【作者】 郑红霞；

【导师】 李钰；

【作者基本信息】 哈尔滨工业大学 ， 生物医学仪器与工程， 2012， 博士

【摘要】 微重力可致航天员在太空飞行过程中生理机能发生改变。这些变化主要包括心血管系统、肌肉系统、免疫系统和骨骼系统的病理性改变，其中心血管系统病理性改变是造成空间飞行过程中航天员不能顺利完成空间作业，甚至猝死的主要原因。因此，探讨微重力对心脏结构和功能的影响及其相关机制，可为进一步建立基于生物医学基础的有效防护措施提供理论基础，对于保证航天员在太空飞行时的健康和有效工作具有重要意义。由于在空间进行微重力实验耗资大，机会有限，因此地面模拟微重力效应是目前空间生物学与航天医学研究的主要研究方式。为了克服模拟微重力效应研究中细胞二维培养的不足，本研究构建了一种新型的海藻酸钠微囊体，使细胞在该载体内呈三维生长，以保证尽可能实现在体细胞的特征。为了选择力学性能稳定，可以对抗回转过程产生的剪切力作用的载体材料，本实验首先利用流变仪检测了不同组成成分的海藻酸钠微囊体力学性能，结果显示1:30:0.05海藻酸钠/胶原/壳聚糖为最佳组成。扫描电镜显示微囊体为内部多孔结构，有利于细胞培养。其次，细胞增殖实验表明心肌细胞在该微囊体内的稳定增殖，原代心肌细胞在微囊体内可形成类心肌组织，并维持长期搏动。因此，海藻酸钠/胶原/壳聚糖微囊体包覆心肌细胞可用于后续微重力效应的相关研究。为了进一步研究海藻酸钠微囊体用于模拟微重力效应研究的可靠性，本研究利用回转仪以转数15rpm回转培养24hr，比较海藻酸钠微囊体和cytodex微球为载体的心肌细胞微丝结构改变，发现两者微丝结构变化相似，此外不同表型的乳腺癌细胞在海藻酸钠微囊体中回转培养48hr后，乳腺癌细胞的生物学特性发生改变，表明海藻酸钠微囊体用于模拟微重力效应研究的可行性。在构建以海藻酸钠微囊体为载体的细胞三维培养模型基础上，本文利用免疫荧光染色、MTT和流式细胞仪等方法研究了模拟微重力效应对心肌细胞结构、增殖和凋亡的影响，结果表明模拟微重力效应24hr导致三维培养的心肌细胞伪足消失，增殖能力降低，36hr出现早期凋亡，并在回转培养72hr时出现DNA损伤。为了深入研究模拟微重力效应对三维培养的心肌细胞功能的影响及其相应机制，本研究还分析了心肌细胞搏动频率的变化，通过显微镜观察海藻酸钠微囊体内心肌细胞的搏动频率，发现模拟微重力效应24hr可致原代心肌细胞收缩频率改变，表明模拟微重力效应改变三维培养的心肌细胞的搏动功能。同时利用实时定量PCR和Western-blot分析了模拟微重力效应与介导兴奋-收缩耦联的缝隙连接蛋白Connexin43以及影响心肌功能的离子通道蛋白（钠离子通道蛋白、L-钙离子通道蛋白、钠钾泵、钠氢交换体）表达变化的关系，研究结果提示模拟微重力效应对编码钠通道的基因SCN5a表达未造成明显影响，而其余通道蛋白均有不同程度的表达变化（Connexin43表达呈一过性改变，可产生适应性恢复；L-钙通道蛋白表达增高；钠钾泵和钠氢交换体表达降低），说明模拟微重力效应改变了动作电位产生的分子基础，从而导致心肌搏动功能改变。此外，尾悬吊大鼠实验也表明模拟微重力效应改变了心肌离子通道蛋白表达，其中Connexin43和钠氢交换体改变与体外培养的心肌细胞改变一致，而L-钙通道蛋白和钠钾泵表达与体实验相反，以上研究结果提示神经体液调控参与了模拟微重力效应下心肌组织离子通道蛋白的表达。在以上研究的基础上，为了深入分析模拟微重力效应下线粒体氧化应激响应的协同变化，利用线粒体特异探针荧光染色、活性氧探针DCFH-DA、Rh123染色以及抗氧化酶活性检测试剂盒研究了心肌细胞氧化应激的发生及相关机制，结果表明模拟微重力效应24hr心肌线粒体分布发生改变，48hr细胞活性氧显著增加，线粒体膜电位改变，导致心肌处于氧化应激状态。同时心肌细胞抗氧化物酶活性增强、热休克蛋白和转录因子NF-κB高表达。抗氧化物SOD活性分析表明回转培养的氧化应激机制可能以超氧化物为主。此外，线粒体的氧化应激研究结果也提示氧化应激参与了心肌细胞模拟微重力效应的响应机制，而且与心肌细胞搏动变化及部分离子通道的变化具有一致性。综上，本研究通过构造新型海藻酸钠微囊体，建立了适用于模拟微重力效应研究的细胞三维培养体系，同时通过系统的探讨心肌细胞对空间微重力环境的响应及其功能变化，证实模拟微重力效应会导致心肌细胞骨架改变，并引起线粒体分布发生变化，活性氧显著增加，线粒体膜电位改变，使心肌处于氧化应激状态。同时引起缝隙连接蛋白和离子通道蛋白的表达改变，最终导致原代心肌细胞搏动功能紊乱。更多还原

【Abstract】 Microgravity caused changes in physiological function of astronaut in spaceflight. These changes include the cardiovascular system, muscle system, the immunesystem and the skeletal system. Among them, the cardiovascular system change ispathological process, which caused the astronauts can’t finish the homework space,and even the main cause of sudden death. Among those studies, the potential risksassociated with cardiovascular system have been a central concern in the study of thehuman physiologic adaptation to the microgravity environment. Therefore, toinvestigate the effects of microgravity on cardiac structure and function and itsmechanism is impotent, which to ensure the health and effective work of astronautsin space flight.It is costly and limited opportunities for microgravity experiments in space;therefore, the ground simulation of microgravity effect is the main way of using. Inorder to reflection effect of simulated microgravity on myocardial cell in vivo, in thisstudy, three-dimensional myocardial cell is used to study effect of simulatedmicrogravity on myocardial cell structure and function. Using tissue engineeringbiotechnique, we provided the new carriers (alginate/collagen/chitosan hydrogel) toinvestigate the effect of simulated microgravity on biological characteristic ofcardiac cells. The results by a rheometer and SEM showed that the new carrierssystem was mechanically stable and porous scaffolds which allow cells to grow inthree-dimensional model. To evaluate whether the carriers are suitable to support thegrowth of mammalian cells, the viability of H9c2cells was assessed by MTT assayand the electrophysiologic characteristics of cardiac myocytes was investigate. Theresults showed that the viability of cells within carriers steadily increased, and thespontaneous and synchronous contraction of the whole cardiac cell-carriers wasmaintained more than two months.In order to prove the alginate carriers encapsulated cell system for the reliabilityof the effect of simulated microgravity, it was observed the simulated microgravityeffect on H9c2microfilament cultured in alginate microcapsules and cytodexes, andthe biological characteristics of breast cancer cells cultured in simulated microgravity effect. The results showed that simulated microgravity effect usedNASA-RCCS with15rpm leaded to microfilament change in two carriers anddifferent biological alteration in two phenotypical dissimilar human breast cancercell lines, which was proved be suitable for alginate encapsulation of cells to studythe effect of simulated microgravity.Furthermore, the effect of simulated microgravity with the establishedthree-dimensional cell culture model on structure, proliferation and apoptosis ofcardiomyocyte was analysed. The results showed that simulated microgravity effectused alginate carriers decreased cardiomyocytes proliferation, lead to cytoskeletondepolymerization, induced apoptosis in72hr. However, under2D rotation systemH9c2cells showd decreased cell area and cytoskeleton damage on day8, while noeffect was observed on cell proliferation;Finally, effect of simulated microgravity on myocardial cells pulse functionwas observed. Simulated microgravity effect changed the frequency of myocardialcontractility. Furthermore, at the molecular level analysis of the impact of channelsprotein expression used transfer sodium, potassium and calcium ion, and signaltransduction of Cx43gap junction protein expression. Study found that cell modelsand animal models of simulated microgravity in the microgravity on the geneencoding sodium channel SCN5a expression did not cause significant impact, whilethe remaining changes the action potential of the structural basis, leading heart theoccurrence of disorders.In this study, the mitochondrial specific probe Mito Tracker Red, reactiveoxygen species probe DCFH-DA, Rhodamine123, and antioxidant enzyme activityassay kits were used. The results revealed that3D rotation caused by simulatedmicrogravity effect mitochondrial distribution, structure and dysfunction, leaded tomyocardial oxidative stress in myocardial cells, increased antioxidant enzymeactivity and heat shock protein and transcription factor NF-κB expression.In summary, this study established a three-dimensional carrier systerm foreffects of simulated microgravity. It indicated simulated microgravity at the cellularlevel changed myocardial structure and function, and impacted expression of ionchannels protein; and that simulated microgravity lead to the occurrence of oxidativestress. And in vivo experiments, simulated microgravity change the expression of iontchannel protein.更多还原