【作者】 高放；

【导师】 余志斌； 张立藩； 马进； 程九华；

【作者基本信息】 第四军医大学 ， 航空、航天与航海医学， 2008， 博士

【摘要】 在航天失重环境停留可引起机体流体静压消失,从而使全身动脉血管系统的跨壁压发生重新分布变化:脑及上半身血压处于较地面1G直立体位时相对升高的“高血压”状态;下半身血管则处于较1G直立时相对较低的“低血压”状态。本实验室首次在地面动物实验系统阐明:模拟失重可引起大鼠脑动脉血管的肌源性紧张度增强,收缩反应性升高,血管中膜肥厚和平滑肌细胞层数增多,以及血管周围神经支配增强等适应性变化。而对于后身中、小动脉,则可引起肌源性紧张度减弱,收缩反应性降低,血管中膜萎缩,以及血管周围神经支配减弱等变化。据此我们曾提出“外周效应器机制假说”,认为失重所引起的心肌与动脉血管平滑肌结构和功能改变很可能是导致飞行后心血管失调的另一个重要原因（首要因素仍是血量减少）。我们还进一步发现:血管平滑肌离子通道机制与血管组织局部肾素-血管紧张素系统（local renin-angiotensin system, L-RAS）可能是介导失重所致血管区域特异性适应变化的重要调节环节。在对抗措施研究方面,我们首次报道了每日短时-GX重力[模拟间断性人工重力（intermittent artificial gravity, IAG）]对防止模拟失重致心肌与动脉血管不良影响有令人惊奇的对抗效果。但还有以下问题需进一步阐明:首先,我们前期工作主要来自大鼠肌型中、小动脉,而对于弹力型大动脉血管的研究则较少,影响我们对失重致血管适应变化的全面了解。例如,大血管壁周向应力集中于靠近管腔的肌层,如果血管跨壁压力分布是引起血管区域特异性适应变化的始动原因,则显著的重塑变化应发生于这些部位。其次,近年开始重视失重是否可引起大血管顺应性降低的问题,故有关弹力型大血管中膜细胞外间质的变化,需加强研究。再者,对于L-RAS的作用仍需通过药理阻断实验等进一步加以阐明。而且以大血管组织为标本,将更加有利于采用多种实验方法进行观察。最后,关于重力性对抗措施的机理问题,我们虽然已观察到,其对于肌型中等大小动脉血管的令人惊奇效果,但仍需进一步观察其对于弹力型大动脉血管重塑及关联的L-RAS改变的效果。为了回答以上问题,我们以“尾部悬吊头低位倾斜大鼠模型”模拟中期（4周）失重对动脉系统的影响,以每日1 h恢复正常体位（站立）模拟IAG的对抗效果。用组织形态测量研究大血管壁各肌层与弹力层的厚度、横截面积及血管内径等参数的变化。用免疫组织化学、蛋白免疫印迹分析、原位杂交及实时PCR等四种方法研究大动脉血管壁组织L-RAS关键成分,血管紧张素原（angiotensinogen, A_O）及血管紧张素Ⅱ1型受体（angiotensinⅡreceptor type 1, AT1R）的表达与定位。最后,我们还利用所建立的血管灌流培养系统,初步观察了每日短时低血管压力是否可以防止长时间异常压力引起的重塑变化,以为进一步的机理研究探索新的途径。本工作的主要发现如下:（1） 28天模拟失重可引起大鼠前/后身中等大小肌型动脉血管结构发生区域特异性适应改变。在光镜下通过组织形态学测量再次验证了我室前期电镜下观察的实验结果:28天模拟失重后,与对照组（CON）相比,悬吊组（SUS）基底动脉的管壁厚度（T）、中膜横截面积（CSA）及基底动脉的平滑肌细胞层数(N_CL)分别增加了25.9%、17.6%和20.5% （P<0.01）,而两组的内腔直径（D）和平滑肌细胞平均横截面积（AC）则没有显著性差别;反之,SUS组胫前动脉的T、CSA和N_CL分别减小了15.2%、17.3%和13.6% （P<0.01）,且两组的D和A_C没有显著差别。（2） 28天模拟失重可引起大鼠前/后身弹力型大动脉血管结构发生区域特异性适应改变。在光镜下测量结果显示: SUS组颈总动脉平均管壁厚度（T_W）较CON组增加了25.6% （P<0.01）。而两种动脉的D、平滑肌层层数（N_M）、弹力膜层数(N_EL)及腹主动脉的T_W,在两组间均无显著差别。颈总动脉管壁有4层平滑肌层和4层弹力膜层。与CON组相比,SUS组大鼠颈总动脉从M₁到M₄的各平滑肌层厚度分别增加了44.0%、42.4%、22.2%及39.8% （P<0.01）。反之,腹主动脉管壁有9层平滑肌层和9层弹力膜层,与CON组相比,SUS组大鼠腹主动脉从M₁到M₄的各平滑肌层厚度分别减少了17.0%、11.7%、11.4% （P<0.01）及10.1% （P<0.05）,而从M₅到M₉的各平滑肌层厚度则没有明显变化。再者,SUS组大鼠颈总动脉4层平滑肌层的CSA均显著增大,且以M₁和M₂的增加最为明显。而SUS组大鼠腹主动脉M₁、M₂和M₃的CSA降低最显著。与平滑肌层厚度变化相反,模拟失重后颈总动脉与腹主动脉各弹力膜层的厚度和CSA则分别显示降低和增加趋势,并且个别层的差别已达显著程度。（3） 28天模拟失重可引起大鼠颈总动脉和腹主动脉管壁组织中的Ao与AT1R蛋白与基因表达分别发生上调和下调改变。免疫组织化学实验显示,A_O与AT1R蛋白染色呈棕色,主要分布在血管外膜和周围组织,而中膜较少。模拟失重后,颈总动脉管壁中膜和外膜中的A_O与AT1R免疫反应性显著增强,而腹主动脉则显著减弱。免疫蛋白印迹分析实验进一步表明:与CON组相比,SUS组大鼠颈总动脉的A_O与AT1R蛋白表达分别增加了130.0%和50.0% （P<0.05）;而SUS组腹主动脉的A_O与AT1R蛋白表达则分别减少了48.9%和36.8% （P<0.05）。原位杂交实验显示,被检测到的A_O与AT1R mRNA信号主要分布于血管组织的中膜与外膜。模拟失重后,颈总动脉管壁中膜和外膜中的A_O与AT1R mRNA信号显著增强,而在腹主动脉则显著减弱。实时PCR实验进一步表明:与CON组相比,SUS组大鼠颈总动脉的A_O与AT1R mRNA表达分别增加了164.7% （P<0.01）和70.2% （P<0.05）;而SUS组大鼠腹主动脉的A_O与AT1R mRNA表达则分别降低了32.6% （P<0.05）和55.1% （P<0.01）。（4）阻断AT1R的条件下,28天模拟失重仍然能引起基底动脉与颈总动脉发生相对的肥厚性改变,并且腹主动脉的A_O与AT1R蛋白表达显著减少。利用losartan慢性阻断AT1R 4周后,两个给药组[对照给药组（C+L）,悬吊给药组（S+L）]的基底动脉、胫前动脉、颈总动脉与腹主动脉的相关参数普遍较未给药组为低。如与对照组（C）相比,C+L组基底动脉的T、CSA、Ac和NCL分别降低了15.5%、17.6%、11.3%和10.3% （P<0.05,或<0.01）,而C+L组胫前动脉的T、CSA、Ac和NCL则分别减少了30.2%、21.1%、16.1 %和23.7% （P<0.05,或<0.01）。与C组相比,C+L组颈总动脉与腹主动脉的T也分别减少了29.9%和8.2% （P<0.01）。但与C+L相比, S+L组基底动脉的T、CSA和NCL仍然分别增加了12.2%、14.3%和5.7% （P<0.05,或<0.01）,而S+L组胫前动脉的上述各项参数则未见有显著改变;S+L组颈总动脉的T增加了23.0% （P<0.05）,而腹主动脉的T则仅呈减小趋势。与C组相比,C+L组颈总动脉A_O和AT1R的表达无显著性变化;而腹主动脉的A_O及AT1R表达均显著减低（P<0.05）。与C+L组相比,S+L组颈总动脉A_O和AT1R的表达均无显著性改变;而S+L组腹主动脉的AT1R表达降低（P<0.01）。（5）每日1 h站立可完全防止模拟失重大鼠弹力型大动脉血管发生适应性重塑及A_O与AT1R表达变化。本实验除了再次验证我们早先关于肌型中等动脉血管的报道,还首次观察到每日1 h站立也可完全防止模拟失重大鼠弹力型大动脉血管的区域特异性重塑变化。与CON组相比,模拟失重+ 1 h/d站立组（SUS+STD1）大鼠颈总动脉的TW、各平滑肌层的厚度和CSA均无显著性改变;但其与SUS组间的差别则达显著程度（P<0.01）。再者,与CON组相比,SUS+STD1组腹主动脉的M1～M4的各平滑肌层厚度和M1～M4的CSA均无显著性改变;但其与SUS组的差别则达显著程度（P<0.05,或<0.01）。此外,每日1 h站立也完全防止了两种大动脉血管组织中相关联的A_O与AT1R蛋白与mRNA表达发生改变。（6）血管灌流培养实验的初步结果支持血管跨壁压是始动因素的假说。我们课题组已初步建立了血管灌流培养系统。在不同的灌流压下培养大鼠颈总动脉血管3天后,免疫组织化学染色显示,血管组织中的c-纤维粘连蛋白（c-fibronectin, c-FN）主要分布于中膜靠近管腔的平滑肌层中,且离管腔越远的平滑肌层染色越淡。在高灌流压（150 mmHg）下血管组织中的c-FN表达增加,而在低灌流压（80或0 mmHg）时表达很少;但在持续高压灌流下间断地降低灌流压,则血管壁组织中的c-FN表达很少,与低压灌流时相似。总之,以上发现进一步支持了我们的假说,即压力本身是失重引发血管区域特异性适应改变的始动因素,并且血管的局部RAS在该调节中发挥了重要作用。我们的工作也为重力性对抗措施IAG提供了重要的生理依据。血管灌流培养技术为进一步的机理研究提供理想的活体实验模型。更多还原

【Abstract】 Terrestrial animals and humans have adapted to a constant force of 1 G. Exposed to microgravity, undoubtedly all gravitational blood pressure gradients from head to feet that are associated with upright posture on Earth disappear, and a redistribution of transmural pressure and blood flows across and within the arterial vasculature is induced by the removal of hydrostatic pressure gradients. Therefore, in humans, vessels in brain and upper body regions are chronically exposed to higher than normal upright 1-G blood pressure, whereas vessels in dependent body regions are exposed to lower than normal 1-G blood pressure. Our previous ground-based animal studies have shown for the first time that simulated microgravity may induce upward and downward regulations in function, structure, and innervation state of the medium- and small-sized muscular arteries from fore （cerebral） and hind body parts of the same animal subjected to tail-suspended head-down tilt （SUS）. On the basis of these findings and the relevant ground-based and spaceflight studies reported recently, we have raised the“peripheral effector mechanism hypothesis”. We suggested that, in addition to hypovolemia, the microgravity-induced adaptation changes in function and structure of cardiac muscle and vascular smooth muscle might be another important factor responsible for postflight cardiovascular dysfunction. We further showed that the ion channel remodeling mechanism of vascular smooth muscle cells （VSMCs） and vascular local renin-angiotensin system （L-RAS） might be involved in vascular region-specific adaptational changes to microgravity. In the study of countermeasure, we have shown for the first time that daily 1-h standing （STD）, which mimics the effect of intermittent artificial gravity （IAG）, can surprisingly prevent the adaptational changes in cardiac muscle and muscular arteries due to microgravity alone.However, there remain several problems to be further elucidated. First, our previous data have been mainly from the studies with medium- and small-sized muscular arteries of rats and little information is available regarding the elastic large arteries. In order to obtain a perfect understanding of vascular adaptation to microgravity, these data are indispensable. For example, numerous studies have demonstrated a direct relationship between the circumferential stress and the structure of the vessel wall itself. Furthermore, at higher pressure loadings, the circumferential stress is highest at the inner wall, and declines across the wall. If redistribution of vascular transmural pressure is the primary stimulus initiating regional adaptation in arteries during microgravity, then the remodeling changes of innermost SM layers would be most significant. Second, recently more attention has been focused on the problem of whether microgravity can induce a decrease in aortic compliance. Therefore, the changes in extracellular matrix in the media of elastic large arteries should be further studied. Thirdly, the role of L-RAS in mediating vascular adaptation to microgravity still need be further clarified by pharmacological blockade and relevant methods. Furthermore, many sophisticated experimental methods can be used with specimens from large arteries. Finally, though we have shown that the gravity-based countermeasure might have a surprising counteracting effect on the adaptation remodeling of medium-sized muscular arteries, it still need be further examined with large elastic arteries.In order to elucidate the aforementioned problems, we used SUS rat model to simulate the microgravity-induced effects on arterial vasculature and daily 1-h standing to simulate the countermeasure effect of IAG. Moreover, we measured the thickness of each smooth muscle layer and elastic lamina, and the cross-sectional area of the wall and luminal diameter of large arteries and etc. by histomorphometry. we measured the expression of the key elements of L-RAS, angiotensinogen （A_O） and angiotensinⅡreceptor type 1 （AT1R） in the wall of large arteries using immunohistochemistry, Western blot analysis, in situ hybridization, and real-time PCR analysis. Finally, using the established ex vivo perfusion artery organ culture system, we made a preliminary observation on the effect of daily short-duration restoration to low pressure in preventing vascular remodeling due to a sustained high pressure.The main findings of the present work are as follows:（1） A 28-d simulated microgravity may induce region-specific adaptation changes in medium-sized muscular arteries from fore and hind body in rats.The results observed with electron microscope in our previous work were again validated with optical microscope by histomorphometry. The results showed that compared with control group （CON）, the media thickness （T）, media cross-sectional area （CSA）, and number of smooth cell layers (N_CL) of basilar artery from tail-suspension group （SUS） increased by 25.9%, 17.6%, and 20.5% （P<0.01）, respectively, after a 28-d simulated microgravity. Both the luminal diameter （D） and mean CSA of smooth muscle cell （AC） showed no significant differences between CON and SUS. Whereas, compared with CON, the T, CSA, and NCL of the anterior tibial artery from SUS decreased by 15.2%, 17.3%, and 13.6% （P<0.01）, respectively, and both D and AC showed no significant differences between the two groups.（2） A 28-d simulated microgravity may induce region-specific adaptation changes in large elastic arteries from fore and hind body in rats.The results observed with optical microscope showed that the average thickness of the wall （T_W） of the common carotid artery in SUS was significantly greater than CON group by 25.6% （P<0.01）, and there were no significant differences in the parameters, such as the D, mean number of smooth muscle layers （N_M）, and mean number of elastic luminae (N_EL) in the two kinds of arteries and in the TW of the abdominal aorta between CON and SUS groups. In the wall of common carotid artery, there are four smooth muscle layers and four elastic laminae. In SUS rats, the thickness of each smooth muscle layer from M1 to M4 increased by 44.0%, 42.4%, 22.2%, and 39.8% （P<0.01）, respectively, as compared with that of CON rats. In the wall of abdominal aorta, there are nine muscle layers and nine elastic laminae. In SUS rats, the thickness of each muscle layer from M1 to M4 decreased by 17.0%, 11.7%, 11.4%, （P<0.01） and 10.1% （P<0.05）, respectively, but from M5 to M9 it did not show significant changes. Furthermore, In the common carotid artery of SUS group, the CSA of each muscle layer increased significantly, with the trend that the maximum enlargement was in M₁ and M₂. While in the abdominal aorta of the SUS rats, the maximum decrease was in M₁, M₂, and M₃ layer. On the contrary with changes of smooth muscle layer, the thickness and CSA of each elastic lamina showed a trend of decrease and increase in the common carotid artery and abdominal aorta, respectively, after SUS, and some of these differences were statistically significant.（3） A 28-d simulated microgravity may induce up- and down- regulation of gene and protein expression of A_O and AT1R in the wall tissue of carotid artery and abdominal aorta in rats.The immunohistochemical results showed that stained A_O and AT1R are in brown color and mainly located in adventitia and perivascular tissue, but less in media. In the wall of the common carotid artery from a SUS rat, more intensive A_O and AT1R immunoreactivity were detected in the media and adventitia as compared with that of a CON rat. On the contrary, in the wall of the abdominal aorta from a SUS rat, these immunoreactivities were scarcely detected as compared with that of a CON rat. The results further indicated by Western blot analysis that in the common carotid artery from SUS rats, the A_O and AT1R protein expression increased by 130.0% and 50.0% （P<0.05）, respectively, as compared with CON rats. Whereas in the abdominal aorta from SUS rats, the A_O and AT1R protein expression decreased by 48.9% and 36.8% （P<0.05）, respectively, as compared with CON rats. The results by in situ hybridization showed that the specific signals of the A_O and AT1R mRNA were located in the media and adventitia of the two kinds of vessels. In the wall of the common carotid artery from a SUS rat, more intensive A_O and AT1R mRNA signals were detected in the media and adventitia as compared with that of a CON rat. On the contrary, in the wall of the abdominal aorta from a SUS rat, these mRNA signals were scarcely detected as compared with that of a CON rat. The results were further indicated by real-time PCR. Compared with CON rats, in the common carotid artery from SUS rats, A_O and AT1R mRNA expression increased by 164.7% （P<0.01） and 70.2% （P<0.05）, respectively. Whereas in the abdominal aorta from SUS rats, A_O and AT1R mRNA expression decreased by 32.6% （P<0.05）and 55.1% （P<0.01）, respectively, as compared with that of CON rats.（4） Chronic blockade of AT1R cannot prevent completely hypertrophic changes in basilar and carotid arteries due to simulated microgravity in rats.After four weeks of treatment by losartan, all the morphometric parameters of basilar artery, anterior tibial artery, common carotid artery, and abdominal aorta from the two treated group [treated control group （C+L） and treated tail suspension group （S+L）] significantly decreased as compared with untreated groups. For example, compared with control group （C）, the T, CSA, Ac, and NCL decreased by 15.5%, 17.6%, 11.3%, and 10.3% （P<0.05 or <0.01） in the basilar artery from C+L, respectively, and these parameters decreased by 30.2%, 21.1%, 16.1 %, and 23.7% （P<0.05 or <0.01） in the anterior tibial artery from C+L, respectively. Compared with C, the T decreased by 29.9% and 8.2% （P<0.01） of common carotid artery and abdominal aorta from C+L, respectively. However, compared with C+L, the T, CSA, and NCL increased by 12.2%, 14.3%, and 5.7% （P<0.05 or <0.01） in basilar artery from S+L, respectively, and these parameters of anterior tibial artery from S+L showed no significant changes. In common carotid artery from S+L the T increased by 23.0% （P<0.05） as compared with C+L. Compared with C, A_O and AT1R expressions of common carotid artery from C+L showed no significant changes, and both expressions of abdominal aorta from C+L significantly decreased. The A_O and AT1R expressions of common carotid artery from S+L showed no significant changes, and the AT1R expression of abdominal aorta from S+L obviously decreased （P<0.01） as compared with C+L.（5） Daily 1-h STD over 28 days fully prevented remodeling and L-RAS changes in large elastic arteries that might occur due to simulated microgravity alone in rats.The surprising effectiveness demonstrated in medium-sized muscular arteries was further confirmed in the present study with large elastic arteries. Compared with CON, the T_W, CSA and thickness of each smooth muscle layer of common carotid artery from suspension for 23 h/day and STD for 1 h/day group （SUS+STD1） showed no significant changes, but these parameters showed significant differences （P<0.01） as compared with SUS. Moreover, compared with CON, the thickness of each smooth muscle layer from M₁ to M₄ and CSA from M₁ to M₄ in SUS+STD1 group showed no significant changes, but these parameters showed significant differences （P<0.05 or <0.01） as compared with SUS. Furthermore, Daily 1-h STD fully prevented changes of protein and gene expression of AO and AT1R in the two kinds of elastic large arteries as well.（6） The hypothesis that pressure is the primary stimulus is supported by the preliminary result from the ex vivo experiment.Segments of rat common carotid artery can be maintained under different perfusion pressure schedules in organoid culture for three days. At 150 mmHg, not 0 or 80 mmHg, a marked expression of c-fibronectin （c-FN） was observed in the innermost smooth muscle layers of the media. However, daily 4-h restoration to 0 mmHg during the sustained high pressure perfusion under 150 mmHg prevented the enhanced c-FN expression.In conclusion, these findings have provided data to further support our hypothesis that pressure itself is the primary stimulus that initiates regional adaptation of arteries during microgravity and vascular L-RAS plays an important role in its regulation. Our work has also provided physiological evidence in support of the effectiveness of gravity-based countermeasure, like the IAG. The artery organoid culture system might provide an ideal ex vivo experimental model for further mechanistic study of mechanism.更多还原