| Objective and SignificanceDuring the aging process of the human body, one of the important functional changes in the microvessel is the alteration of the vascular permeability directly involved in the change of the endothelial barrier function which is closely related to the distributing of the filamentous actin (F-actin), the chief component of the cytoskeleton. The close relationship between advanced glycation end products (AGEs) and vascular hyperpermeability is well documented. Our current study tries to investigate the pathophysiological facts underlying the vascular complications associated with aging by observing the effects of AGE on the proliferation activity and the morphological changes of F-actin in human umbilical vein endothelial cell (HUVEC). Meanwhile, we attempt to establish a stable D-galactose aging model in endothelium, and observe the morphological influence of aging stimulation on the F-actin in endothelium in order to supply some evidence on the mechanism of human natural aging process theoretically and experimentally.Materials and methodsThe experiment was divided into two parts, functional and morphological effect on endothelium by AGE and D-galactose were studied respectively.In the first part the HUVEC were cultured with normal, AGE-HSA modified culture medium respectively. Cell viability curves were drawn using MTT Cell Proliferation Assay (MTT-CPA). Then, the two groups of cells were incubated in petri dishes; F-actins in the cells were detected with fluorescence staining of rhodamine-phalloidin everyday during the following six days.In the second part the HUVEC were cultured with normal, D-glucose modified, and D-galactose modified culture medium respectively. Cell proliferation curves were drawn using typran elimination method. Then, the three groups of cells were incubated in petri dishes and cultured for nine to twelve days; F-actins in the cells were detected with fluorescence staining ofrhodamine-phalloidin.The F-actin fibers were fluorescently stained as follows. Cells were fixed by 2% formaldehydum polymerisatum for 10 min at 20 ℃, then incubated with 0.5%Triton X-100 for 30 min and 0.5% bovine serum albumin for 45 min and 2U/ml rhodamine-phalloidin for 40 min at 4℃, the cells rinsed well between each step. Finally the Petri dishes were examined on the phase difference microscope and the imAGE were recorded by photography, the excitation wave-length being 565 nm.Results and analysesIn the first part of the experiment, the viability curve of the AGE-treated group decline shapely compared with that of the normal group. One sample t-test showed that the P value is less than 0.01 comparing the OD value of the two endothelium group, showing that the AGE inhibited the proliferation activity of the endothelium greatly.Observation by fluorescence microscopy showed that the fibers of F-actin in the cells of the normal group enriched adjacently along the cell membrane, forming some kind of typical cobblestone model and no intensive fibers were observed in the cytoplasm, while the one in the cells of AGE-treated group resided in a changed manner. There appear three kinds of abnormal forms of F-actin fibers, the stress fibers, the filopodia and the board like pseudopodia. Most of the peripheral F-actin fibers disappeared and dense stress fibers were observed in the cytoplasm during the late stage, intercellular gap formed and spread. The phenomena indicated that the F-actin in the DPB of the endothelium rearranged under the stimulation of AGE, i.e. they diffused into the cytoplasm and formed the stress fibers which induced the shrinkage and morphological changes of the cells, finally resulted in the lesion of the endothelium barrier and vascular hyperpermeability.We observed similar phenomena in the second part of the experiment. The proliferation curve of D-galactose-treated group descended notably compared with that of the normal group and the D-glucose-treated group. The differences in the two comparisons were showed to be of statistical significance...
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