mechanical tension after the first pull of the smooth muscle remained at a stable level, and the reaction of the GF109023 and the ML-7 was the same as the control group, while the Y27632 and the REGN152 significantly inhibited the mechanical stretching of the airway smooth muscle in the contraction The data showed that only ML-7 did not affect the recovery of the contractile force, while the ROCK inhibitor and the PKC inhibitor significantly reduced the recovery and contraction of the airway smooth muscle after being stretched. 3) The structural basis of airway smooth muscle maintenance and contraction force is the polymerization of myosin, and the ROCK is the control of the process. In order to study the ultrastructure of airway smooth muscle maintenance and contraction and to counter the mechanical tensile capacity in the course of the contraction, and to study the mechanism of regulating the change of the structure, the ROCK inhibitor Y27632 and the PKC inhibitor GF10 were also used. 9023x, when the muscle reaches the peak of the contraction force, the tissue is fixed and the muscle section of the muscle is made into the ultrathin section, and the muscle ball of the different experimental group is analyzed after the image is obtained. The results showed that there was no significant difference in the density of myosin heavy chain in the GF109023x and the control group, while the inhibition of the ROCK pathway significantly reduced the myosin in the cross-section of the airway smooth muscle. The present study found that the passive stiffness of smooth muscle can be regulated independently of the active contraction; the aggregation and assembly of myosin filament can help smooth muscle to maintain its contractile force, all of which are R The results of the above results improved the model of the contraction of the smooth muscle and found the mechanism of the change of the stiffness of the smooth muscle cell, and further explained the airway of the smooth muscle. The principle of smooth muscle contraction. Rho kinase signaling pathway plays an important role in the physiological function of airway smooth muscle and the pathological mechanism of asthma, and the discovery of related regulation mechanism is new to the development of asthma in the future.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R562.25

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