| I. ObjectiveTo observe the effects of different shear stresses on the morphological changes in cochlear microvascular endothelial cells cultured in vitro and the possible mechanisms so as to enrich the mechanism of the permeability regulation of the blood-labyrinth barrier.II. Methods1. Through culture in vitro, cochlear microvascular endothelial monolayer cells, brain microvascular endothelial cells and human umbilical endothelial ECV-304 in monolayer state were obtained.2. We designed a set of hydrodynamic system, including roller pump, peristalsis pump, liquid container, and various catheters so that shear stress can be inflicted on the above-mentioned cultured cells by different forces generated through the regulation of the flow.3. As to cochlear microvascular endothelial cells in guinea pigs, we designed four different shear stresses, i.e. =0.883 dyn/cm2, 0.976 dyn/cm2, 1.184 dyn/cm2 and 2.929 dyn/cm2, under which the morphological changes of the cells were observed under an inverted phase contrast microscope at time points of 0.5, 1, 2, 4, 8 16, and 24 h: at the corresponding time point, the cellular morphology was photographed and the morphological parameters such as Pyx and Q were detected with Tiger image analysis system. Similarly, we also designed three different shear stresses of 0.276 dyn/cm2, 0.476 dyn/cm2 and 1.069 dyn/cm2 acting on brain microvascular endothelial cells and 5 dyn/cm2 on ECV-304 cells with identical observing time points and contents to those of cochlear microvascular endothelial cells. Then the data obtained at the corresponding time points were included in a table and the corresponding results were analyzed statistically and hence a diagram of curves was drawn.4. Fixation and staining were performed on cochlear microvascular endothelial cells after the action of shear stress by using phalloidin and F-actin was observed by laser scanning confocal microscopy after fluorescent staining. Then curves of changes were drawn based on the diagram of F-actin fluorescence contents under different shear stresses. The relationship with different time points was analyzed statistically.II. Results1. Free cells in cochlear microvascular tissue section, found at 3 h after enzymatic digestion with 0.5% collagenase type I, partially formed into mass. At 24 h after culture, "cell islets" attaching the walls could be seen and 4 days later, "cell islets" gradually proliferated and extended, showing the tendency of monolayer cells with the appearance of ellipse, triangle and pebble. After exclusion of the suspected mixed cells, highly dense monolayers formed with the appearance of "paving stone". Cells in brain vascular fragment in guinea pigs were found to attach the walls at 2 h after enzymatic digestion with collagenase and highly dense monolayers formed 3 days later with the appearance of "paving stone". EVC-304 cells grew into monolayers on day 3 after culture. After evaluation of the above cells by immunohistochemistry, over 95% cells were VIII antigen positive, and hence confirmed to be endothelial cells.2. As to cochlear microvascular endothelial cells in guinea pigs, no morphological changes of the cells were found when shear stresses of 0.883 dyn/cm2 and 0.976 dyn/cm2 acting for 24 h. However, when shear stress was 1.184 dyn/ cm2 acting for 8 h, compliant changes of the cellular morphology began to be present: single cell changed from former ellipse into long shuttle and the cellular arrangement changed from former disorderliness into orderliness following the direction of the flowing liquid. The changing tendency was in a time-dependent manner. However, no morphological changes of brain microvascular endothelial cells were found when shear stress was 0.276 dyn/cm2 acting for 24 h, and the single cell is from blunt round shape to long fusiform shape gradually when shear stress was 0.476 dyn/cm2 acting for 4h, but when shear stress reached 1.069 dyn/cm2 acting for 16h, enlarged intercellular space and abscission were found. As to human umbilical V c...
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