| Objective:1.To obtain purification rat dental pulp cells and set up a rat dental pulp cells cultural method stably and high efficiently.2.To construct an adenoviral vector encoding cadherin11 gene,infect the rat dental pulp cells in vitro,and detect the expression of cadherin11.To investigate the effect of adenovirus expressing cadeherinll(Ad-Cadherinll) on biological characteristics of rat dental pulp cells.Providing a potential approach to the clinical application of cadherin11 impact on pulp dentin regeneration.Methods:1.Separation, cultivation and identification of rat dental pulp cells. Sacrificed the 2-month-old wistar rats by cervical dislocation, split crown and removed the pulp tissue under sterile conditions.Cut off 1mm apical pulp tissue and pulp tissue was cut into small pieces, placed 1cm spacing evenly spread on the bottom, add DMEM medium containing 200ml/L fetal bovine serum,37℃,5% CO2 incubator. Cell fusion into a single post (80 to 90% confluence),250g/L trypsin digestion and passage the rat dental pulp cells and observed cells morphology, size, attachment and growth. Take the 3rd generation rat pulp cells, cell samples were collected, embedded in paraffin, and vimentin, cytokeratin immunohistochemistry staining, observed under the light microscopy.2.Transfection into rat dental pulp cells with cadherin11.The replication adenoviral vector encoding cadherin11 gene was constructed by using homologous recombinant modality.Cultured the rat dental pulp cells in vitro and cells transfected with virus,the effect of transfection was observed under the fluorescence microscope, and to detected the virus transfection efficiency by cell counting.The exogenous gene modified rat dental pulp cells were comprehensively studied on their biological features, in terms of morphology.and draw the cell growth curve,detected the CDH11 expression by immunocytochemistry and RT-PCR.Results:1.Separation, cultivation and identification of rat dental pulp cells.(1)Primary rat dental pulp cells were round strong refraction cells,after adherent, the cells were fusiform shape, fibroblast like, a colony like growth, closely arranged, were spindle shaped cells around, smaller volume, can be single-layer fusion about 10d.(2)Passaged rat dental pulp cells were fibroblast-like, similar in size, distribution, tightly packed, had a strong ability to grow in vitro, at the time of 5 days 90%cells were fusions. (3) From the growth curve can be drawn that, 1～2d was the cell residence time, cell growth was slower,3-6d cells proliferated rapidly, was the logarithmic growth phase,7～10d cells showed a steady state, was the period of inhibition cell growth, P10 generation cells gradually aging, the cell doubling time was 6.71 h.(4)Vimentin was positive expression, cytokeratin was negative expression, it indicated that the cultured cells derived from dental pulp cells mesoderm.2.Transfection into rat dental pulp cells with Cadherin11. (1) Constructed adenovirus carrying the cadherin11 target gene expression vector,measured the titer is 1×1010TU/ml. (2) Higher transfection efficiency could be obtained at MOI of 50.Transfected dental pulp cells after 48h, more than 70% of the rat dental pulp cells appeared fluorescent expression. (3) Growth curve showed that cadherin11 on the growth of dental pulp cells have significant role in promoting. (4) The immunocytochemistry and RT-PCR were detected in the experimental group showed high expression of cadherinl 1.Conclusion:1.The rat dental pulp cells which isolated from rat pulp tissue have strong growth in vitro, and the cells could be passaged for a long time without affecting its steady growth, which laid the foundation for the rat dental pulp cells transfected with cadherin11. the experiment of transfection into rat dental pulp cells with Cadherin11.2.Constructed the cadherin11 adenovirus expression vector successfully.Cadherin11 gene modified rat dental pulp cells had significant promoting effect on the growth. |
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