| It was thought that human dental pulp stem cells (hDPSCs)were progenitors existing in dental pulp.They could be activated by dental damage and terminally differentiated into functional odontoblasts. Neurotrophins (NTs) were important growth factors and of multiple functions, and thay are proved to be expressed during tooth development. NTs is an important part of network regulation of tooth development and plays an important role in the development of tooth. Studies showed that NTs promoted proliferation, differentiation and calcification of dental pulp cells in vitro,and they may play a role in the differentiation of progenitors of odontoblasts. However, whether human dental pulp stem cells express NTs, and whether NTs could enhance the proliferation and differentiation of human dental pulp stem cells in vitro had been poorly investigated. In this study, we preliminarily cultured human dental pulp stem cells, investigated the expression of NTs, and explored the effects of NTs on the proliferation and differentiation of human dental pulp stem cells. It provided experimental evidence for further research of role of NTs in the tooth development and regeneration ,as well as proliferation and differentiation of human dental pulp stem cells.This study includes three parts:1. Cell culture and identification: Dental pulp tissue of healthy third molars was digested with mixture of collagenase and dispase and the stem cells were isolated by limited dilution of cultured cell for single cell clone. Their morphology and growth condition were observed under inverted light microscope. Immunohistochemistry procedure was employed to detect surface marker of the cloning cells. Results: hDPSCs exhibited self-renewal and clonogenic cell population.The incidence of colony forming cells was 4-28 colony/104 cell plate. Immunohistochemical analysis showed that cultured cells expressed vimentin and STRO-1 while keratin were absent in hDPSCs.2. Expression of NTs in hDPSCs: RT-PCR, immunohistochemistry and western blotting were employed to detect mRNA and protein expression of NTs in hDPSCs, respectively. Results: RT-PCR, immunohistochemistry and western blotting showed that hDPSCs expressed BDNF in the level of both mRNA and protein. Western blotting showed NT-4/5was also expressed in hDPSCs, while other NTs were absent.3. Effects of NTs on proliferation and differentiation of hDPSCs: Cells between the third and fifth passages were used in this study. The optical density was obtained by MTT assay in cells stimulated by NTs in different concentrations. Effect of NTs on differentiation of hDPSCs was indirectly measured by the activity of alkaline phosphatase of the cells.Results: When compared with control group, NGF of 25ng/mL and BDNF of 25ng/mL stimulated the cell proliferation significantly (P<0.05) .However, NT-3 and NT-4/5 showed no effect on cell proliferation. When compared with control group, the ALP activities of hDPSCs were obviously increased with NGF of 100ng/mL(P<0.05), BDNF of 100 ng/mL(P<0.05). NT-3 and NT-4/5 of 25ng/mL(P<0.05). It is concluded that different concentrations of certain NT and different kind of NTs have different effects on proliferation and differentiation of hDPSCs.
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