| Nicotine is one of the most important substance in the tobacco toxic elements. Nicotine, as an active element, both by mainstream smoke and sidestream smoke,can enter the mother's blood stream and distribute through placenta. Fetal nicotine affects the developing fetus. In this study we established experimental model in vivo,including effect of nicotine on Balb/c mouse tooth germ development. We observed the effects of nicotine on mouse tooth germ development by methods of morphology, immunohistochemistry, Western Blot,RT-PCR and cell culture. A series of investigation about the probable mechanism were done. 1. Morphological change of mouse tooth germ development.At first, experimental animal model of nicotine was established, Balb/c mouse 54, ratio of male and female was 3:1, in the same box. 26 pregnant mice were divided into two groups, experimental and control group. Pregnant mice of experimental group were given intraperitoneal injections of nicotine at a dose of 1.67mg/kg/d from the 6th through the 20th gestational days. Mothers were sacrificed on the 16th, 18th and 20th gestational day. Specimens from 3-5 of nicotine treated fetuses were stained with H-E and observed by light microscope. Specimens from fetuses of 20th pregnant mice were observed with transmission electron microscope.Nicotine group: The vital rate of fetuses and birthweight were decreased. The quantity of tooth was reduced, the tooth germ development was delayed, number of cells was decreased and differentiation degree was weaker.Electron microscopy: In the control group: the plasm of odontblasts was abundant, nucleole of nucleus was clear; plasm of mitochondnia was abundant, and neural crest was clear. In the experimental group, volume of odontoblast was reduced, nucleole of nucleus was not clear, plasm of mitochondnia in enamalepithelium was swelling, the plasm was light, and neural crest was disappeared.There was vesicle in the cell. The cell began to degenerate.lt is suggested that nicotine has toxic effect on fetal mouse tooth germ development.2. Expressing of nicotine acetylcholine receptors in nicotine treated mouse tooth germ development.Pregnant mice were sacrificed on 16th, 18th and 20th day. 3-5mice were selected in experimental and control group for testing. Nicotine acetyleholine receptors by method of immunohistochemistry. Tooth germs were taken from 3 mice of each group, extracting tooth germ protein and RNA, for Western blot and RT-PCR. Immunohistochemically, the expression of nAChR hi normal developing tooth germ was found. After nicotine stimulating, expression of nAchR was elevated. With Western blot, expression of nAChR could be seen in various period of normal developing mouse tooth germ. Compared with control group, after nicotine stimulating, expression of nAchR protein was obvious enhanced on 16th, 18th, 20th mouse tooth germs.In RT-PCR, there was nAchR a 7mRNA in normal developing mouse tooth germ. After nicotine stimulating,expressing of nAchR a 7mRNA was enhanced. It is suggested that the effect on mouse tooth germ developing was apparently mediated by nicotinic acetycholine receptors. There was expression of nAChR in normal developing mouse tooth germ.3. Study on nicotine treated mouse ectomesenchymal cells proliferation, cell cycle and Ca2+ ionIn this study we established experimental model of ectomesenchymal cells in vitro by enzymatic method. The expression of nAChR in ectomesenchymal cells was tested with immunohistochemistry methed. By MTT method, FCM, fluore scent and laser scanning confocal microscopy, we observed the changes in cell proliferation, cell cycle and Ca2+ion by adding a -Bgt, an antagonist of nicotine acetylcholine receptors into the medium of ectomesenchymal cellsTo determine the effect of nicotine on ectomesenchymal cells mediated by a -Bgt, the following experiments were performed. (1) Undifferentiated ectomesenchymal cells of Balb/c mice model in vitro was established.(2) Expression of nAChR was examined by immunohistochemistry. (3) Theinhibito...
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