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The Function of Protein SH3BP2 in the Differentiation of Osteoclast-like Cell

Author: LiuTianYun
Tutor: MengXueMei
School: Tianjin Medical University
Course: Clinical Stomatology
Keywords: SH3BP2 Osteoclasts Differentiation RANKL M-CSF FLT-3
CLC: R782
Type: Master's thesis
Year: 2011
Downloads: 14
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Abstract

The giant jaw disorder (Cherubism), is a genetic disease, is characterized by a kind of collar bone resorption distribution in which a large number of osteoclast. Its of pathogenic genes SH3BP2 encoded proteins is an adapter protein, which plays an important role in osteoclast differentiation process, when its expression is blocked in mouse osteoclast precursor cells Raw264.7 RAW264 .7 can not differentiate into mature osteoclasts. Giant jaw disorder in the the SH3BP2 mutation is a mutation of a class of functions, it is able to raise osteoclasts before the body cells of osteoclast differentiation factor (receptor activor of, NF-kB we hypothesize that the hemophagocytosis, of RANKL) and macrophage cells set off the stimulus factor (macrophage the response of the colony-stimulating factors, M-CSF). RANKL is induced to generate the factor of osteoclast necessary, in combination with M-CSF induced monocytes to osteoclast differentiation of osteoclast cells to produce classical pathway. Vivo osteoclast cells and mesenchymal cells mixed, a small number, are difficult to separate, so research usually by in vitro induced monocytic differentiation of osteoclasts, but because of limited sources of monocytes or induced difficulties, resulting in vitro induction obtained The number of mature osteoclasts become hindering research osteoclast Some features of a bottleneck. Currently through protein SH3BP2 research, it has been found that interact with proteins SH3BP2 signal molecule involved in the regulation of osteoclast differentiation (Syk, Src, PLCy), but only through these signaling molecules, and not enough to clarify SH3BP2 right breaking regulation of bone cell differentiation, there may be other proteins that can interact. Therefore, to identify these proteins, can further improve the signaling pathways regulating osteoclast differentiation protein SH3BP2 participation, to reveal the occurrence of the disease process, develop a reasonable plan to provide a basis for gene therapy. Objective: ① establish an effective induced monocytes to osteoclast differentiation induction system to solve the problem of the source of osteoclast. ② Construction SH3BP2 mutant plasmids, and expressed in monocytes, osteoclast differentiation mechanism to establish a platform for study protein SH3BP2 regulator. ③ find new may be involved in osteoclast differentiation and may protein interaction SH3BP2, in accordance with the function of structural characteristics of this protein are studied SH3BP2 regulating osteoclast differentiation. Method the: ① RANKL and M-CSF induced by osteoclast precursor cells (monocytic cell line THP-1 cells, peripheral blood mononuclear cells PBMCs and cord blood mononuclear cells UBMCs) multinucleated osteoclast-like cell differentiation, compare osteoclasts cell morphology and differentiation time, choose easy vitro differentiation of monocytes. ② Select four SH3BP2 gene mutant construct a plasmid vector. And try the plasmid was transfected into the mononuclear cells. ③ the bioinformatic predictions may SH3BP2 interacting protein. Immunohistochemical staining to detect the protein in lesions of osteoclasts and monocytes to the expression of osteoclast differentiation process. Expression by immune coprecipitation experiments to detect whether there is interaction of the two proteins in osteoclasts. Results: ① in THP-1, PBMCs the UBMCs three comparison, the number of the nucleus of the osteoclast-like cells generated in morphology UBMCs up, followed by THP-1, at least PBMCs of. On the induction time, THP-1 differentiation fastest completed. (2) Construction of wild-type and the four mutant SH3BP2 plasmid. Transfected plasmid to THP-1 cells, but not expressed protein. UBMCs can be performed at the 14th day to the osteoclast differentiation plasmid transfection. ③ FLT-3 and SH3BP2 the giant jaw disorder osteoclasts and monocytes to the process of osteoclast differentiation are expressed. Conclusion: ① THP-1 cells are capable of differentiation joint stimulate RANKL and M-CSF generate osteoclast-like cell morphology. ② was constructed of four mutations SH3BP2 plasmid and detect protein expression in 293 cells. However, the THP-1 cells can not express the plasmid encoded SH3BP2 protein. (3) cord blood monocyte differentiation to a certain extent after a (lipofectimaine2000 in five days), the application of liposomal transfection methods can be transfected plasmid into the cells. ④ FLT-3 may be involved in the process of the SH3BP2 regulate osteoclast differentiation.

The Function of Protein SH3BP2 in the Differentiation of Osteoclast-like Cell

Abstract

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