| Objectives DMSO is widely used in clinical and research,but its influence on cells is often neglected.This study was to investigate the effect of low concentration of DMSO on the RAW264.7 cells mediated by RANKL,to reduce the working concentration of DMSO,and providing experimental basis for low concentration DMSO in clinical and research.Methods 1 Cell proliferation : We seeded the cells in the 96-well plates,the cells were mediated by 50ng/ml RANKL for 24 h and selected 0,0.001%,0.005%,0.01%,0.05%,0.1%,0.15%,0.2%,eight concentrations of DMSO acted on the cells.When the cells were stimulated at 1st,3rd,5th,7th,and 9th days,the number of cells in each group was tested.2 TRAP activity:The cells were seeded in the 96-well plates and mediated by 50ng/ml RANKL for 24 h,then selected 0,0.001%,0.005%,0.01%,0.05%,0.1%,0.15%,0.2% eight concentrations of DMSO acted on the RAW264.7 for 4 days.According to the TRAP Activity Kit instructions,we used the Enzyme-linked immunosorbent to detect the value of absorbance for each group,and used the BCA protein kit to detect the concentration of protein in each group.The value of absorbance was divided by the concentration of protein as the TRAP activity.3 TRAP staining: We seeded the cells in the 96-well plates,the cells were mediated by 50ng/ml RANKL for 24 h and selected eight concentrations of DMSO acted on the RAW264.7 for 4 days.Then we stained the cells by the TRAP staining kit.After drying at room temperature,we randomly collected 20 photos from each plate by microscope.We measured the picture by Image-Pro Plus 6 software and recorded the total number of TRAP stained positive cells of osteoclast cells,the total area and the number of nuclei in a single cell.4 The detection of relative gene expression: We seeded the cells in the 6-well plates,then the cells were mediated by 50ng/ml RANKL for 24 h and selected eight concentrations of DMSO acted on the RAW264.7 for 4 days.The total RNA was extracted,and the c DNA was synthesized.Then Real-Time quantitative PCR detection was carried out by the Real-Time Quantitative PCR Kit.5 Calcium phosphate resorption assay of osteoclast cells: We seeded the osteoclast cells in the Osteoclast Activity Assay Substrate(OAAS)plates,after mediated by 50ng/ml RANKL for 24 h,we selected eight concentrations of DMSO acted on the RAW264.7 cells with 50ng/ml RANKL for 10 days.After drying at room temperature,we randomly collected 20 photos from each plate by microscope and measured each picture by Image-Pro Plus 6 software.Results 1 Osteoclast proliferation:The 1st days,the number of cells in each group was similar,and there was no statistical difference(P>0.05).The 3rd day,the number of osteoclast cells in the DMSO and RANKL groups were 1.5 times more than the RANKL group,there was statistical difference between each group and RANKL group(P<0.01).The 5th day,compared with the RANKL group,the number of cells in 0.001% DMSO group increased significantly(P<0.01),but the promoting effect in 0.005% and 0.01% DMSO group was reduced.The 0.2% DMSO group had the greatest promoting effect on the proliferation of cells.The effect of DMSO on the proliferation of cells for 7th days is roughly the same as that of 5th days,the cells of each group proliferated obviously.At the 9th day,the number of cells in each group increased more than 7th day.The number of cells in the 0.1% DMSO group reached the maximum.But there was no significant difference between the 0.1% DMSO group and other DMSO groups.2 The TRAP activity: The TRAP activity in all DMSO groups were lower than the RANKL group,and the TRAP activity decreased with the increase of DMSO concentration.There was statistical difference between RANKL group and DMSO groups(P<0.01).3 Morphological analysis:1)The total surface area:Compared with the RANKL group,the total area of osteoclast cells formed in 0.001% and 0.005% DMSO groups increased significantly(P<0.01).The total surface of osteoclasts reached the maximum at 0.005% DMSO group.When the concentration of DMSO was further increased to 0.1%,0.15%,and 0.2%,the total surface area of osteoclast decreased with the increase of concentration.There was no significant difference between 0.001% DMSO group,0.05% DMSO group,0.1% DMSO group and RANKL group(P>0.05).2)The number of osteoclast cells: Compared with RANKL group,the number of osteoclast cells in 0.001% DMSO group was more than RANKL group,there was no significant difference between the two groups(P>0.05).When the concentration of DMSO was 0.05%,0.1% and 0.15%,the number of osteoclasts increased significantly(P<0.01).And the number of osteoclasts in 0.2% DMSO group increased,but there was no significant difference between the two groups(P>0.05).3)The average surface area: Compared with RANKL group,the average surface of osteoclasts increasd significantly in 0.005% and 0.01% DMSO group(P<0.01).When the concentration of DMSO was further increased to 0.05%,0.1%,0.15% and 0.2%,the average surface area decreased with the increase of concentration,and all these groups were statistically different from RANKL group(P<0.01).4)The number of nuclei: Compared with RANKL group,the number of cells with less than 3 nuclei contained in single osteoclasts in 0.001% DMSO group was least,but in 0.15% DMSO group was most.There was statistical difference between these two groups and the RANKL group(P<0.01).Compared with RANKL group,the number of cells with 3 to 20 nuclei contained in single osteoclasts in 0.005% DMSO group was least,but in 0.1% DMSO group was most.There was statistical difference between these two groups and the RANKL group(P<0.01).Compared with RANKL group,the number of cells with more than 20 nuclei contained in single osteoclasts in 0.005% DMSO group was most,but in 0.2% DMSO group was least.There was statistical difference between these two groups and the RANKL group(P<0.01).4 The relative gene expression of osteoclast cells:1)Acp5: Compared with RANKL group,except for 0.001% DMSO group promoted the expression of Acp5 genes.The other DMSO groups inhibited the expression of Acp5 gene.2)Calcr: Compared with RANKL group,0.001% and 0.01% DMSO groups promoted the expression of Calcr gene.But there was no significant difference between these two groups and RANKL group(P>0.05).3)Ctsk: Compared with RANKL group,except for 0.001% DMSO group promoted the expression of Ctsk genes.The other DMSO groups inhibited the expression of Ctsk gene.5 The Cap resorption capacity of osteoclast cells: Compared with the RANKL group,the Cap resorption area in 0.001% DMSO group was significantly increased(P<0.01),the Cap resorption area of 0.005% DMSO group was bigger than the RANKL group,but there was no significant difference between two groups(P>0.05).Conclusions 1 DMSO has the effect of promoting osteoclast proliferation,and inhibits TRAP activity and osteoclast related gene expression with increasing concentration.2 The surface area of osteoclasts appeared to be a bell shaped curve with the increase of DMSO concentration.The concentration of 0.005% and 0.01% DMSO showed a promoting effect compared with the RANKL group,but with the increase of DMSO concentration,it gradually turned to inhibition.3 The TRAP activity of osteoclasts and the size of TRAP staining positive cell area cannot be directly equal to the calcium phosphate absorption capacity and the ability of bone resorption of such cells. |
PDF Full Text Request