The Preliminary Study Of CTLA-4-Ig Inhibition On The Osteoclast Differentiation And Maturation

Objective:To observe the effect of CTLA-4-Ig on the U937 cell differentiation into osteoclast-like cells induced by phorbol ester and 1,25（OH）₂VD3,and to provide a potential target for the further study of drugs that can accelerate tooth movement.Method:The experiment was divided into three groups:control group,induction group,and experimental group.Each time,each group was inoculated with6-well plate cells in 6 wells and repeated three times.The induction process was first induced by phorbol ester.And on the 2nd and 5th days,an inducer containing phorbol ester and 1,25（OH）₂VD3 was added,and add CTLA-4-Ig at the same time.Identification and counting of the model were performed by using light microscope and tartrate-resistant acid phosphatase（TRAP）staining.And the osteoclast marker gene of MMP-9,TRAP,CTSK,RANK were determined for the osteoclast-like cell maturation through Real-time PCR.Then the osteoclast marker protein of TRAP and CTSK were determined for the change of osteoclast-like cell maturation through ELISA.Results:1.Morphological observation of U937 cells is small and round,and it had been growing in suspension.After phorbol ester and 1,25（OH）₂VD3induced 2 days,the cells became adherent and the volume gradually became larger and spindle-shaped,polygonal and other different forms.TRAP staining showed that the control group was negative,and the induction group appeared positive after 8 days.The positive staining cells were also seen in the experimental group,but the number was significantly reduced（P<0.05）.2.Apply Real-time PCR to test the mark gene expression(2^-ΔCT)of the the induction group compared the control group of MMP-9,TRAP,CTSK,RANK for 1.26,21.78,1.74,1.60 times higher.There are statistics significance in different groups（P<0.05）,which can demonstrate the high expression of osteoclast-like cells.Also the induction group compared the experimental group with MMP-9,TRAP,CTSK,RANK for 0.25,2.50,0.64,0.10 times higher,which revealed that the induction group had significant difference compared with experimental group（except RANK,P<0.05）.3.Apply ELISA to detect the CTSK/TRAP protein in different groups of osteoclast-like cells,which is the same with the expression of mRNA.The concentrations of the induction group compared the control group of CTSK,TRAP for 2.72,2.37 times higher（P<0.05）,Also the induction group compared the experimental group with CTSK,TRAP for 0.74,0.29 times higher,which revealed that the induction group had significant difference compared with experimental group（P<0.05）.Collusion:CTLA-4-Ig can partially inhibit the differentiation of U937 cells induced by phorbol ester and 1,25（OH）₂VD3 into osteoclast-like cells。And it provides a potential target for the further study of drugs that can accelerate tooth movement during orthodontic treatment.