Abstract：Abstract：pEF-neu-CHIP vector was transfected to MC3T3-E1 cells. Then cells were cultured with α-mimal essential medium supplemented with 1.0mg/ml Geneticin(G418). The surviving (Geneticin-resistant ) cells were cloned. Finally overexpression of CHIP protein was detected by Western blot in MC3T3-E1-HA-CHIP stable cell line. MC3T3-E1 wild type cell line and CHIP-overexpressing cell lines were induced towards osteoblast with condition medium (10mM/mlglycerophosphatesodium and 50ug/ml ascorbic acid). Differentiation and mineralization of two cell lines were verified by assay of Alkaline phosphatase （ALP） activity , ALP staining, Von Kossa staining and Alizarin Red staining. The results showed that ALP activity of CHIP-overexpressing cell lines were lower than that of MC3T3-E1 wild type cell line. The same results were testified by ALP staining. Mineralization of CHIP-overexpressing cell lines were also lower than that of MC3T3-E1 wild type cell line . This result was proved by Von Kossa staining and Alizarin Red staining. All above results demonstrated that overexpression of CHIP inhibited differentiation and mineralization of MC3T3-E1 cell line.
