Study On The Traction Force And Related Protein Activity Of Stem Cells And HSCs Under Different Substrate Stiffness

For understanding the role of cell traction forces in the interactions of the mechanical signals generated by different substrate stiffness and the chemical signals generated by the activation of cytokines and protein kinases,furthermore,to clarify the interaction between mechanical signal and other signal molecules in the process of the stem cell therapy of liver fibrosis,and its mechanism,in this study,based on some references,a polyacrylamide hydrogel(PA)cell culture system was constructed in vitro to simulate the stiffness of normal liver tissue,the early and late stages of fibrotic liver tissues respectively.Observing cell growth and cell proliferation of Bone marrow mesenchymal stem cells(BMSCs)and Hepatic stellate cells(HSCs)on different stiffness substrates,then using immunofluorescence staining to investigate the effect of substrate stiffness on cytoskeletaland FAs assembly.Using traction force microscopy technique to characterize the cell traction forces on different stiffness substrates,and observe the effects of TGF-?1and sFRP-1 on cell traction forces of both cell on substrates with different stiffness,then through the phosphoproteomics analysis to figure out signaling pathways and cell courses which were involved in the cell responses to substrate stiffness.Both of cells could growth well on all substrate stiffness in this study,and the results of immunofluorescence staining showed that the stiffness of the substrate significantly cytoskeleton and FAs assembly in both of cells,and the high-stiffness substrate promoted cytoskeleton maturation assembly and FAs formation.The results of cell traction forces measurement showed that mean modulus of substrate elements stress(MSES),elastic strain energy absorbed by elastic substrates(ES),and cell prestress index all increased with the increasing of substrate stiffness,and the effect of substrate stiffness on cells was coincident in both of cells.Though adjusting the cytoskeletal assembly and FAs formation,cells would regulate its cell traction forces,to perceive the mechanical signal from substrates,and then adjust the cell morphology and function.In the HSCs groups,sFRP-1 had no significant effect on MSES on three substrate stiffness,and its effect on elastic strain energy and cellular prestress was various,it would be decreased on 3.4 kPa and increased on 8.8 kPa indicated that the Wnt signaling pathway in HSCs cells was involved in the regulation of cell traction forces,and there would exist a feedback regulation of Wnt signals by the effect of substrate stiffness.TGF-?1 consistently promoted MSES,elastic strain energy and cell prestress increased in the three stiffness levels,indicating that TGF-?1 could promote the generation of cell traction forces.In the MSCs groups,sFRP-1 significantly reduced those three mechanical indexes at 8.8 kPa and decreased MSES at 3.4 kPa,no significant difference at 17.6 kPa MSCs at 8.8 kPa was more sensitive to sFRP-1,suggesting that the activation of Wnt signaling pathway in MSCs cells was more significant on 8.8 kPa.The effects of TGF-?1 on MSES at three stiffness levels were various,decreased at 3.4 kPa,8.8 kPa but increased significantly at 17.6 kPa.Elastic strain energy and cellular prestress were significantly increased on 3.4 kpa and 17.6 kpa,suggesting that TGF-?1 could induce different cell responses on soft and stiffness substrates,but it improved the energy output capacity of stem cells to the substrates and their own contraction capacity generally.At 3.4 kPa,MSES decreased may because the substrate could not provide adequate resistance,and the cell response on 8.8 kPa have some contradiction with other stiffness.Combined with the results of sFRP-1,this stiffness maybe has particular significance and probably have relation with Wnt signaling pathway.Phosphoproteomics analysis showed that in the MSCs groups,with the increase of substrate stiffness,the cell traction forces could be regulated through the focal adhesion signaling pathway,microfilament cytoskeleton regulation pathway,smooth muscle cell contraction pathway and other related signaling pathways,and the G1 phase arrest of stem cells could be relieved to promote proliferation.HIF-1 signaling pathway is up-regulated with increasing substrate stiffness,suggesting that it may promote liver fibrosis by promoting relevant factors expression including TIMP-1.In the HSCs groups,TGF-? signaling pathway,cell cycle pathway,NF-kappa B signaling pathway,Wnt signaling pathway and other related signaling pathways were activated with the increasing of substrate stiffness,the common results were the initiation of cell cycle,the reduction of apoptosis and the promotion of HSCs cell survival and proliferation.Since the HSCs applied in this study were activated HSCs,and the increased substrate stiffness promoted their proliferation and survival.It can be considered that the increased substrate stiffness promoted the process of liver fibrosis and was related to above signaling pathways.