The Effect Of 50 Hz EMF On Proliferation, Differentiation And C-myc MRNA Of Dental Papilla Mesenchymal Cells

Tissue engineering consists of three parts: the study of seed cells, scaffold and factors adjusting cell growth. Both dental papilla mesenchymal cells and tooth germ is seed cells or seed organ in bone tissue engineering and tooth tissue engineering. Some of the mechanism of tooth development was found. But research showed tooth germ cultured in vitro grow much more retardation than that cultured in vivo, and the engineering tooth had not the nature structure and shape as a normal tooth. How to find ways to solve the big problem, the ways not only repair the tooth germ injury but also promote the growth hormone, and the ways not only promote the form of original blood capillary in tooth transplantation but also develop the method of cultivate to promote the survival rate of tooth germs. Thus the method to adjust the development of tooth germ is very important in bone tissue engineering and tooth tissue engineering, and it is also the key issue of engineering tooth applied in clinic. In this study the electro-magnetic field (EMF) was recommend to establish electro-magnetic cultured model of dental papilla mesenchymal cells to elucidate the effect of EMF on the proliferation and differentiation of dental papilla mesenchymal cells in cellular and molecular biology mechanisms. This study involved four parts:1. In this part the dental papilla mesenchymal cells were separated from the tooth germ in maxillary and mandibular bone of 17～18d fetal rat and cultured in enzymatic digestion and tissue cultivate. The shape and growth condition of primary cells was observed in the inverted phase contrast microscope. Identification of cell was made in immuohistochemistry method. The result showed: the cells cultured in this study had the biology properties of dental papilla mesenchymal cells, that vimentin (+) and keratin(-). Both enzymatic digestion cultivate and tissue cultivate was used in the dental papilla mesenchymal cells successfully. The depurated cells for the study were obtained from the several passion by enzymatic digestion.2. In order to study the effect of 50 Hz EMF on the proliferation of dental papilla mesenchymal cells, MTT and LCM were used to analysis the effect of 50 Hz EMF on the proliferation of dental papilla mesenchymal cells in 3d and 5d. The result showed:①50 Hz EMF effected the proliferation of dental papilla mesenchymal cells. It was "windows effect".②50 Hz/0.6mT repressed cell proliferation both in 3d and in 5d. 50 Hz/1.8mT promoted cell proliferation , but 50 Hz/0.2mT inhibited the cell proliferation in 5d.③EMF effected the proliferation of dental papilla mesenchymal cells by inhibited or promoted at S-phase(DNA duplication) of cell cycles.3. In order to study the effects of electromagnetic fields (EMFs) on intercellular cyclic AMP (cAMP) in rat dental papilla mesenchymal cells in vitro, The third passage were divided into 4 groups and stimulated with EMFs. The intercellular cAMP level was investigated at different time points by Radiation immunity assay. The results showed that EMF (50Hz 0.6mT ) increased the intercellular cAMP level (P < 0.05) not only in the 3 days, but also in 5 days. We are led to conclude that the cAMP may be involved in the mediation of the growth inhibitory and differentiation-inducing signals of specific EMFs in vitro.4. In order to study the effect of EMF (50Hz/0.6 mT) on c-myc mRNA level in rat dental papilla mesenchymal cells in vitro, For semi-quantitative RT-PCR, total RNA from dental papilla mesenchymal cells prepared with Trizol was amplified by Superscript II and Taq polymerase. Primer sequences of rat c-myc and GAPDH were designed and synthesized. The PCR program was as follows: 40 cycles for c-myc and GAPDH at 95℃for 20 sec, 55℃for 30 sec, and 72℃for 45 sec. Fluorescence of PCR product was detected by means of an image analyzer. Signals of c-myc and GAPDH mRNA were quantitated and normalized with the respective GAPDH mRNA expression levels, for calculation of relative intensity, with the use of an mage analyzer. The result showed EMF (50Hz/0.6 mT) not effect c-myc mRNA evel in dental papilla mesenchymal cells.