Effects Of Simulated Microgravity With RCCS On Growth Features And Secretory Function Of Thyroid Follicular Epithelial Cells In Rat

Background and Objective China's manned space project has now been in a critical period,and is essential to maintain the astronauts'health,safety and efficiency in order to achieve the goal of long-duration space flights.The weightlessness in space flight has adverse effects to human being and could induce a series of physiological and pathological alterations including osteopenia,muscle atrophy,cardiovascular disorders and immune suppression,and also has impacts on the endocrine system.Thyroid gland is the largest endocrine gland in human body and the growth,development and metabolic activities of the body are affected and regulated by thyroid hormones.Foreign scholars have confirmed that aerospace weightlessness has important effects on thyroid function and morphology.As far as we know that there is no relevant studies reported to date in China.This study was designed to investigate the effects of simulated microgravity with RCCS on thyroid follicular epithelial cells?FRTL-5 cells?in rat,and aimed to provide theoretical basis for the management of spaceflight related medical problems.Methods The RCCS culture system was used to simulate the microgravity environment.The rat thyroid follicular epithelial cells?FRTL-5 cell lines?were randomly divided into simulated microgravity group?SMG?and normal gravity control group?NG?.After being cultured for 6,12,24 and 36 hours respectively,FRTL-5 cell's the proliferation and cell cycle were assayed by MTT and flow cytometry,the levels of Tg,TPO,T₃,T₄,FT₃ and FT₄ in cell supernatant were detected by ELISA and chemiluminescent immunoassay.The cell's surface morphology after 6,12,24,and 36 hours'RCCS culture was observed under inverted phase contrast microscope,and the cell ultrastructure after 12 and 36 hours'RCCS culture was observed under transmission electron microscope.The fluorescence intensity of actin cytoskeleton in FRTL-5 cells after 36 hours'RCCS culture was observed by laser confocal microscopy.Results?1?MTT results showed that the proliferation of FRTL-5 cells in SMG group was significantly inhibited compared with NG group?P<0.05?at 6,12,24 and 36hours?P<0.01?.Among them,the inhibition of proliferation at 24 hours was most obvious?P<0.01?.At 36 hours,there are two situations,one was the recovery of cell proliferation in SMG group and the other was the rapid increase of cell proliferation rate in NG group.?2?The cell cycle of flow cytometry showed that the percentage of cells in G1 phase of FRTL-5 cells was significantly increased after 6,12,24 and 36hours of microgravity culture compared with NG,except that the proportion of S phase cells was significantly reduced at 6 hours.The proportion of cells in G2/M phase of each phase showed a decrease in the early period of simulated weightlessness with a lowest value at 12 hours,a significant increase at 24 hours and a recovery at 36 hours.The results suggest that DNA synthesis of FRTL-5 cells was decreased in the early phase and became more active at 24 hours under RCCS culture,and the proportion of DNA synthesis after 36 hours culture showed a downward trend and approached to NG.?3?The chemiluminescent immunoassay showed that FT₃,T₄ and FT₄ levels in SMG group were decreased significantly after 6 hours'RCCS culture compared with that in NG group?P<0.05?.FRTL-5 cells secretion of FT₃,T₄ and FT₄ in the rest of experimental phases was not affected significantly.?4?ELISA assay showed that Tg and TPO in cell supernatant were significantly increased in SMG compared with that in NG?P<0.01?,with a significant increase at 6 hours,followed by a downward trend,and the an increase again after 24 to 36 hours'RCCS culture.There was a significant difference between 6 and 24 hours,and 24 and 36 hours in SMG group?P<0.01?.?5?The inverted phase contrast microscope showed that the morphology of FRTL-5 cells changed significantly under simulated weightlessness,and tended to die at early phase of culture,and then the number of the cells was increased gradually after 24hours culture.?6?The transmission electron microscopy showed that the ultrastructure of FRTL-5 cells was changed significantly after 12 hours culture under simulated weightlessness.?7?The laser scanning confocal microscopy revealed that the FITC-labeled FRTL-5 cells showed significant changes after 36 hours RCCS culture,i.e.,the microfilament skeleton was partially depolymerized,the tension fibers were reduced,the structure and arrangement were disorganized,and the pseudopodia were rare and irregular.Conclusion The study showed that the RCCS simulated microgravity could inhibit the cell cycle transformation and proliferation of FRTL-5 cells,and affect the secretory metabolism of FT₃,T₄,FT₄ and the production of Tg and TPO in FRTL-5 cells.At the same time it could also induce pathological alterations of cellular morphology,cytoskeleton,and ultrastructure of FRTL-5 cell.