The Growth And Orientation Of Cells And The Mechanism Of Cytoskeleton Reassembly In Different Courses Of Substrate Tension

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 mainstress 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 stressfibers 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...