氣道平滑肌被動剛度和收縮力維持及相關(guān)調(diào)控機(jī)制的研究
發(fā)布時間：2019-05-15 12:18
【摘要】：哮喘�。ˋsthma）是目前世界上最常見的慢性呼吸道疾病之一，并已經(jīng)被公認(rèn)為是人類公共衛(wèi)生健康的重大威脅，但令人遺憾的是，我們至今還不能很好地解釋哮喘病的發(fā)病機(jī)理和發(fā)展過程。哮喘病最主要的病理學(xué)特征是氣道受到刺激之后的過度收縮反應(yīng)，又被稱為氣道的高反應(yīng)性(Airway Hyperresponsiveness,AHR)。氣道平滑肌作為主導(dǎo)氣道收縮的因素，它的力學(xué)行為及其收縮性能，包括其剛度，收縮力，力-速率關(guān)系等力學(xué)特性，對于氣道的力學(xué)反應(yīng)和哮喘病研究具有重要的生理病理學(xué)意義，然而目前我們對于平滑肌力學(xué)性質(zhì)的了解目前還存在很大的空白，近期研究結(jié)果顯示，哮喘病人的氣道平滑肌在受到拉伸之后，維持收縮力和恢復(fù)收縮力的能力顯著高于健康人；另一方面，氣道平滑肌的剛度，作為調(diào)節(jié)氣道可擴(kuò)張性的重要因素，一直以來被作為哮喘研究的重要目標(biāo)。因此，本研究在眾多的氣道平滑肌力學(xué)性質(zhì)之中選擇了氣道平滑肌剛度和維持收縮力的能力作為研究方向，采用了氣道平滑肌組織，探索了剛度和維持收縮力作用以及其背后的信號通路和超微結(jié)構(gòu)，主要的研究內(nèi)容和結(jié)果如下： 1）氣道平滑肌剛度可以獨(dú)立于主動收縮力被調(diào)控，而Rho激酶（ROCK）是調(diào)控這一機(jī)制的信號通路。為了觀察細(xì)胞骨架剛度的變化，我們建立了細(xì)胞膜透化的氣道平滑肌組織，分離了肌球蛋白磷酸化和主動收縮對剛度產(chǎn)生的影響，實驗中搭配使用鈣離子和乙酰膽堿作為調(diào)控因子，觀察記錄并分析被動剛度變化。結(jié)果表明細(xì)胞骨架剛度并非完全被動,在pCa=10-9的情況下10-4M乙酰膽堿仍然可以顯著提高通透化平滑肌的剛度，更重要的是，在平滑肌剛度增加的同時，并沒有檢測到主動收縮力的產(chǎn)生，肌肉繼續(xù)維持靜息張力，與此同時未偵測到任何肌球蛋白輕鏈（MLC20）磷酸化。進(jìn)一步研究顯示， ROCK抑制劑H1152(1μM)完全沉默了這一被動剛度變化，而調(diào)控主動收縮過程的肌球蛋白輕鏈激酶（MLCK）抑制則對這一剛度反應(yīng)沒有影響。 2）氣道平滑肌維持收縮力的能力，特別是在收縮中對抗機(jī)械拉伸的機(jī)制是由ROCK通路調(diào)節(jié)的。為了研究平滑肌維持收縮力的能力及其信號通路，本試驗中使用了四種不同的抑制劑，分別是：蛋白激酶C（PKC）抑制劑GF109023x(15μM),肌球蛋白輕鏈激酶（MLCK）抑制劑ML-7(5μM)，Rho激酶（ROCK）抑制劑Y27632(3μM)以及H1152(3μM)，這些抑制劑分別抑制了平滑肌收縮中最重要的三個通路。為了排除不同抑制劑對主動收縮力的影響，試驗中的每一組肌肉收縮力都通過不同濃度的抑制劑（實驗組）或乙酰膽堿（對照組）匹配到50%的最大等長收縮力（Fmax）。實驗記錄氣道平滑肌產(chǎn)生的等長收縮力達(dá)到峰值之后100s內(nèi)維持等長收縮力的能力，數(shù)據(jù)結(jié)果顯示GF109023x以及ML-7沒有影響平滑肌維持等長收縮力的能力，然而兩個不同的ROCK抑制劑都顯著的降低了平滑肌維持收縮力的能力。為了模擬深呼吸（DI）對氣道的舒張（Bronchodilation effect）作用，進(jìn)一步試驗對正在收縮的平滑肌加入了25%Lref的機(jī)械拉伸，100s連續(xù)的周期拉伸數(shù)據(jù)顯示了和之前試驗相似的數(shù)據(jù)：對照組數(shù)據(jù)顯示平滑肌在第一個次牽拉之后收縮力對抗力學(xué)拉伸的反應(yīng)停留在一個穩(wěn)定的水平，，GF109023以及ML-7的反應(yīng)與對照組相同，而Y27632和H1152顯著地抑制了氣道平滑肌在收縮中對抗力學(xué)拉伸的能力。進(jìn)一步研究集中于力學(xué)拉伸之后收縮力的恢復(fù)過程，數(shù)據(jù)表明只有ML-7沒有影響收縮力的恢復(fù)，而ROCK抑制劑和PKC抑制劑都顯著減緩了氣道平滑肌受到拉伸后恢復(fù)收縮力的能力。 3）氣道平滑肌維持收縮力的結(jié)構(gòu)基礎(chǔ)是肌球蛋白的聚合，而ROCK是調(diào)控這一過程的信號通路。為了研究氣道平滑肌維持收縮力和在收縮過程中對抗機(jī)械拉伸能力的超微結(jié)構(gòu)基礎(chǔ)，并研究調(diào)節(jié)該結(jié)構(gòu)變化的機(jī)制，試驗同時使用了ROCK抑制劑Y27632和PKC抑制劑GF109023x，我們在肌肉達(dá)到收縮力峰值時固定組織并做肌肉橫截面的電鏡超薄切片，取得圖像后分析不同實驗組的肌球蛋白絲密度。結(jié)果顯示在等長收縮力峰值相同的前提下，GF109023x抑制組和對照組的肌球蛋白重鏈密度沒有顯著差異，而ROCK通路的抑制顯著地降低了氣道平滑肌橫截面中肌球蛋白重鏈的密度。 本研究發(fā)現(xiàn)了平滑肌被動剛度可獨(dú)立于主動收縮被調(diào)節(jié)；肌球蛋白絲的聚合組裝能幫助平滑肌維持其收縮力，而這些機(jī)制均由ROCK通路調(diào)節(jié)。上述結(jié)果完善了平滑肌的收縮單元模型，發(fā)現(xiàn)了平滑肌細(xì)胞骨架剛度的變化機(jī)理，進(jìn)一步解釋了氣道平滑肌收縮原理。Rho激酶信號通路在氣道平滑肌生理學(xué)功能和哮喘病病理學(xué)機(jī)制中扮演重要角色，相關(guān)調(diào)節(jié)機(jī)制的發(fā)現(xiàn)對未來開發(fā)哮喘新藥具有重要意義。
[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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R562.25
本文編號：2477500
[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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R562.25
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