摘要
本文在总结以往研究者工作的基础上，使用生物力学，细胞生物学、免疫组织化学、计算机图象处理等多种研究方法，对培养的原代血管平滑肌细胞、EC’V-304细胞株施以不同大小、方向和频率的周期性基底拉伸(10％，0．67Hz；0．15％，0．4Hz)，同时结合使用静态基底拉伸和静态基底松弛的不同组合，研究在不同的力学过程中细胞生长和取向的调整；微丝、微管骨架的重排；生长和生理行为的变化等。通过总结实验结果对细胞取向和细胞骨架重排的力信号机制进行了探讨，强调指出了力学信号引起细胞响应并不完全依赖于化学信号机制，而可通过对细胞骨架张力的调整直接引起细胞在形态、取向方面的主动响应。主要工作和得出的相应结论如下：
1．对应用于本文研究的弹性膜圆管膨胀加载装置、四点弯曲梁单向交变等应变加载装置进行了力学分析，并对其中可能产生不利于实验研究影响的地方作了校正和处理。针对对各装置加载特点改进对照组设置，对照消除基底拉伸以外力学因素的影响。
2．用组织培养法原代培养兔血管平滑肌细胞，并利用免疫组织化学方法对其进行了鉴定。
3．对血管平滑肌细胞施以不同应变率的周期拉伸(最大应变分别为lO％、0．1 5％)，利用放射免疫法分析血管平滑肌细胞血管紧张素II的分泌：微管吸吮系统分析细胞与基底粘附力；免疫组织化学方法分析应力纤维的排布；流式细胞仪分析细胞分裂指数；利用计算机图象分析系统分析细胞形态和取向结果表明：
(1) 周期拉伸使平滑肌细胞取向垂直于主应变方向，周期拉伸引起细胞取向调整存在应变阈值。
(2)周期拉伸对血管平滑肌细胞增殖、表型分化、分泌行为、基底粘附等生理行为均产生影响。
(3)周期拉伸下细胞取向的发生，与周期拉伸的其他一些生理效应有不同的机制
4．改变周期拉伸的加载方向，研究血管平滑肌细胞取向的再调整过程，结果表明：细胞对周期拉伸所引起的取向调整有一定的“记忆”现象。
5．对不同接种形态(圆斑、半平面)的ECV-304细胞施以最大应变10％，0．67HZ的周期拉伸，用FITC．鬼笔环肽标记微丝、FITC-微管仅亚基抗体标记微管，并利
用计算机图象处理系统分析细胞取向、长短径的变化趋势和不同方向上的增殖和迁移情况。结果表明：
1) 周期拉伸条件下，细胞群体形态的重建表现出与细胞个体取向相似的变化形式，垂直于主应变方向上细胞增殖速度快于应变方向；垂直于主应变方向上的迁移速度快于主应变方向。
2) 周期拉伸条件下细胞取向的调整与细胞长短径比的变化表现出明确的相关性；周期拉伸使细胞在应变方向上的截距缩短，在应变垂直向上截距增长；应力纤维的重排与细胞取向的调整直接相关。
6．基于上述结果本文提出了周期拉伸下细胞取向重排的机制可能是周期拉伸中的松弛过程使应力纤维在应变方向上解聚，而相对加强了应力纤维在垂直于应变方向上聚合的假想。
7．为验证上述假想，并分别研究不同力学状态下细胞的取向和骨架重排，将周期拉伸分解成不同的拉伸和放松过程，设计了一系列对细胞进行拉伸和松弛的实验，结合参考文献和这些实验的结果可以得出：
1)细胞应力纤维装配在没有其他因素干扰时倾向于沿细胞长轴进行的。周期拉伸作为刺激因素能激发应力纤维、粘着斑的装配(可能通过力敏感离子通道和整合素信号系统等化学信号机制)。
2)周期拉伸可看作拉伸过程和放松过程的组合，当细胞受到拉伸时，骨架(应力纤维)做出相应的调整，应力纤维的原始长度被延长。被延长后的应力纤维，在周期拉伸的放松过程中会处于松弛状态。
3)当应力纤维受到松弛作用时，会发生解聚和破坏。
4)在周期拉伸过程中，倾向于与拉伸方向平行的应力纤维，松弛的程度明显高于那些倾向于与拉伸方向垂直的应力纤维，于是它们因松弛而破坏的概率也相应的高。
5)在松弛破坏应力纤维的同时，周期拉伸促进它们的再装配(这两个效应是并存的)，这样反复的装配一选择性破坏．再装配的结果，最终使垂直于拉伸方向的应力纤维占优势，并使得细胞发生取向的调整。
8．提出应力纤维松弛过程的理想模型，并通过分析指出放松后的应力纤维的张力状态与应力纤维的原始应变率、应力纤维弹性模量、应力纤维与主应变方向的夹角、应变率有关。
Cyclic stretch of substrate at different magnitude,direction and frequency(1 0％，0．67Hz；0．1 5％，0．4Hz)and diverse combination of static stretch and relaxation was exerted on vascular smooth muscle cells(VSMCs)and ECV-304 cells．The effect of different mechanical courses on the cell morphology,orientation，cytoskeleton rearrangements，and the changes in growth and physiological behavior during was studied by using biomechanics，cell Biology,Immunohistochemistry and computer image processing methods．The abbrevi ative detail s as follow：
1．Elastomeric·tube—expanding apparatus and four-point—bend loading apparatus，
which were used for exerting cyclic substrate stretch on cells was standardized based on mechanical analysis．
2．Primary culture of rabbit vascular smooth muscle cells(VSMC)were used in the study were identified by immunohistochemical method
3．Exerted cyclic stretch(the largest strain of 10％and O．15％)with different frequency on vascular smooth muscle．Radioimmunochemical method was used for analyzing the angiotensin II secretion of VSMC；immunochemical method was used to study the distribution of stress fiber；flow cytometer was used to study cell division index and computer image analyzing system was used to study cell morphology and orientation．The results showed that：
(1)Cyclic stretch made orientation of smooth muscle cell perpendicular to main
stress direction There was a strain threshold for stimulating the orientation adjustment．
(2)Cyclic stretch had effects on physiological behavior like proliferation，phenotyping，secretion and substrate adhesion of vascular smooth muscle．
(3)Under cyclic stretch，the mechanism involved in cell orientation differed from other physiological effects．
4． In order to study the readjustment process in VSMC，the direction of loading was altered 90。．Results showed that the cells had sort of“memory'’phenomenon of orientation adjustment caused by periodic stretch．
5．Exerted cyclic stretch(maximal strain 1 0％，0．67Hz)on ECV-304 cell planted in various original shapes(round dot，half plane)．Marked microfilament with FITC—phalloidin and microtubules with FITC-a-tubulin antibody．The computer imaging software was used to analyze cell orientation，changes in long／short axis ratio， proliferation and migration in different direction．The results showed：
(1)Under cyclic stretch，cell proliferated faster in the direction vertical to main strain than in strain direction while migrated faster in the direction vertical to strain than in strain direction．So the reconstruction of cell colony morphology appeared similar with individual cell’S orientation under cyclic stretch．
(2)Under cyclic stretch，obvious relativity existed between cell orientation
adjustment and change of the ratio of cellular short／long axis．Intercept in the strain direction was shortened but in the direction verti cal to strain was increased．
Rearrangement of stress fibers correlated directly with cell orientation adjustment．
6．Based on these results it is hypothesized that assembly&disassembly of stress
fibers is involved in the cell orientation rearrangements．It appeared that under cyclic stretch there was disassembly of stress fibers in the direction of strain whereas there was assembling of stress fibers in the vertical direction．
7．In order to validate this hypothesis a series of experiments to stretch and relax cells were designed．Results of these experiments showed：
1)Cell’S stress fiber assembly tended to take place along the direction of cellular long axis when there weren’t any disturbing factors．As a stimulating agent，
periodic stretch could activate assembly of stress fibers and adhesion foci(probably owning to chemical signaling mechanisms such as force—sensitive ion channel and integrin signaling system，etc)．
2)When cells are stretched，cytoskeleton(stress fiber)adjusted accordingly, which elongated the original length of stre
引文
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