| Dental pulp injury and repair remains the center of interest of dental pulp biological research, with studies of dental pulp stem cells as an important component in this facet. Work in this line will help to elucidate dental developmental scenario and unravel molecular mechanisms underlying induced differentiation of cells. The present work aimed to culture dental pulp stem cells with consequent characterization and induction of cellular differentiation, thus providing reliable models for dental pulp stem cell research as well as new approaches toward biological functional of Notch signaling in dental pulp stem cell.We have successfully isolated multipotential dental pulp stem cells by single cell culture from mouse dental pulp, and observed several propertiesof these cells in vitro. The results are as follows: 1) The dental pulp stem cells exhibit self-renewal and multipotentiality, and clonogenic cell population appeared. The incidence of colony forming cells was 1.6-2.5 colony/104 cells plate. 2) H.E. (Hematoxylin-Eosin) staining revealed small cytoplasmic to nuclear ratio, with many cells possessing two nuclei and blurred cellular boundaries. 3) Endogenous alkaline phosphatase activity is strong. 4) Immunohistochemical analysis showed that dental pulp stem cells exhibited expression of markers such as type I collagen, osteonectin, osteopontin, type HI collagen, fibroblast growth factor-2 and bone sialoprotein, while dentin sialophosphoprtein was absent in DPSC cultures. 5) The telomerase activity of dental pulp stem cells was at low levels, which indicated that life-span of the stem cells was short, they were significantly different from cancer cells, which are immortalized cells. 6) Electrpn-microscrope showed there were junctions between the dental pulp stem cells. The junctions between cells profoundly influence cell proliferation and differentiation.To determine their differentiation in vitro, dental pulp stem cells were cultured in the medium which added BMP.TGF- 0 , FGF growth factors. On the 8d, DSPP mRNA was analysised by RT-PCR. Our findings indicated that BMP,TGF- 3 , FGF growth factors contributed to the differentiation of dental pulp stem cells toward odonblasts.In the last part of the study, we constructed and identified retrovirus expression vector, which expressed recombinase ere, and attempted to infect the conditional knock-out mouse Notch-RBP-J dental pulp stem cells in vitro. It indicated that the proliferation of the infected dental pulp stem cells in vitro were suppressed by attenuation of Notch signaling,Therefore we tended to come the conclusion that Notch pathway molecules could be essential for the maintenance of dental pulp stem cells. This system might be of some help hi studying the function ofNotch gene hi dental pulp stem cells.
|
PDF Full Text Request