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Effect of quercetin on the cytoskeleton of rat cardiac myocytes cultured in vitro under simulated microgravity

XIONG Jiang-Hui LI Ying-Hui ** NIE Jie-Lin DING Bo ZHANG Xiao-You HUANG Zeng-Ming BI Lei (Institute of Space Medico-Engineering, Beijing 100094,China)  
In order to explore effect of quercetin on cytoskeleton distribution in cardiac myocytes under simulated microgravity, we observed the distribution of actins and tubulins, which are the constituent proteins of cardiac myocyte microfilaments and microtubules respectively, using immunocytochemistry and fluorescence microscope.Enriched cultures of cardiac myocytes were obtained from 2-day-old Wistar rats by stepwise trypsin dissociation, the dispersed cells were incubated in cell culture coverslips fixed in cell culture flasks.Cells were divided into three groups: a simulated microgravity group(flasks were set up in a clinostat and rotated at 30 r/min for 48 hours in a 37℃ incubator), a control group(cultured in stationary flasks)and a quercetin group(cells were inculated in 100 ug/ml quercetin under clinorotation).The coverslips was washed in PBS, and fixed with 3.7% paraformaldehyde for 30 minutes.After another wash in PBS and blocking in PBS containing 5% normal sheep serum for 30 minutes, the coverslips were incubated with a 1∶50 dilution of mouse anti rat α-tubulin monoantibody for 90 minutes.The coverslips were rinsed with PBS and then incubated with rabbit anti-mouse IgG conjugated with FITC for 150 minutes at room temperature.A wash of PBS was performed and the coverslips were incubated with Texas Red-X phalloidin(1∶500)for 120 min, then the coverslips were rinsed, mounted in crystal mount medium with mowiol and photographed using laser scanning confocal microscope.Microfilaments and microtubules were stained with green and red fluorescence respectively when observed under a confocal microscope.Compared to the control group, the microfilaments and microtubules of rat cardiac myocytes cultured in vitro under simulated microgravity were more heavily concentrated near the nucleus.Treatment with quercetin could partly inhibit the effect of simulated microgravity on the redistribution of microfilaments and microtubules and there was little difference in the thickness of microfilaments between the quercetin and control group.These results suggest that treatment with simulated microgravity results in the redistribution of microfilaments and microtubules of rat cardiac myocytes cultured in vitro.Quercetin shows its protective effect via partial inhibition of the cytoskeleton redistribution in cardiac myocytes induced by simulated microgravity [Acta Zoologica Sinica 49(1):98-103, 2003].
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