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Objective The aim of this study was to investigate the effects of the ERK5 pathway on bone morphogenetic protein?9(BMP9)?regu?lated osteogenic/odontogenic differentiation of immortalized stern cells from the apical papilla(iSCAP). Methods BMP9 was introduced into the iSCAP by using recombinant adenoviruses,and the P?ERK5 protein expression was measured via western blotting. Then,the osteogenic/odontoblas?tic changes were analyzed by alkaline phosphatase(ALP)staining and Alizarin red staining,and the expression of osteogenic/odontoblast?associat?ed genes,such as Runx2,OCN,OPN,and DMP1 was measured by RT?PCR. Results BMP9 could up?regulate the phosphorylation of ERK5 in iSCAP. After using BIX02189,which was an inhibitor of ERK5,the phosphorylation of ERK5;the activity of ALP;the expression of Runx2, OCN,OPN,and DMP1;and the calcium deposition were all significantly inhibited. Conclusion The ERK5 signaling pathway plays an important role in BMP9?regulated osteogenic/odontogenic differentiation of iSCAP.
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Objective To establish the strain of immortalized human precartilaginous stem cells (PSCs) which can be a stable cell resource for study of the molecular mechanism of gene targeting on differ-entiation of PSCs. Methods Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs using lipofeetin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Immunohistochemistry, RT-PCR and Southern blot were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. Results The positive colonies were isolated and subcultured, named as immortalized precartilaginous stem cells (IPSCs), which were confirmed as positive to fibrnblast growth factor receptor-3 (FGFR-3). The existence of SV40Tag cDNA was detected by Southern blot and the expression of SV40Tag mRNA and protein by RT-PCR and immunohistochemistry. Conclusion IPSCs strain with SV40Tag can be constructed successfully.
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Recent evidence has increasedly supposed cancer stem cell theory.that cancers can be viewed as an abnormal organ in which tumor growth is driven by a population of cancer stem cells,with self-re-hewing and muti-differentiation potentiality.CD133 has been repofled sequentially in many malignan-cies.Although the biological function of CD133 is not well understood.the CD133 epitope cueeently senves as a useful marker for the isolation of cancer stem cells.Research has discovered that CD133+ tumor cells are in re-lation to tumorigenesis,invasion,metastasis,drug-resistance and recurrence.So,understanding the molecular bi-ology of the CD133+ tumor cells is essential for developing more effective anti-cancer tIeatments,especiallv tar-geting cancer stem cells.
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Objective To investigate the ostengenie potential of adipose-derived stem cells(AD- SCs)when exposed to adenovirns containing hBMP-2 cDNA(Adv-hBMP-2)and offer a choice of cell source for gene therapy and tissue engineering.Methods Human adipose tissues were obtained from patients who received orthopaedic surgery or liposuction.ADSCs were obtained by digesting the adipose tissues.Firstly,flowcytometric analysis was performed for the confirmation of mesenchymal stem cell ori- gin and the surface markers including CD34,CD44,CD68,CD71,CD90,and CD105.The ADSCs were transfected by Adv-hBMP-2 and the effects were tested in vitro,lmmunoprecipitation and Western blotting and ELISA were performed for confirming BMP gone transduction and its stable expression.The transform of ADSCs was assessed by extracellular ALP staining,intracellular ALP spectrophotometry,von Kossa staining and RT-PCR.In the in vivo experiment ADSC-Adv-hBMP-2 cells were injected into the hind limb of nude mice and analyzed radiographically and histologically.Results ADSCs were successfully isolated from human adipose tissues.The isolated ADSCs expressed CD44,CD71,CD90 and CD105 and CD34 and CD68 were absent.The result confirmed the mesenchymal stem cell origin of the cells.West- ern blotting and ELISA confirmed successful and persistent hBMP-2 production by ADSC-Adv-hBMP-2 cells.Extracellular ALP staining,intracellular ALP spectrophotometry,yon Kossa staining and RT-PCR revealed that ADSCs treated with Adv-hBMP-2 had a tendency of transfering into osteoblast.X-ray and H&E sections from hind limb of nude mice injected with ADSC-Adv-hBMP-2 cells confirmed bone forma- tion at 2 weeks.Conclusions Liposuction aspirates contain abundant ADSCs that can be transduced with hBMP-2 gene,and the tranduced ADSCs differentiate into the osteoblast.ADSCs may be an ideal source of mesenchyme-lineage stem cells for gone therapy and tissue engineering.