The Function Of Protein SH3BP2 In The Differentiation Of Osteoclast-like Cell

Cherubism is an autosomal dominant inherited syndrome characterized by excessive bone degradation and a large number of osteoclast at the surface of the bone. The gene responsible for cherubism named SH3BP2 code an adaptor protein which plays a critical role in the differentiation of osteoclast, When the expression of protein SH3BP2 was blocked, RAW264.7 are unable to differentiate into mature osteoclast. SH3BP2 mutations in cherubism are function-gained mutations. They can up-regulate the response of monocytes to RANKL and M-CSF. RANKL is indispensable for the differentiation of osteoclasts. The formation of osteoclast induced by RANKL and M-CSF is the canonical differentiation pathway. In vitro, osteoclast is difficult to be separated and purefied. The lack of osteoclast is becoming a problem that prevents the progress of research on SH3BP2 in osteoclast differentiation. It has been reported that during the process of osteoclast differentiation protein SH3BP2 can phosphorylate signal molecules (Syk,Src,PLCy) by interacting with them directly. But that is not enough to profile the function of protein SH3BP2 in this way. There must be some other proteins. To find out these proteins would be helpful to clarify the role of SH3BP2 in osteoclast differentiation, which could provide the theory basis for the clinical therapy of cherubism.Objectiveâ‘ To establish an inducing system that can induce monocyte to differentiate into osteoclast efficiently.â‘¡Construct SH3BP2 plasmid vector, then transfect them into monocytes, to establish a cell model for the research the function of protein SH3BP2 in the differentiation of osteoclast.â‘¢To find a new protein which could be interact with SH3BP2 and involve in the differentiation of osteoclast. Which could be helpful to explain the role of the protein SH3BP2.Methodsâ‘ Induce different monocytes (THP-1, PBMCs, UBMCs) to differentiate into osteoclasts by RANKL and M-CSF. Comparing the morphology of osteoclasts from different monocytes. Then choose the one that is easier to be induced in vitro.â‘¡Select 4 types of SH3BP2 mutations to construct plasmid vector. And try to transfect them into monocytes.â‘¢Bioinformatics forecast the proteins that can interacte with protein SH3BP2. Detect the expression of the protein in cherubism osteoclasts and the pre-osteoclast in the different stage of differentiation by immunohistochemistry. If there is a positive result then detect the interaction of SH3BP2 and the protein by CO-IP.Resultsâ‘ The osteoclast-like cells that differentiated from THP-1 cells have more nuclui than PBMCs, but less than UBMCs. The inducing time that THP-1 needs is shorter than the other two cells.â‘¡The plasmids were transfected into THP-1 cells successfully, but the cells failed to overexpress protein SH3BP2. Plasmid vector can be transfected in to UBMCs at the forteenth day of differentiation induced by RANKLand M-CSF.â‘¢Protein FLT-3 and SH3BP2 can be detected in the cherubism osteoclast specificly and the progenitor osteoclasts in the different stages of differentiation.Conclusionsâ‘ THP-lcell can differentiate into osteoclast-like cell induced by RANKL and M-CSF.â‘¡The plasmid vector can express protein in 293 cells, but failed in THP-1 cells.â‘¢Using lipofectimaine2000 can transfect plasmid vector into UBMCs after induced by RANKL and M-CSF for 5 days.â‘£Protein FLT-3 maybe involve in the osteoclast differentiation which is regulated by SH3BP2.