Wild-type Smad3 gene promotes osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro
Wang, Y.-T.; Zheng, Q.-X.; Guo, X.-D.
Zhonghua Yi Xue Za Zhi 84(18): 1523-1527
2004
ISSN/ISBN: 0376-2491 PMID: 15500712 Document Number: 579799
To observe the effect and mechanism of wild-type Smad3 gene on the proliferation and osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cell (MSC). Rat bone-derived MSC were cultured and transfected with the complexes of pcDNA3.0-Myc-Smad3 or pcDNA3.0-Myc-Smad3DeltaC and LipofectAMINE reagent. Immunofluorescence technique was performed to evaluate the c-Myc expression. The cell proliferation was detected by MTT method. In order to observe the osteoblastic characteristics in stable expression MSC, alkaline phosphatase (ALP) mRNA and core binding factor alpha1 (cbfa1) mRNA were investigated by RT-PCR technique, and ALP activity and mineralization were examined by PNP method and alizarin red staining respectively. PD98059 was used to interdict selectively ERK pathway in order to investigate the effect of ERK pathway on Smad3 regulating MSC. c-Myc protein was expressed in Smad3-MSC and Smad3DeltaC-MSC. The proliferation of Smad3-MSC was slower than that of Smad3DeltaC-MSC or V-MSC. The relative levels of ALP mRNA and cbfa1 mRNA in Smad3-MSC were higher markedly than those in Smad3DeltaC-MSC or V-MSC, as well as ALP activity and mineralization. However, without a significant difference ALP activity and mineralization was after PD98059 treatment (both slightly decreased P > 0.05). The wild-type Smad3 gene, which is a crucial component promoting bone formation, can inhibit the proliferation of MSC and promote the osteoblastic differentiation and maturation of MSC independent of ERK pathway.