Study On The Epigenetic Mechanisms Of Bone Damage In Mice Exposed To Fluoride

Fluoride is an important trace element in the human body,fluoride and human life activities and teeth,bone tissue is closely related to the metabolism,it is a double edged sword,an appropriate amount of good for the body control,will result in excess body injury.Long-term intake of excess fluoride can cause to skeletal fluorosis and dental fluorosis as the main feature of chronic systemic diseases.Epigenetics refers to changes in the absence of DNA sequence,the gene function heritable changes have taken place,leading to phenotypic changes.In recent years,a growing number of documented,many environmental factors that affect human health through epigenetic mechanisms,but the bone damage epigenetic fluoride has been reported.Objective:1.To modify the conventional fluorine measuring device on basic level to construct trace fluorine measuring device,providing the measuring device for subsequent trials.2.At the animal level,build the establishment of fluorosis model,to study the effects of different concentrations of fluoride and the parameters related to bone metabolism in mice.3.To study the impact from various concentration of fluoride on the methylation of the upstream CpG island of the genes BMP-2,DPYSL2,PTP,OT in mouse bone cells on a cellular level.Method:1.Obtaining the trace fluorine measuring device by combining the self-made salt-bridge and micro loading platform with regular device.Detecting the indicators including linearity range,accuracy,percent recovery,reproducibility and so on.Taking toothpaste with fluoride as sample and making a comparison with the data from the regular measuring device to detect the accuracy.2.On an animal level,taking KM mouse as experimental subject to construct the model of drinking water endemic on fluorosis.Dividing mouse into four groups based on the fluoride concentration 0,10,50,100mg/L in water,measuring the fluorine in blood and urine on every Thursday during the period of 20 weeks.After the modeling,puncturing the hearts to obtain blood as alternate,taking grey matter of mouse bone to measure fluorine and detecting the quantity of BMP-2,DPYSL2,PTP,OT in serum from each group by ELISA.3.On cellular level,taking parietal bone osteoblasts from 3d old mouse to cultivate and to conduct fluorine stain in vitro.With the concentration of sodium fluoride,dividing into 3 dye fluoride group(10,50,100mg/L)and a blank normal group(Omg/L),each group was divided into three periods(24,48,72h),each time period set 3 holes.Extracting total RNA of cells from each hole,and conducting MSP,detecting methylation by gel electrophoresis.Results:1.Modified trace fluorine measuring device met the criterion of methodology with the indicators including linearity range,accuracy,percent recovery,reproducibility and so on.The results from toothpaste with fluorine compared with regular device showed no significant difference,while the modification was accurate and effective,the required sample volume as low as 25 ?L,comparison with conventional measuring fluorine device,greatly reducing the amount of sample required.2.After modeling,performance and general observations and blood skeletal fluorosis fluorouracil data based on external behavior of mice fluorine combined analysis,drinking water fluorosis mice successfully constructed.Group comparison,mouse blood and urine fluoride fluorine content,and increased with time.Different groups within the same point in time,the mouse blood and urine fluoride fluorine in drinking water with elevated fluoride concentrations increased.Comparison between the two groups,with increasing fluoride content of bone fluoride concentrations and increased,and serum BMP-2,DPYSL2,OT concentrations increased with increasing concentration of fluorine,PTP with increasing concentrations of fluorine concentration decreases,indicating fluoride on BMP-2,DPYSL2,OT,PTP expression.3.Consumer Law enzyme culture mouse primary osteoblasts,after transfection in vitro by different concentrations of fluorine,detect BMP-2,DPYSL2,OT,PTP upstream CpG island methylation results are shown,PTP,BMP-2,DPYSL2 upstream of the gene CpG island methylation with increasing concentration of fluorine was not detected exhibiting high methylation state,OT gene upstream because it did not contain CpG island methylation its PCR.Conclusion:1:Datas from modified trace fluorine measuring device were accurate and effective,the minimum quantity of the sample to be tested was 25uL,which largely reduced the amount of the sample and laid foundation for the subsequent trails.2.With the increasing of fluorine concentration,fluorine in blood,urinate and bones gradually increased,and fluoride promoted the expression of BMP-2,DPSLY2 and OT,while it suppressed the expression of PTP.3.As the fluoride concentration increased,PTP,DPYSL2,BMP-2 gene upstream CpG island methylation increased the degree of undetected.