血管钙化在模拟失重大鼠脑动脉重建中的作用及间断人工重力干预研究

The functional and structural cerebral arterial remodeling induced by weightless environment is one of the major causes of postflight orthostatic intolerance. Up to now, mechanisms underlying these changes have not been fully elucidated and no effective countermeasures have been raised. Vascular calcification is an important risk factor of cardiovascular events, with increased vascular stiffness and decreased vascular compliance as the main performances. Recent studies have shown increased cerebral arterial stiffness by long-term space flight. Besides, animal studies also demonstrated hypertrophic changes, increased stiffness and myogenic tone of the cerebral artery by simulated microgravity, all of which might be related with cerebral arterial calcification. As reported, intermittent artificial gravity (1-h/d－Gx prevention) could reverse the aforementioned changes induced by simulated microgravity, but the mechanisms are still not fully understood. Our study applies techniques such as hindlimb unweighted rat model preparation, in vitro vessel culture, vascular smooth muscle cells culture and cell transfection, to investigate cerebral arterial calcification induced by simulated microgravity and the following effects on the mechanical properties, function and structure of cerebral artery from multiple levels, including in vivo, organ, cell and gene levels. Moreover, we also intend to investigate the possible role of vascular calcification in the molecular mechanism of the prevention effects of 1-h/d－Gx on cerebral arterial remodeling, in order to determine the molecular target mediating the countermeasure effects of intermittent artificial gravity. Our study supplies novel knowledge for mechanisms underlying cerebral arterial remodeling induced by microgravity, and proposes new reference for the countermeasure studies against postflight orthostatic intolerance.

失重环境下脑动脉功能和结构重建是航天后立位耐力不良发生的重要原因之一，其机理尚未完全阐明，也缺乏有效对抗措施。血管钙化是心血管事件发生的危险因素，主要表现为血管僵硬度增加和顺应性降低。近年发现，长期航天飞行引起脑动脉僵硬度增加；模拟失重亦引起脑动脉肥厚性改变、僵硬度增加和肌源性紧张度增强，这些变化可能与脑动脉钙化有关。短时间断人工重力（1-h/d－Gx干预）可以改善模拟失重所致上述变化，其作用机制尚不清楚。因此，本研究拟采用模拟失重大鼠模型制备、细胞培养、血管培养和细胞转染等技术从动物整体、器官和细胞基因水平检测模拟失重大鼠脑动脉钙化及其对动脉力学特性、功能和结构的影响，并研究血管钙化参与1-h/d－Gx干预对抗脑动脉重建的可能分子机制，以期找到短时间断人工重力对抗脑动脉重建的分子作用靶点。本研究可为阐明失重环境脑动脉重建的机理提出新的见解，并为航天后立位耐力不良防护措施研究提供理论依据。

失重环境下动脉功能和结构重建是航天后立位耐力不良发生的重要原因之一，其机理尚未完全阐明，也缺乏有效对抗措施。血管钙化是心血管事件发生的危险因素，长期航天飞行引起的前身动脉僵硬度增加可能与此有关。本研究证实，1）模拟失重引起大鼠脑动脉和颈总动脉发生钙化，具体表现为：模拟失重引起大鼠血浆钙和磷的水平显著升高；模拟失重引起大鼠脑动脉和颈总动脉钙含量增加、钙化结节增多、ALP活性增强、成骨样细胞表型标志物的蛋白和基因表达增加、平滑肌细胞收缩表型标志物SM22α的蛋白和基因表达减少。2）模拟失重大鼠的颈总动脉钙化涉及OPG/RANKL/RANK系统改变。通过蛋白免疫印迹证实：模拟失重引起大鼠颈总动脉RANKL及其受体RANK的蛋白表达显著增强，而“诱饵”受体OPG蛋白表达没有显著变化；通过动脉体外培养发现激活RANK通路可调节性增强模拟失重大鼠颈总动脉增强的RANK基因表达；通过激活或抑制RANK通路改变OPG/RANKL/RANK系统可显著改变模拟失重增强的成骨样细胞表型标志物的基因表达。3）跨壁压升高引起的颈总动脉钙化涉及OPG/RANKL/RANK系统改变，亦与下游NF-κB通路激活有关。我们将正常大鼠的颈总动脉进行常压和高压灌流培养以模拟失重条件下的跨壁压力改变，发现高压显著增强大鼠颈总动脉RANKL、RANK的蛋白和基因表达，改变OPG/RANKL/RANK系统可显著抑制高压增强的NF-κB活性和成骨样细胞表型标志物的蛋白表达。4）间断人工重力干预可通过改变OPG/RANKL/RANK系统对抗模拟失重引起的颈总动脉钙化。在模拟失重过程中进行1h/d-Gx干预可显著抑制模拟失重增强的RANK蛋白表达和颈总动脉钙化。总之，本项目通过分子生物学技术、血管体外灌流培养等技术从动物整体、器官、细胞和基因以及药理学等多个水平发现模拟失重可引起大鼠脑动脉和颈总动脉发生钙化并且涉及OPG/RANKL/RANK系统的改变，间断人工重力干预可能通过此通路延缓模拟失重引起的动脉钙化。本研究可对航天飞行后心血管失调现象提出新的见解，并可为航天后立位耐力不良防护措施研究提供理论依据。