The Synergistic Effect Of Intermediate Filament Vimentin And Actin Stress Fiber In HASCs Osteogenic Differentiation

Part 1 The differences in the expression of intermediate filament vimentin and the actin stress fibers with their associated proteins in hASCs osteogenic differentiation.Objective:Human adipose derived stem cells were induced into osteoblasts differentiation,to observe the expression changes and morphological changes of vimentin and actin stress fibers and their related proteins in osteogenesis.Methods:The 3?6th generation of hASCs was planted in tissue culture dish with the density of 7000/cm2.When the fusion degree of the cells reached 80%,the growth medium was replaced with the osteogenic medium,which was changed every two days.Flow cytometry was used to detect hASCs surface markers.Western blot used to detect osteogenic markers(Runx2 and OPN),the cytoskeletal marker proteins(vimentin,tubulin,and actin),in addition,cytoplasmic heat shock protein 27,heat shock protein 90,and so on.The morphological changes of vimentin,tubulin protein,actin and nesprin3/Lamin A were observed by immunofluorescence experiment.The results of alkaline phosphatase staining and alizarin red staining were used to verify the effect of osteogenic differentiation.Results:The western blot experiment suggested that the expression of vimentin and nesprin3 were decreased,while the expression of actin was gradually increased.The immunofluorescence images showed the loose three-dimensional network of vimentin around the nucleus can drift to the top of the cell and forms a bamboo-like or clavate shape.While the actin stress fibers become thicker and more layered around the nucleus.Conclusions:In the process of osteogenic differentiation of hASCs,the cytoskeletons are remodeled,the expression of vimentin is decreased,and its position is shifted from the nuclear to the top of the cell.The expression of actin is elevated,and its filaments are thickened and replaced with a hierarchical arrangement around the nucleus.Their changes are opposite in quantity and space.Part 2 The role of actin stress fibers in the differentiation of human adipogenic stem cells.Objective:By interrupting the actin stress fiber to study the impact of actin stress fiber on intermediate filaments protein vimentin and the ability of hASCs to osteogenic differentiation.To further analyze the role of actin stress fibers in osteogenesis of hASCs.Methods:For the growth medium and osteogenic inducing medium,0.1?g/ml of Cyto D was added to treat the hASCs.Using the qRT-PCR experiment to test the gene expression changes of osteogenesis markers(OPN,Runx2),cytoskeletal protein,(actin,vimentin),cytoplasm protein(heat shock protein 27,heat shock protein 90),etc,at 7 day.The morphological changes of vimentin,tubulin and microfilament actin protein were observed by immunofluorescence experiment.On the 7th day and 14th day of treatment,alizarin red staining and oil red O staining were used to observe the effect of osteogenesis and adipogenesis,respectively.Results:0.1 ?g/ml concentration of Cyto D can successfully disrupt the polymerization of actin stress fibers and make it in a depolymerization state.The depolymerization of actin can reduce the gene expression of actin,shock protein 27 and heat shock protein 90,while the gene expression of vimentin is increased.However,when the cells dealled with osteogenic medium that contains 0.1 ?g/ml Cyto D,we found that even the stress fibers are interrupted,the osteogenic medium can still improve the total RNA expression of actin and vimentin,but,it was lower than osteogenic medium,in general.However,the gene expression of adiponectin in the group of osteogenic medium+Cyto D is increased sharply.The gene expression of OPN and Runx2 in osteogenic medium was higher than in osteogenic medium that contains 0.1?g/ml Cyto D.That is,to a certain extent,the increase of actin monomers induced by continuous depolymerization of cytoplasmic actin is able to change the osteogenic state of adipose stem cells,The pathological staining showed that on the 7th day,we find some lipid droplets in group of osteogenic medium that contains 0.1?g/ml Cyto D.This means that in some cells,after the depolymerization,the actin monomers are achieved to a certain extent,and then trigger the formation of lipid droplets.The immunofluorescence results showed that the vimentin did not drift upward after the stress fiber was interrupted,nor did it form a stick shape.Conclusions:Depolymerization of actin stress fiber reduce the expression of vimentin,affects vimentin to exert different space functions,and change the status of cell differentiation,continuous actin depolymerization reduces the ability of hASCs to osteogenesis,at the same time,can trigger the formation of lipid droplets.Both osteogenesis and lipogenesis exist simultaneously.Part 3 The role of intermediate filaments in the differentiation of human adipogenic stem cells.Objective:By upregulate or downregulate the expression of vimentin to study the effect of vimentin on actin and the ability of hASCs to osteogenic differentiation.To further analyze the role of intermediate filaments vimentin in the differentiation of human adipose-derived stem cells.Methods:Using lentivirus to infect human adipose-derived stem cells,to make vimentin down-regulated,and then induce them osteogenesis.Using qRT-PCR experiment to test the gene expression changes of osteogenesis markers at 7 day.Using lentivirus to infect human adipose-derived stem cells,to make vimentin up-regulated,and then induce them osteogenesis.Using qRT-PCR experiment to test the gene expression changes of osteogenesis markers at 7 day.Results:The down-regulation of VIM caused the corresponding increase in the related gene expression of ?-actin,HSP27 and HSP90,compared with the control group.The expression level of ALP was slightly increased,while the expression of Runx2 and OPN was slightly decreased.The gene expression of OPN and Runx 2 was increased after VIM was down-regulation.The immunofluorescence image shows that vimentin is still able to form a three-dimensional network,even when VIM is down-regulation,and actin stress fiber can still exist in short rod.However,In the process of differentiation,the vimentin can still form a shape of bamboo-like raft,but the thickness of floating in the top of the cell is decreased,while actin stress fiber is spread out at the bottom of the nucleus,and then spiralled up to the top of the cell.qRT-PCR experiment showed that when VIM was increased,some related genes are decreased,such as ?-actin and HSP90.The osteogenesis in the cells have reached a new balance.In the process of differentiation,we found that Runx2,OPN and other osteogenic related gene expression were decreased,compared with the control group.The immunofluorescence data showed that the vimentin formed less bamboo-like raft structure,but mostly in a fiber state.Conclusions:The increase of VIM disrupts the homeostasis of intracellular actin and its associated proteins,which is not conducive to osteogenesis.The decrease of VIM can provide space for the function of actin and activate the expression status of osteogenic genes in the cell and eventually promote osteogenesis.