Effects Of Simulated Microgravity With RCCS On Growth Features And Metabolomics Of Human Epidermal Stem Cells

Background and Objective With the rapid development of manned spaceflight technology,the astronaut stay in space will continue to extend,and the risk of accidental injury will also increase.The injury is one of the major threats to astronauts in space missions,and most of them are the injuries of skin and soft tissue.Studies have showed that there are a variety of cells involved in the repair process of skin injury.The epidermal stem cell,which is the skin tissue-specific stem cells with self-renewal and differentiation potential,plays an important role in the repair process of skin injury.Therefore,it is of great significance to study the effects of microgravity environment on the growth characteristics and cell metabolomics of epidermal stem cells.There are limited reports on the effects of weightlessness on human epidermal stem cells.This study was designed to study the effects of microgravity simulated using the rotary cell culture system（RCCS）on the cell proliferation,cell cycle,ultrastructure and metabonomics of human epidermal stem cells（h Ep SCs）,and aimed to provide a theoretical basis for the management of stress injury under weightlessness environment and the application of human epidermal stem cells in research of space medicine.Methods Human epidermal stem cells（h Ep SCs）were cultured in vitro and the microgravity cell culture was simulated by RCCS system.The h Ep SCs of 4～10generations in logarithmic phase were randomly divided into normal gravity control group（NG）and simulated microgravity group（SMG）.After 1 d,3 d and 5 d culture,The h Ep SCs of two groups were collected.The cell proliferation detected by CCK-8,the cell growth cycle detected by flow cytometry,the related proteins and gene expression detected by western blot and RT-PCR were performed respectively,the cell ultrastructure was observed under transmission electron microscopy,and the metabolomics was analyzed by UHPLC-LTQ-MS technique and KEGG database.There were three biological duplicates in each group.Results（1）The CCK-8 test results showed that after 1 d,3 d and 5 d of culture under weightlessness environment,the h Ep SCs proliferation capacity in SMG group was significantly inhibited compared with that in NG group（P<0.05）.At the same time,the cell proliferation ability in SMG group showed a decreasing trend,and there were significant differences between 1 d and 3 d,1 d and 5 d,and 3 d and 5 d（P<0.05）.（2）The flow cytometry results showed that the cell ratio in G₀/G₁ phase under mictogravity culture at 1 d and 5 d was decreased,while the cell ratio in S phase was increased compared with that in NG group（P<0.05）.In addition,the G₀/G₁ phase cells were increased,while the cell ratio of S phase and G₂/M phase was decreased in SMG group at 3 d significantly（P<0.05）.（3）The results of western blot and RT-PCR showed that there was no significant difference in cyclin B1 expression between SMG group and NG group at 1 d and 5 d（P>0.05）.However,the cyclin B1 expression in SMG group at 3 d was increased significantly compared with that in NG group（P<0.05）.The cyclin D3 expression in SMG group was increased at 1 d and 5 d,while it was decreased at 3 d compared with that in NG group significantly（P<0.05）.（4）The results of transmission electron microscopy showed that there were abundant secondary lysosomes,mitochondria and vacuolar structures in cytoplasm of h Ep SCs in SMG group after 1 d and 3 d culture,while they were decreased after 5 d culture compared with NG group.（5）The metabolomics analysis showed that after 1 d and 3 d culture,there were 57 common differential metabolites including 23 metabolites down-regulated and 34 metabolites up-regulated in SMG group compared with that in NG group.Further analysis revealed that these altered metabolites were mainly the phosphatidylserine phosphatidylethanolamine,lysopecithin ceramide D-tryptophan,docosahexaenoic acid,etc.The KEGG compounds classification was conducted and displayed that the altered metabolites were mainly divided into phospholipids,glycolipids,fatty acids and amino acids.Then the KEGG pathway enrichment analysis revealed that there were multiple pathways involved including amino acid metabolism pathway,lipid metabolism pathway,membrane transport pathway,and neurotrophic protein signaling pathway.Conclusion This study showed that the RCCS simulated microgrvity could affect h Ep SCs’proliferation,cell cycle and ultrastructure,induce fluctuated expression of cyclin B1 and cyclin D3.The expression profiles of cellular metabolomics involved in cellular metabolic networks and gene regulation were significantly altered.