【作者】 朱杰；

【导师】 王国栋；

【作者基本信息】 西北农林科技大学 ， 生物物理学， 2010， 博士

【摘要】 肌节周期性的收缩力可经由近邻的胞外基质和心肌纤维传导至整个心脏组织,从而迫使心脏壁收缩并将血液压出心室,是心脏搏动的原动力。肌纤维和胶原纤维超微结构及生物力学特性的病理性变化往往会影响纤维对外界刺激信号的应答,从而影响心脏搏动的规律性,造成心脏功能紊乱。而胶原蛋白纤维是肌内膜的主要成分,要深入研究心肌纤维的功能,还必须研究心肌外层的胶原纤维膜的超分子结构与力学特性,这些信息将有助于人们更好地了解胶原蛋白结构影响肌细胞-肌细胞、肌细胞-基质通讯的途径,以及胶原蛋白在心肌组织生长、发育、病变和修复/再生过程中所起的作用,为临床治疗提供科学依据。本研究以肌纤维和胶原纤维为主要研究对象,采用原子力显微镜高分辨成像和纳米压痕技术,研究了牛心心肌纤维、昆虫飞行肌纤维、鼠尾肌腱胶原蛋白纤维在不同生理状态的表面超微结构、压弹性和粘弹性,尝试找到肌纤维收缩过程中肌节长度、纤维直径等参数之间的关系;找到抗I型胶原蛋白抗体在胶原蛋白纤维上的结合位点并得到二者之间的特异性作用力;从而建立心肌纤维和胶原蛋白纤维的超微结构、力学特性和生理状态之间的内在联系。通过研究,获得了以下主要进展:(1)不同生理状态下,牛心肌纤维的肌节长度大致在1.22μm~1.33μm,其中松弛状态的肌节长度相对于僵直态有大约9%的增长,该结果提示,肌动蛋白和肌球蛋白会随着生理条件的变化而不断交替形成横桥,从而形成力学传导以应对外界环境的变化和信号刺激;与位置相关的压弹性曲线与AFM高度图的剖面曲线在外形上具有很高的相似性,相邻最大弹性系数点之间的距离与剖面分析得到的肌节长度非常接近,该结果显示了肌节的空间周期性和力学周期性的统一;源于AFM研究的肌纤维结构和弹性参数将会推动人们对生物结构、生物力学和生理功能之间关系的认识,为心脏疾病的基础研究和临床治疗提供更多的线索。(2)飞行肌肌原纤维在僵直态、松弛态和三种活化态的肌节长度分别为:2.10±0.05μm(僵直态)、3.10±0.10μm(松弛态)、2.50±0.15μm(活化态, 2mM Ca2+)、2.60±0.25μm(活化态, 5mM Ca2+)和2.55±0.15μm(活化态, 10mM Ca2+),并得到得到5mM Ca2+浓度为合适的肌纤维活化浓度;另外,不论飞行肌纤维处于什么生理状态,A带的长度始终保持在1.50μm左右,而I带的长度则有较大的变化(0.7μm~1.6μm),该现象符合肌动蛋白纤维与肌球蛋白纤维在重叠区相对滑移的力学和空间结构模型。AFM高度图剖面分析的结果显示,不同生理状态下,肌原纤维内部肌动蛋白粗纤维之间的最小距离分别为53nm(松弛态)、58.5nm(僵直态)、56.7nm(活化态, 2mMCa2+)、54.8nm(活化态, 5mM Ca2+)和55.6nm(活化态, 10mM Ca2+),进而结合同根纤维的肌节长度变化建立了肌纤维的钙离子诱导等体积伸缩模型;根据沿肌球蛋白纤维纵向的剖面曲线分析图,找到了横桥在肌球蛋白纤维表面的位置及其周期性间距为37.5±0.5nm;另外,在同一生理状态下,肌原纤维的弹性系数和杨氏模量的大小关系满足:Z线>M线>重叠区>I带;同一肌节位置在不同活化态下的弹性系数和杨氏模量的大小关系基本满足:活化态(5mM Ca2+)>活化态(2mM Ca2+)>活化态(10mM Ca2+)。(3)固定在云母片上的胶原纤维在空气中自然干燥时,胶原纤维的D单元长度在干燥的过程中并无显著变化,基本稳定在66.67nm;而间隙带的深度则有一定的增加,从4.4nm逐渐增加到4.7nm,增幅达7%;变化最为明显的则是纤维的直径,从201nm降到了187nm,减幅达到7%,尽管如此,由于D单元长度并无变化,因而该方法(样品制备和空气中成像)依然适于研究胶原纤维的纵向精细结构。文中采用抗I型胶原蛋白抗体偶联金纳米颗粒对胶原蛋白纤维成功实现了定位标记,AFM研究发现,抗体作用时间不同的纤维样品表面上的金纳米颗粒分布密度有较大差别。抗体作用时间越长,金颗粒的密度越大;其中,抗体作用时间等于2h的金纳米颗粒密度与厂家提供的标准结合密度很接近,但从实际的AFM图像质量可知,抗体作用时间为1h的金颗粒密度更适合做抗体结合位置的统计分析。以抗体作用时间为1h的样品作为研究对像的实验结果显示,与抗体结合的金颗粒主要分布在胶原纤维的重叠区,显示出抗I型胶原蛋白抗体对胶原纤维重叠区的结合特异性。(4)用未加修饰的探针对抗体结合时间不同的胶原纤维样品进行了成像,分别得到了高分辨的高度图和低分辨的摩擦图,二者分别从结构细节和总体力学特性上反映出抗体在胶原纤维上的结合位点主要集中在重叠区。而未加修饰探针的单点力学测量结果显示,胶原纤维表面结合的抗体对肌原纤维压弹性的测量几乎没有影响;但对于粘附力的收集结果却有显著的影响,特别是在重叠区,随着抗体结合时间的增加,探针与胶原纤维间的粘附力相对于探针与天然纤维样品之间的粘附力分别增加了17.8%、48.9%和82.2%,而间隙带的粘附力则保持相对稳定,说明抗体主要分布在重叠区,与前述高度图和摩擦图的结果保持一致。最后,本论文重点研究了抗体与胶原蛋白的结合力。主要用抗I型胶原蛋白抗体对探针进行了修饰,并用修饰过的探针进行探针-天然胶原纤维间的粘附力(结合力)测量,统计结果显示,单个抗I型胶原蛋白抗体与胶原纤维重叠区的结合力约等于250pN。更多还原

【Abstract】 Cardiac muscle fibers consist of bundles of myofibrils in which runs a series of sarcomeres where the contractile forces are produced by cyclic interactions between actin (thin filament) and myosin (thick filament) in overlap, and then transmitted to extracellular matrix and adjacent fibers and to the heart wall to squeeze out the blood filled in the heart cavity through a series of sarcomeres. Since the elastic properties influence the response of fibers to applied forces and may cause cardiac dysfunction, it is of importance to study the elastic behavior of fibers in response to forces in both directions. As the main component of endomysium, collagen fiber should be studied to make sure the function of cardiac muscle. The ultrastructural and biomechnical studies on collagen fiber would help us to understand how collagen affect on the conmunication pathway between cardiac muscle cells and extracellular matrix, and how collagen play a key role on tissue growth, development, disease, repaire and regeneration. Both of them will provide a scientific basis for clinical treatment.In this study, surface ultrastructure and biomechanics of bovine cardiac muscle fibers, insect flight muscle and rat tail tendon collagen I fibers in different physiological conditions were examined with high resolution atomic force microscopy and nanoindentation in order to find the relation among fiber ultrastructure, biomechanics and physiological function. Through the studies above, several important results were achieved as follows:(1) Sarcomere length of 1.22±0.02μm (n=5) in rigor with a significant 9% increase in sarcomere length in relaxing state (1.33±0.03μm, n=5), indicating that overlap move with the changing physiological conditions. Compression elasticity curves along with sarcomere locations have been taken by AFM compression processing. Coefficient of Z-line, I-band, Overlap, and M-line are 25±2pN/nm, 8±1pN/nm, 10±1pN/nm, and 17±1.5pN/nm respectively in rigor state, and 18±2.5pN/nm, 4±0.5pN/nm, 6±1pN/nm, and 11±0.5pN/nm respectively in relaxing state. Young’s Modulus in Z-line, I-band, Overlap, and M-line are 115±12kPa, 48±9kPa, 52±8kPa, and 90±12kPa respectively in rigor, and 98±10kPa, 23±4kPa, 42±4kPa, and 65±7kPa respectively in relaxing state. The elasticity curves has shown a similar appearance to the section analysis profile of AFM height images of sarcomere and the distance between adjacent largest coefficient and Young’s Modulus is equal to the sarcomere length measured from the AFM height images using section analysis, indicating that mechanic properties of fibers have a similar periodicity to the topography of fibers.(2) Sarcomere length of insect flight muscle fiber in rigor, relaxed and 3 activated states were 2.10±0.05μm(rigor), 3.10±0.10μm(relaxed), 2.50±0.15μm(activated, 2mM Ca2+), 2.60±0.25μm(activated, 5mM Ca2+) and 2.55±0.15μm(activated, 10mM Ca2+) respectively. A-band kept 1.50μm in length whatever the physiological state the flight muscle fibers be at when A-band had changed from 0.7μm to 1.6μm,which was consistent with the slip model of the mechanical and space structure of actin and myosin filaments (i.e. thin and thick filaments) in overlap zone. The spacing between the adjacent thick filaments were 53nm(relaxed)、58.5nm(rigor)、56.7nm(activated, 2mM Ca2+)、54.8nm(activated, 5mM Ca2+) and 55.6nm(activated, 10mM Ca2+), then a equal volume contractile model was introduced according to the change of sarcomere length. And the distance of 37.5±0.5nm between adjacent crossbridges on myosin filament was achieved. And the Young’s Modulus of the same muscle fiber in the same physiological state in different sarcomere locations were in the order of Z-line>M-line>overlap>I-band;the value in the same loaction were in the order of activated(5mM Ca2+)>activated(2mM Ca2+)> activated (10mM Ca2+)。(3) D-spacing length of air-dried collagen fiber kept around 66.67nm in different drying time, when the gap depth increased from 4.4nm to 4.7nm (~7%) and the diameter decreased from 201nm to 187nm (~7%). 1h antibody binded collagen fibers were chosed to analysis the binding position of antibody according to the image quality, and the overlap was determined to be the binding location of anti-collagen I antibody on collagen I fiber through the amplifier(gold nanoparticles) and high resolution atomic force microscopy.(4) Friction and hieght images of labeled samples using unfunctionized tips showed us the overlaps were the binding location of antibody on collagen fibers. And the adhesive force on overlap increased by 17.8%、48.9% and 82.2% in 20min, 1h and 2h labeling time respectively,which demonstrated that the antibody were fixed to overlap zone. And single interaction between antibody and collagen fiber was ~ 250pN acording to the results from the force-distance curves recorded with the tip functionized by anti-collagen I antibody.更多还原