The Connection Of The Changes Of Cytoskeleton And Adhesion Of Mesenchymal Stem Cells With Their Proliferation And Osteogenic Differentiation Under Simulated Microgravity

The significant effects of microgravity on the human bodies make them research hotspot in the field of space life science and space medicine. Inhibition of bone marrow mesenchymal stem cells’ proliferation and osteogenic differentiation under microgravity may be one of the causes for the bone loss in space. This paper explores the effects of simulated microgravity on the cytoskeleton, adhesion, proliferation and osteogenic differentiation of mouse mesenchymal stem cells, and elucidates the mechnisms by analysis specific genes expression that related to cell growth and differentiation.In this study, mouse mesenchymal stem cells(BMSCs) were cultured using 3D biaxial gyroscope to simulate microgravity environment and establish the cell culture system. To observe the effect of SMG on cell proliferation, The growth curve was drawn and western blot was used to detect the expression of cell cycle related protein named Cyclin B1 and mTOR pathway protein named Raptor and Rictor; The changes of the cytoskeleton and adhesion under SMG of BMSCs were observed by immunofluorescence staining with Tubulin, Actin and Vinculin, and the cell adhesion related proteins Vinculin, paxillin and FAK were detected by western blot; Total RNA was extracted using reverse transcription polymerase chain reaction(RT-PCR) to detect the expression of osteoblast differentiation associated genes osteopontin and Osteonectin. The activity of key protein RhoA was determined by Rho-GLISA and the protein expression was detected by Biot Western, which was used to study the effect of the protein in the simulated microgravity environment; The method of RT2 ProfilerTM PCR Array was used to detect the expression of 84 genes related to cell proliferation and differentiation, and some important genes were screened by western blot to detect the expression of the proteins.The results show that the simulation of cytoskeleton structure under microgravity conditions changed significantly, not only microtubules becoming disorganized, also microfilaments becoming shorter and abnormal distributed. The capacity of cell proliferation and osteogenic differentiation were decreased, and the expression of proliferation related protein Cyclin B1, Raptor, rictor and osteogenic differentiationrelated genes Osteopontin, Osteonectin were all decreased; Also the activity andexpression of RhoA protein were decreased. The results of RT2 ProfilerTMPCR Array shows that among the 81 genes detected, the expression of Csf2, Gdf5, Il1 b,Sox2 and other genes were up-regulated, and Bmp4, Igf1, Sox9 and other genes were down regulated.The results indicate that the adhesion, proliferation and osteogenic differentiation of mouse BMSCs significantly changed under SMG; RhoA activity change may lead to the changes of cytoskeleton and the adhesion that ultimately affected cell proliferation and osteogenic differentiation; The bone loss of the astronauts in space environment may result from the interaction of osteogenic differentiation decrease and osteoclast differentiation enhance.