| There is a high incidence of local fluorosis in china, and skeletal fluorosis had effected many patients exposed to the excessive fluoride. However, the mechanisms of action of fluoride on bone remain unclear. As the vital cell, the osteoblasts have an important function on bone - forming stage.The aim of the present study was to analyse the toxicity in rat osteoblasts induced by fluoride during bone - forming stage. In order to exclude the influence of local factors in vivo, observe the direct effects of fluoride on the single cell, we established the methods of isolating, culturing, and identifying the osteoblasts from the newborn rat calvaria. The proliferation and differentiation of osteoblasts was discussed so that we can learn the effects of fluoride on bone - forming stage from the cellular level; The influence of fluoride on cell growth, cell cycle and apoptosis was investigated through the experiments in vitro and in vivo , and this can offer the basic data for further discussing the mechanisms of the sekeletal damage induced by fluorosis. The responses to fluoride ?induced geno-toxicity was studied for evaluating the toxicity of fluoride completely.MethodsIn vitro experiments:1. Isolation and Culture of OsteoblastCalvariae from 2d - old newborn rats were removed aseptically. The perios-teal layers on both sides were carefully stripped off with tweezers under D -Hanks. Then, the bone specimens were treated by trypsinization for 20 min at37℃ , and the supernatant was discarded. After that the specimens were digested by collagenase for 90 min, the supernatant was collected and centrifuged. The collagenase - release cells were plated in 199 medium supplemented with 15% fetal bovine serum (FBS). The medium was changed twice per week until the cells reached confluence. The differential adherence method was used to purify the osteoblasts. The immunohistochemical method of type I collagen was used to identify the osteoblasts.2. Measurements of proliferation and differentiation of osteoblasts Osteoblasts were collected using 0. 25% trypsin and replated at a density of5 × 104/ ml in 96 - well flat - bottomed plates. The medium was changed to serum - free medium after 24h. The next day sodium fluoride with different concentration (0, 0.01, 0.05, 0. 1, 0.5, 1, 2, 4mmol/L) was administrated. The proliferative response to fluoride was determined by the percents of reduced Alamarblue; The activity of alkaline phosphatase (ALP) was measured by Elisa method.3. Analysis the cell cycle and apoptosisOsteoblasts were plated in flasks at a density of 1 × 105 cells/ml, and allowed to attach for 3d. After that the medium was changed to serum - free medium. Sodium fluoride with different concentration (0, 1, 2, 4mmol/L) was administrated and the cells cultured continuously for 24h. Cells were collected by trypsinization, afte washing with PBS twice, PI was supplemented, and cell cycle and apoptosis was analyzed by Flowcytometry ( FCM).4. Detecting the DNA damageWhen the cells in flasks had reached confluence, Their medium were changed to serum - free medium for 24h. After that sodium fluoride with different concentrations (0, 0.5, 1, 2, 3mmol/L) were administrated and the cells were cultured continuously for further 24h. After that the cells were collected fay trypsinization, washed with PBS, replated at a density of 1 x lOVml, the single cell gel electrophoresis assay was used to study the DNA damage.In vivo experiments:In stock diets condition, Wistar female rats drank distilled water containing0,50,100,150mg/L NaF for 2 months, then they are mated with normal rats. The calvarium of offsprings was used to investigate the effects of fluoride on ul-trastructure by LM and TEM. The osteoblasts of offsprings was used to analysis cell cycle and apoptosis by FCM.StatisticsEXCEL and SPSS 10.0 was used to analysis the results.Results1. Cell IsolationAfter isolating and culturing for 24 h, the active osteoblasts adhered to the wall of flask, and showed the s...
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