模拟失重对心血管系统的影响及机制研究

发布时间：2017-12-31 07:46

本文关键词：模拟失重对心血管系统的影响及机制研究　出处：《第四军医大学》2009年博士论文　论文类型：学位论文

【摘要】： 失重/模拟失重可导致心血管系统功能失调,心脏、动脉血管平滑肌结构重塑和功能变化是心血管系统功能失调的重要原因。然而,目前国内尚无有关长期模拟失重导致心血管系统功能失调的研究报道;此外,失重/模拟失重下心肌、血管结构重塑和功能变化的机制并不清楚。因此,本课题的目的是: (1)通过60天头低位卧床模拟失重实验获取中国人群的心血管系统实验数据,同时对阻抗振动和中药太空养心方两组对抗措施的防护效果进行评价; (2)阐明模拟失重下心肌萎缩的分子调控机制; (3)阐明模拟失重下后身动脉血管收缩功能变化的调控机制; (4)阐明失重和模拟失重对血管平滑肌细胞增殖的影响及β-catenin信号调控机制。为达到以上研究目的,本课题采用以下实验方法进行研究: (1)以60天头低位卧床模拟失重,采用彩色多普勒超声技术检测心脏和血管的结构和功能;以静脉阻断体积描记术检测下肢静脉顺应性及静脉血流阻力,同时评价对抗措施的防护效果; (2)以尾吊鼠为模拟失重模型,以太空养心方为防护措施,采用Langendorff离体心脏灌流技术检测离体心脏功能;以FITC-Lectin对心肌细胞进行免疫荧光染色,分析心肌细胞横截面积的大小;采用Western blot技术检测心肌组织TnI、Bcl-2、Hsp20蛋白表达及Akt、GSK3-β、FAK、P38 MAPK和Hsp27蛋白磷酸化水平的变化; (3)采用离体血管灌流技术,以Powerlab生理记录仪检测模拟失重后股动脉和腹主动脉对NE或PE的收缩反应;通过给予ERK/CaD、MLCK、PI3K、P38 MAPK、L-型钙通道特异性抑制剂PD98059、ML-7、LY-294002、SB203580、Nifedipine及肌动蛋白细胞骨架破坏剂Cytochalasin D,评价这些通路在尾吊模拟失重后血管收缩反应中的作用; (4)采用48小时回转模拟失重和航天飞行真实失重环境培养血管平滑肌细胞,观察失重和模拟失重对血管平滑肌细胞增殖的影响;采用Western blot和免疫荧光技术检测模拟失重后β-catenin、N-Cadherin、Cyclin D1、P21Cip1蛋白表达及Akt、GSK-3β蛋白磷酸化水平的变化;采用免疫荧光技术检测航天飞行后血管平滑肌细胞核的变化及β-catenin、N-Cadherin、Cyclin D1、P21Cip1蛋白表达变化。本课题获取的实验结果如下: 一、头低位卧床实验 (1)58天卧床导致心肌萎缩、LDVD、SV等心功能指标下降。阻抗振动和太空养心方对卧床导致的心肌质量和心功能下降均有部分防护效果,并且太空养心方的防护效果优于阻抗振动; (2)58天卧床对颈动脉和股动脉血管直径无明显影响,但却可降低三组的门静脉横截面积,并且下降幅度在对照组达到了统计学显著性差异; (3)58天卧床影响了血管的收缩功能:肺动脉、颈动脉和股动脉收缩速度在三组均发生下降,其中除颈动脉外,肺动脉收缩速度下降在阻抗振动组和中药组达到统计学显著性差异;三组的股动脉收缩速度在卧床后均显著低于卧床前的水平;股动脉RI在对照组和阻抗振动组有下降的趋势,而在中药组却有轻微升高趋势;胫前动脉RI在对照组和中药组有升高趋势,而在阻抗振动组则有下降趋势; (4)卧床38天导致下肢静脉顺应性显著下降,但与对照组和阻抗振动组相比,中药组的下降幅度明显更小,太空养心方对38天卧床导致的下肢静脉顺应性下降有明显的防护效果;52天卧床后,对照组和中药组下肢静脉顺应性进一步下降,而阻抗振动组下降幅度减缓,略低于卧床38天的水平,说明阻抗振动对52天卧床导致的静脉顺应性下降有更好的防护效果;此外,中药组下肢静脉顺应性虽然发生了进行性下降,但与对照组相比,下降幅度显著降低,说明太空养心方对52天卧床导致的下肢静脉顺应性下降仍具有较好的防护效果; (5)卧床后下肢静脉血流阻力呈进行性升高,在38天,对照组的升高幅度与阻抗振动组持平,均高于中药组,说明太空养心方对38天卧床导致的下肢静脉血流阻力升高的防护效果高于阻抗振动;但在52天后,对照组和中药组的血流阻力呈进一步升高趋势,而阻抗振动组血流阻力的增幅却低于卧床38天,说明阻抗振动对52天卧床导致的下肢静脉血流阻力升高具有比中药太空养心方更好的防护效果。二、模拟失重对心脏结构功能的影响及防护措施研究 (1)尾吊7天模拟失重导致大鼠心脏收缩和舒张功能下降,太空养心方对心功能的下降具有较好的防护效果; (2)尾吊7天模拟失重导致大鼠心肌萎缩,TnI蛋白表达下调,Akt/GSK-3β信号通路显著受抑制,而太空养心方能够对抗尾吊模拟失重导致的心肌细胞萎缩,TnI蛋白表达下调及Akt/GSK-3β信号通路受抑; (3)尾吊7天、14天和21天可导致心肌组织TnI蛋白表达进行性下降;7天、21天可显著抑制Akt/GSK-3β信号通路;而尾吊14天则能增强Akt/GSK-3β信号通路活性; (4)尾吊14天模拟失重可显著抑制心肌组织中P38 MAPK/Hsp27信号通路。三、模拟失重对血管功能的影响及机制研究 (1)尾吊模拟失重降低了大鼠股动脉的收缩反应并抑制了ERK/CaD信号通路; (2)尾吊7天模拟失重对股动脉MLCK/MLC20信号通路影响不大,但尾吊14天却可显著抑制MLCK/MLC20信号通路; (3)尾吊7天、14天可显著抑制股动脉L-型钙通道的功能; (4)尾吊14天可降低腹主动脉收缩反应;同时抑制P38 MAPK/Hsp27信号通路;但对股动脉中的P38 MAPK/Hsp27信号通路无影响; (5)尾吊7天、14天可抑制股动脉PI3K信号通路; (6)尾吊模拟失重对股动脉血管中的细胞骨架聚合功能没有影响。四、失重和模拟失重对血管平滑肌细胞增殖的影响及β-catenin信号通路研究 (1)失重和模拟失重可导致VSMC增殖能力下降,β-catenin信号调节通路在其中起着重要作用; (2)失重和模拟失重导致了β-catenin信号调节通路上游信号分子N-Cadherin表达、Akt和GSK-3β活性的下降; (3)失重和模拟失重抑制β-catenin信号,导致VSMC增殖下降的直接原因是Cyclin D1表达下调,细胞周期停滞于G1期;以及P21Cip1表达过高或过低,首次发现模拟失重和失重对P21 Cip1表达存在不同方式的调控。本课题得到以下结论: (1)首次在中国志愿者身上发现60天头低位卧床可导致心肌萎缩、功能下降;动脉血管收缩功能下降,下肢静脉顺应性下降和血流阻力升高;太空养心方和阻抗振动锻炼对60天卧床导致的心血管功能失调具有部分防护效果; (2)尾吊模拟失重可通过抑制调控心肌细胞肥大的Akt/GSK-3β信号通路导致心肌萎缩,太空养心方能够增强Akt/GSK-3β信号通路的活性,抑制尾吊导致的心肌萎缩;同时还能提高心肌组织Hsp20蛋白表达水平,增强心肌的保护作用。此外,尾吊模拟失重还能通过抑制P38 MAPK/Hsp27信号通路导致心肌萎缩; (3)尾吊模拟失重降低了大鼠股动脉和腹主动脉的收缩反应,抑制了股动脉收缩反应中ERK/CaD信号通路、MLCK/MLC20信号通路、PI3K信号通路及L-型钙通道的功能;抑制了腹主动脉收缩反应中P38 MAPK/Hsp27信号通路功能; 4)首次发现失重可导致VSMC增殖能力下降,失重和模拟失重可抑制β-catenin及其上游信号分子N-Cadherin的表达;模拟失重还可导致Akt/GSK-3β活性下降;导致VSMC增殖下降的直接原因是Cyclin D1表达下调,细胞周期停滞于G1期以及P21 Cip1表达过高或过低。首次发现模拟失重和失重对P21 Cip1蛋白的表达存在不同的调控方式。
[Abstract]:Weightlessness / simulated weightlessness can lead to cardiovascular deconditioning, cardiac remodeling and function changes of vascular smooth muscle structure artery is an important cause of cardiovascular dysfunction. However, there is no domestic long-term cardiovascular research reports simulated weightlessness system dysfunction; in addition, weightlessness or simulated weightlessness on myocardial remodeling and function mechanism, structure change of blood vessel is not clear. Therefore, the purpose of this project is to:
(1) the cardiovascular system simulation experimental data obtained by Chinese population loss of 60 days of head down bed, vibration resistance and traditional Chinese medicine Taikong Yangxin prescription two countermeasures protection effect evaluation;
(2) to elucidate the molecular mechanism of myocardial atrophy under simulated weightlessness.
(3) to clarify the regulatory mechanism of simulated changes hindquarter arterial systolic function of weightlessness;
(4) to elucidate the effect of weightlessness and simulated weightlessness on the proliferation of vascular smooth muscle cells and the regulation mechanism of beta -catenin signal.
In order to achieve the above research purposes, the following experimental methods are used in this study.
(1) to 60 head down tilt simulated weightlessness, detected by color Doppler ultrasound technology structure and function of the heart and blood vessels; with venous occlusion plethysmography for detecting venous compliance and venous blood flow resistance, and to evaluate the protective effect of counter measures;
(2) in tail suspended rats to simulate weightlessness model, the space Yangxin prescription for protective measures, using Langendorff isolated heart perfusion technique of isolated heart function; on myocardial cells by immunofluorescence staining with FITC-Lectin analysis, cardiomyocyte cross-sectional area size; using Western blot technique to detect myocardial TnI, Bcl-2. The expression of Hsp20 protein and Akt, FAK, GSK3- beta, P38 change MAPK and phosphorylation of Hsp27 protein;
(3) using isolated vascular perfusion technique, loss of contractile response after femoral artery and abdominal aorta of NE or PE in Powerlab physiological recorder detection simulation; by giving ERK/CaD, MLCK, PI3K, P38, MAPK, L- type calcium channel specific inhibitor of PD98059, ML-7, LY-294002, SB203580, Nifedipine and actin cytoskeleton damage agent Cytochalasin D, evaluation of these pathways of vasoconstriction in simulated weightlessness effect in the tail suspension;
(4) the 48 hour rotating simulated weightlessness and space flight real weightlessness culture of vascular smooth muscle cells, observe the microgravity effects on the proliferation of vascular smooth muscle cells; using Western blot and immunofluorescence detection of simulated weightlessness after beta -catenin, N-Cadherin, Cyclin, D1, P21Cip1 and protein expression of Akt, GSK-3 protein phosphorylation changes level; detected by immunofluorescence technique after spaceflight in vascular smooth muscle nuclei and beta -catenin, N-Cadherin, Cyclin, D1, P21Cip1 protein expression.
The experimental results obtained in this study are as follows:
First, low head bed test
(1) 58 days of bed rest resulted in myocardial atrophy, and cardiac function indexes such as LDVD and SV decreased. Impedance vibration and space nourishing heart prescription had some protective effects on myocardial mass and heart function decline due to bed rest, and the protective effect of space Yangxin Fang was better than that of impedance vibration.
(2) 58 days' bed rest had no significant effect on the diameter of carotid artery and femoral artery, but it could reduce the cross sectional area of portal vein in three groups, and the decrease in the control group reached a significant difference.
(3) 58 days in bed affected the contractile functions of vascular: pulmonary artery, carotid artery and femoral artery systolic velocity occurred in the three groups were decreased, which in addition to the carotid artery, pulmonary artery systolic velocity decreased statistically significant difference in the vibration resistance group and Chinese medicine group; the three group of femoral artery systolic velocity in the bed were significantly lower than the level before the bed; the femoral artery RI in the control group and the vibration resistance group has a downward trend, while in the Chinese medicine group was slightly increased; RI of anterior tibial artery in control group and Chinese medicine group increased, and the vibration resistance group decreased;
(4) in bed for 38 days resulted in lower extremity venous compliance decreased significantly, but compared with the control group and the vibration resistance group, Chinese medicine group decreased significantly smaller, Taikong Yangxin prescription leads to 38 days in the lower extremity venous compliance decline has obvious protective effect; 52 days after bed rest, control group and Chinese medicine group of lower extremity venous compliance decreased further, and the vibration resistance group decreased slow, slightly lower than the level in bed for 38 days, that led to 52 days of vibration resistance in venous compliance decreased has a better protective effect; in addition, the Chinese medicine group of lower extremity venous compliance despite the progressive decline, but compared with the control group decreased significantly, that lead to Taikong Yangxin prescription for 52 days in lower extremity venous compliance decreased still has a good protective effect;
(5) in venous blood flow resistance was progressively increased, in 38 days, the control group increased and flat vibration resistance group, Chinese medicine group were higher than that, Taikong Yangxin prescription leads to 38 days in lower extremity venous flow resistance increased the protective effect of higher vibration resistance; but in 52 days, and the control group the traditional Chinese medicine group the blood flow resistance was further increased, and the vibration resistance group flow resistance increase was lower than that in bed for 38 days, 52 days due to the vibration resistance in lower extremity venous flow resistance increased compared with the protective effect of Chinese herbal medicine Taikong Yangxin prescription better.
Two, the study of the effect of simulated weightlessness on cardiac structure and function and the protective measures
(1) the tail suspension 7 days simulated weightlessness resulted in the decrease of the systolic and diastolic function of the rat heart.
(2) tail suspended for 7 days simulated weightlessness resulted in myocardial atrophy, TnI protein expression was down regulated, Akt/GSK-3 beta signaling pathway was significantly inhibited, and space Yangxin Fang could resist myocardial atrophy induced by simulated weightlessness, TnI protein expression was down regulated and Akt/GSK-3 beta signaling pathway was inhibited.
(3) tail suspension for 7 days, 14 days and 21 days can lead to myocardial TnI protein expression was decreased; 7 days, 21 days can significantly inhibit the Akt/GSK-3 beta signaling pathway; and tail suspension 14 days can enhance the Akt/GSK-3 beta signaling pathway activity;
(4) 14 days of simulated weightlessness can significantly inhibit the P38 MAPK/Hsp27 signaling pathway in myocardial tissue. Three, the effect of simulated weightlessness on vascular function and its mechanism
(1) tail suspension simulated weightlessness reduced the contractile response of the femoral artery in rats and inhibited the ERK/CaD signaling pathway.
(2) tail suspension 7 days simulated weightlessness had little effect on the MLCK/MLC20 signal pathway of femoral artery, but the tail suspension could significantly inhibit the MLCK/MLC20 signaling pathway for 14 days.
(3) tail suspension for 7 days, 14 days can significantly inhibit the function of the L- type calcium channel of the femoral artery.
(4) tail suspension for 14 days can reduce the contraction response of the abdominal aorta and inhibit the P38 MAPK/Hsp27 signaling pathway, but it has no effect on the P38 MAPK/Hsp27 signaling pathway in the femoral artery.
(5) tail suspension for 7 days and 14 days can inhibit the PI3K signal pathway of femoral artery.
(6) tail suspension simulated weightlessness has no effect on the cytoskeleton polymerization in the femoral artery.
Four, the effect of weightlessness and simulated weightlessness on the proliferation of vascular smooth muscle cells and the study of beta -catenin signaling pathway
(1) weightlessness and simulated weightlessness can lead to the decrease of VSMC proliferation ability, and the signaling pathway of beta -catenin plays an important role in it.
(2) weightlessness and simulated weightlessness resulted in the N-Cadherin expression of the upstream signal molecule of the beta -catenin signaling pathway and the decrease of Akt and GSK-3 beta activity.
(3) weightlessness and simulated weightlessness inhibit the expression of beta -catenin. The direct reason for the decline of VSMC is the downregulation of Cyclin D1, the arrest of cell cycle at G1 stage, and the excessive or low expression of P21Cip1. For the first time, weightlessness and weightlessness are the first time to control P21 Cip1 expression in different ways.
The following conclusions are obtained:
(1) for the first time in Chinese volunteers found that 60 day HDT can lead to myocardial atrophy, decreased function; arterial systolic function decreased, lower extremity venous compliance decreased and blood flow resistance increased cardiovascular function; Taikong Yangxin prescription and impedance of vibration exercise leads to 60 days in disorder with partial protective effect;
(2) after simulated weightlessness by Akt/GSK-3 beta signaling regulation of myocardial hypertrophy induced cardiac atrophy, Taikong Yangxin prescription can enhance Akt/GSK-3 beta signaling activity, inhibition of tail suspension induced myocardial atrophy; at the same time can increase the myocardial tissue Hsp20 protein expression, enhanced myocardial protective effects. In addition, tail suspension simulated weightlessness can inhibit P38 MAPK/Hsp27 signaling pathway leading to myocardial atrophy;
(3) simulated microgravity decreased the contractile response of rat femoral artery and abdominal aorta, the inhibition of ERK/CaD signaling pathway in femoral artery contraction, MLCK/MLC20 pathway, PI3K pathway and L- type calcium channel function; inhibition of P38 MAPK/ function of Hsp27 pathway in response to constriction of abdominal aorta;
4) for the first time found that weightlessness can decrease the proliferation ability of VSMC, weightlessness and simulated weightlessness can inhibit the expression of -catenin beta and its upstream signaling molecule N-Cadherin; simulated weightlessness can also lead to decreased Akt/GSK-3 activity; a direct cause of the proliferation of VSMC is decreased Cyclin D1 expression, cell cycle arrest in G1 phase and P21 Cip1 expression is too high or too low for the first time found that simulated weightlessness and microgravity on the expression of P21 Cip1 protein in the presence of different control methods.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R85

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