| [Objective] Stem cells are multipotent cells which could differentiate into dinstinct populations of cells under a certain of growth conditions in vitro. Thus the idea of culturing stem cells to form alternative organ transplant has been put forward and placed a great importance in life science in the past decades. Based on the definition of stem cells, Gronthos put forword to the conception of dental plup stem cells in2000, developing a potential new way for tooth regeneration. However, the first step of tooth regeneration from stem cells is how to identify the dental cells which involves in the determination of the specific surface markers to isolate the dental pulp stem cells. The study of dental pulp stem cells is still at an early stage in China and abroad, and the surface markers of DPSCs are rarely reported. It is known that Thy-1/CD90and Ringlb are markers of mesenchymal stem cells, and both are expressed posstively in bone pulp stem cells. However, could we use Thy-1/CD90、 Ring1b as markers for DPSCs? What are the roles of these two genes if they are expressed in DPSC? These questions showed a new direction for dental pulp stem cells studies, and waiting for the more investigation.[Methods] There are two parts for this thesis, one is screening and selecting for DPSC markers, another is identification. In the first part, the expression of Thy-1/CD90and Ringlb in the dental pulp was examined with in situ hybridization and FACS for the dental pulp stem cell markers screening. In the second part, there are function identification and biological characteristics identification for Thy-1/CD90and Ringlb as the DPSC markers. For function identification, Thy-1/CD90-/-and Ringlb-/-mouse were used as working model, CD1mouse collected as control group, tooth bar from three types null mice was cultured for7days in vitro to study the effect of Thy-1/CD90and Ring1b on the mouse tooth development. In addition, paraffin section of mice mandible was made for HE staining to compare the histology of incisors between Thy-1/CD90-/-and Ring1b-/-mouse and CD1mouse. Thy-1/CD90+, Ringlb+, Thy-1/CD90+&Ring1b+and BMSC cells were collected by FACS. Miural’s method was followed to do dental cells digestion and culture, then the ways as CFU calculation, MTT and OD values determination, detection of cell surface antigens were used for the identification of Thy-1/CD90and Ringlb as DPSC markers. [Results]In the first part in the thesis, the results of in situ hybridization showed that Thy-1/CD90and Ringlb both mainly expressed in cervical loop of the dental pulp where the detan pulp stem cells niche located. Meanwhile, FACS results showed the percentage for Thy-1/CD90expressing in the whole dental pulp of P5mouse incisor was12.2%, in root of dental pulp was31.5%, in body of dental pulp was2.8%. The percentages for Ring1b were11.7%,28.6%and3.1%. In the second part, found that the development of Thy-1/CD90-/-and Ring lb-/-mice tooth were both lag behind of the normal mice during the stage from E14.5to P2while the tooth cultured in vitro. H-E staining showed the very similar results with organ culture. After cell collection by FACS, got Thy-l/CD90+, Ringlb+, Thy-l/CD90+&Ringlb+and BMSC cells as four working groups for further studies. The results of CFU calculation for these four groups are0.68%,0.73%,0.59%and0.44%. OD values for these selected cells culture from the3rd day to15th day showed there was obviously fast cells growth from5th day to9th day. The analysis for cell surface markers of each group showed, positive express in four groups with Vimtine, CD44and SHH. Negative express was with DSPP and DSP. ON and FGF only negative expressed on Thy-1/CD90+cells, BSP only positive expressed on BMSC cells.[Conclusion]1. Thy-1/CD90and Ringlb are both expressed on dental pulp cells surface.2. The characteristics for Thy-1/CD90and Ring1b express on dental pulp cells are very similar with that of DPSC markers express features.3. The tooth development of Thy-1/CD90-/-and Ringlb-/-mouse is both lag behind with normal mouse, revealed Thy-1/CD90-/-and Ringlb-/-should affect mouse tooth growth and development.4. Thy-l/CD90+, Ringlb+, Thy-1/CD90+&Ringlb+cells have very similar characteristics with BMSCs.5.Thy-1/CD90and Ringlb could be used as the candidate biological surface markers for DPSCs. |
PDF Full Text Request