Chronic Fluorosis Patients And Rats Blood And Liver Tissue DNA Repair Gene Methylation Change Research

Objective In order to investigate the relationship between DNArepair gene methylation and fluorosis development, the rate of methylation of repairgene O6-methylguanine-DNA-methyltransferase gene MGMT and mismatch repairgene MLH1were analysed by utilizing the method of methylation specific PCR andthe mRNA and protein levels were deteted by real-time PCR and western blot,respectively. Methods Choose the research object in the endemic fluorosis region,divided the patients into three groups: mild group, moderate group and severe groupaccording to the degree of dental fluorosis and skeletal fluorosis, at the same timeselect the normal villagers as a control group. Utilized the phenol-chloroform methodto extract DNA, and the methylation specific PCR method to detect the genemethylation situation of MGMT, MLH1from the research object. Extract the RNAand detect the variation of the expression level on MGMT, MLH1gene mRNA byreal-time fluorescence quantitative PCR. Establish the model of chronic fluorosis rats,divide the rats into two groups-control group and fluorosis group, each group has20.By adding sodium fluoride50μg/L to the drinking water of the fluorosis rats, raisethem for6months then kill them by bleeding femoral artery, collect their arterialblood and extract the DNA with phenol chloroform method, utilize MSP to detect thepositive rate of MGMT, MLH1gene methylation in every group. Extract the RNA,detect mRNA expression level of MGMT, MLH1gene from each group by real-timefluorescence quantitative PCR. In addition, extract the organization DNA of livertissue from the rats, utilize MSP to detect the positive rate of MGMT, MLH1genemethylation in liver tissue. Extract the liver tissue protein of rats by cracking process, analysis the MGMT, MLH1protein expression change between the different groupwith Western blot technology. Results (1) The MGMT, MLH1gene from thepatients in fluorosis region suffer an obvious methylation, and the methylationpositive rate increases with the fluorine poisoning deepen. The difference hasstatistical significance P<0.05.(2) The mRNA expression level of MGMT、MLH1gene from the patients in fluorosis region is lower than control group, and has anegative correlation with fluorine poisoning degree. The difference has statisticalsignificance P<0.05.(3) The methylation positive rate of MGMT, MLH1gene fromthe rats blood in fluoride group is higher than control group, The difference hasstatistical significance P<0.05.(4) The mRNA expression level of MGMT, MLH1gene from the rats blood in fluoride group is lower than control group, The differencehas statistical significance P<0.05.(5) The methylation positive rate of MGMT,MLH1gene from the rats liver tissue in fluoride group is apparently higher thancontrol group. The difference has statistical significance P<0.05.(6) The mRNAexpression level of MGMT, MLH1gene from the rats liver tissue in fluoride group islower than control group. The difference has statistical significance P<0.05.(7) Theprotein expression level of MGMT, MLH1gene from the rats liver tissue in fluoridegroup is lower than control group. The difference has statistical significance P<0.05.Conclusion The methylation degree of MGMT, MLH1gene from patients’ bloodsamples in fluorosis region has a correlation with fluorosis disease, and the expressionlevel of transcription drops. The methylation positive rate of MGMT, MLH1genefrom rats’blood samples in fluoride group is apparently higher than control group, andthe expression level of transcription drops, this indicates the expression of repair genemay have a certain relationship with fluoride at least in human and animals. Themethylation positive rate of MGMT, MLH1gene from rats’liver samples in fluoridegroup is higher than control group, and the expression level of transcription drops, sodoes the protein expression, this indicates the expression of repair gene may relate toliver damage caused by fluoride.