本文關鍵詞：模擬失重對心血管系統(tǒng)的影響及機制研究　出處：《第四軍醫(yī)大學》2009年博士論文　論文類型：學位論文

  更多相關文章： 頭低位臥床 模擬失重 失重 血管平滑肌細胞 心肌萎縮 股動脈 Akt/GSK-3β β-catenin

【摘要】： 失重/模擬失重可導致心血管系統(tǒng)功能失調,心臟、動脈血管平滑肌結構重塑和功能變化是心血管系統(tǒng)功能失調的重要原因。然而,目前國內尚無有關長期模擬失重導致心血管系統(tǒng)功能失調的研究報道;此外,失重/模擬失重下心肌、血管結構重塑和功能變化的機制并不清楚。因此,本課題的目的是: (1)通過60天頭低位臥床模擬失重實驗獲取中國人群的心血管系統(tǒng)實驗數(shù)據,同時對阻抗振動和中藥太空養(yǎng)心方兩組對抗措施的防護效果進行評價; (2)闡明模擬失重下心肌萎縮的分子調控機制; (3)闡明模擬失重下后身動脈血管收縮功能變化的調控機制; (4)闡明失重和模擬失重對血管平滑肌細胞增殖的影響及β-catenin信號調控機制。 為達到以上研究目的,本課題采用以下實驗方法進行研究: (1)以60天頭低位臥床模擬失重,采用彩色多普勒超聲技術檢測心臟和血管的結構和功能;以靜脈阻斷體積描記術檢測下肢靜脈順應性及靜脈血流阻力,同時評價對抗措施的防護效果; (2)以尾吊鼠為模擬失重模型,以太空養(yǎng)心方為防護措施,采用Langendorff離體心臟灌流技術檢測離體心臟功能;以FITC-Lectin對心肌細胞進行免疫熒光染色,分析心肌細胞橫截面積的大小;采用Western blot技術檢測心肌組織TnI、Bcl-2、Hsp20蛋白表達及Akt、GSK3-β、FAK、P38 MAPK和Hsp27蛋白磷酸化水平的變化; (3)采用離體血管灌流技術,以Powerlab生理記錄儀檢測模擬失重后股動脈和腹主動脈對NE或PE的收縮反應;通過給予ERK/CaD、MLCK、PI3K、P38 MAPK、L-型鈣通道特異性抑制劑PD98059、ML-7、LY-294002、SB203580、Nifedipine及肌動蛋白細胞骨架破壞劑Cytochalasin D,評價這些通路在尾吊模擬失重后血管收縮反應中的作用; (4)采用48小時回轉模擬失重和航天飛行真實失重環(huán)境培養(yǎng)血管平滑肌細胞,觀察失重和模擬失重對血管平滑肌細胞增殖的影響;采用Western blot和免疫熒光技術檢測模擬失重后β-catenin、N-Cadherin、Cyclin D1、P21Cip1蛋白表達及Akt、GSK-3β蛋白磷酸化水平的變化;采用免疫熒光技術檢測航天飛行后血管平滑肌細胞核的變化及β-catenin、N-Cadherin、Cyclin D1、P21Cip1蛋白表達變化。 本課題獲取的實驗結果如下: 一、頭低位臥床實驗 (1)58天臥床導致心肌萎縮、LDVD、SV等心功能指標下降。阻抗振動和太空養(yǎng)心方對臥床導致的心肌質量和心功能下降均有部分防護效果,并且太空養(yǎng)心方的防護效果優(yōu)于阻抗振動; (2)58天臥床對頸動脈和股動脈血管直徑無明顯影響,但卻可降低三組的門靜脈橫截面積,并且下降幅度在對照組達到了統(tǒng)計學顯著性差異; (3)58天臥床影響了血管的收縮功能:肺動脈、頸動脈和股動脈收縮速度在三組均發(fā)生下降,其中除頸動脈外,肺動脈收縮速度下降在阻抗振動組和中藥組達到統(tǒng)計學顯著性差異;三組的股動脈收縮速度在臥床后均顯著低于臥床前的水平;股動脈RI在對照組和阻抗振動組有下降的趨勢,而在中藥組卻有輕微升高趨勢;脛前動脈RI在對照組和中藥組有升高趨勢,而在阻抗振動組則有下降趨勢; (4)臥床38天導致下肢靜脈順應性顯著下降,但與對照組和阻抗振動組相比,中藥組的下降幅度明顯更小,太空養(yǎng)心方對38天臥床導致的下肢靜脈順應性下降有明顯的防護效果;52天臥床后,對照組和中藥組下肢靜脈順應性進一步下降,而阻抗振動組下降幅度減緩,略低于臥床38天的水平,說明阻抗振動對52天臥床導致的靜脈順應性下降有更好的防護效果;此外,中藥組下肢靜脈順應性雖然發(fā)生了進行性下降,但與對照組相比,下降幅度顯著降低,說明太空養(yǎng)心方對52天臥床導致的下肢靜脈順應性下降仍具有較好的防護效果; (5)臥床后下肢靜脈血流阻力呈進行性升高,在38天,對照組的升高幅度與阻抗振動組持平,均高于中藥組,說明太空養(yǎng)心方對38天臥床導致的下肢靜脈血流阻力升高的防護效果高于阻抗振動;但在52天后,對照組和中藥組的血流阻力呈進一步升高趨勢,而阻抗振動組血流阻力的增幅卻低于臥床38天,說明阻抗振動對52天臥床導致的下肢靜脈血流阻力升高具有比中藥太空養(yǎng)心方更好的防護效果。 二、模擬失重對心臟結構功能的影響及防護措施研究 (1)尾吊7天模擬失重導致大鼠心臟收縮和舒張功能下降,太空養(yǎng)心方對心功能的下降具有較好的防護效果; (2)尾吊7天模擬失重導致大鼠心肌萎縮,TnI蛋白表達下調,Akt/GSK-3β信號通路顯著受抑制,而太空養(yǎng)心方能夠對抗尾吊模擬失重導致的心肌細胞萎縮,TnI蛋白表達下調及Akt/GSK-3β信號通路受抑; (3)尾吊7天、14天和21天可導致心肌組織TnI蛋白表達進行性下降;7天、21天可顯著抑制Akt/GSK-3β信號通路;而尾吊14天則能增強Akt/GSK-3β信號通路活性; (4)尾吊14天模擬失重可顯著抑制心肌組織中P38 MAPK/Hsp27信號通路。三、模擬失重對血管功能的影響及機制研究 (1)尾吊模擬失重降低了大鼠股動脈的收縮反應并抑制了ERK/CaD信號通路; (2)尾吊7天模擬失重對股動脈MLCK/MLC20信號通路影響不大,但尾吊14天卻可顯著抑制MLCK/MLC20信號通路; (3)尾吊7天、14天可顯著抑制股動脈L-型鈣通道的功能; (4)尾吊14天可降低腹主動脈收縮反應;同時抑制P38 MAPK/Hsp27信號通路;但對股動脈中的P38 MAPK/Hsp27信號通路無影響; (5)尾吊7天、14天可抑制股動脈PI3K信號通路; (6)尾吊模擬失重對股動脈血管中的細胞骨架聚合功能沒有影響。 四、失重和模擬失重對血管平滑肌細胞增殖的影響及β-catenin信號通路研究 (1)失重和模擬失重可導致VSMC增殖能力下降,β-catenin信號調節(jié)通路在其中起著重要作用; (2)失重和模擬失重導致了β-catenin信號調節(jié)通路上游信號分子N-Cadherin表達、Akt和GSK-3β活性的下降; (3)失重和模擬失重抑制β-catenin信號,導致VSMC增殖下降的直接原因是Cyclin D1表達下調,細胞周期停滯于G1期;以及P21Cip1表達過高或過低,首次發(fā)現(xiàn)模擬失重和失重對P21 Cip1表達存在不同方式的調控。 本課題得到以下結論: (1)首次在中國志愿者身上發(fā)現(xiàn)60天頭低位臥床可導致心肌萎縮、功能下降;動脈血管收縮功能下降,下肢靜脈順應性下降和血流阻力升高;太空養(yǎng)心方和阻抗振動鍛煉對60天臥床導致的心血管功能失調具有部分防護效果; (2)尾吊模擬失重可通過抑制調控心肌細胞肥大的Akt/GSK-3β信號通路導致心肌萎縮,太空養(yǎng)心方能夠增強Akt/GSK-3β信號通路的活性,抑制尾吊導致的心肌萎縮;同時還能提高心肌組織Hsp20蛋白表達水平,增強心肌的保護作用。此外,尾吊模擬失重還能通過抑制P38 MAPK/Hsp27信號通路導致心肌萎縮; (3)尾吊模擬失重降低了大鼠股動脈和腹主動脈的收縮反應,抑制了股動脈收縮反應中ERK/CaD信號通路、MLCK/MLC20信號通路、PI3K信號通路及L-型鈣通道的功能;抑制了腹主動脈收縮反應中P38 MAPK/Hsp27信號通路功能; 4)首次發(fā)現(xiàn)失重可導致VSMC增殖能力下降,失重和模擬失重可抑制β-catenin及其上游信號分子N-Cadherin的表達;模擬失重還可導致Akt/GSK-3β活性下降;導致VSMC增殖下降的直接原因是Cyclin D1表達下調,細胞周期停滯于G1期以及P21 Cip1表達過高或過低。首次發(fā)現(xiàn)模擬失重和失重對P21 Cip1蛋白的表達存在不同的調控方式。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R85

【引證文獻】

相關碩士學位論文 前1條

1 馮惠;模擬微重力效應對成骨細胞紡錘體結構的影響[D];哈爾濱工業(yè)大學;2011年

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